**Part 1**

## **Applied Environmental Microbiology: Isolation, Characterization and Identification of Bacteria**

### **Rapid and Efficient Methods to Isolate, Type Strains and Determine Species of** *Agrobacterium* **spp. in Pure Culture and Complex Environments**

Malek Shams, Tony Campillo, Céline Lavire, Daniel Muller, Xavier Nesme and Ludovic Vial *Université de Lyon, Ecologie Microbienne Lyon, UMR CNRS 5557, USC INRA 1193 France* 

#### **1. Introduction**

*Agrobacterium* are Alphaproteobacteria common in most soils that closely interact with plants in two respects. Firstly, and as a general trait of the whole taxon, they are rhizospheric bacteria saprophytically living in the root environment (i.e. rhizosphere) of numerous plants. Rhizospheric interactions are generally considered to be of commensal type with no detrimental effect to the plant, but in most instances they are likely beneficial to plants. For evident agronomic purposes it is worthwhile to explore whether agrobacteria are themselves plant growth-promoting rhizobacteria (PGPR) or not. However, this requires an expert determination of the *Agrobacterium* taxonomy. Indeed our current investigations suggest that only some agrobacterial species from the abundant soil *Agrobacterium* guild are selected in the rhizosphere of a given plant. Secondly, but only when they harbor a dispensable Ti plasmid (i.e. tumor inducing plasmid), agrobacteria are plant pathogens able to cause the crown gall disease to most dicots and gymnosperms and some monocots (Pitzscke & Hirt, 2010). Ti plasmids are conjugative and can easily spread in indigenous soil agrobacteria. As a result transconjugant agrobacteria become in turn pathogenic, contributing both to disease spread and perennial soil contamination. An epidemiological survey of crown gall thus also requires expert determination of the *Agrobacterium* taxonomy. A set of biochemical and molecular methods were thus set up to facilitate the taxonomic assessment of agrobacteria either in pure culture or directly from complex environments such as soils, rhizospheres or tumours. After a presentation of the present state of *Agrobacterium* taxonomy, this work provides efficient methods to : i) isolate agrobacteria from complex environments thanks to elective media; ii) determine the *Agrobacterium* genus status of newly isolated strains on the basis of a minimal set of biochemical tests; iii) determine species and type novel isolates of *Agrobacterium* by sequence analysis of relevant marker genes; iv) determine amplicon content and genome architecture; v) detect the presence of *Agrobacterium* and Ti or Ri plasmid directly in complex environments by PCR using selected primers and metagenomic DNA extracted from whole bacterial communities.

#### **2. Current state of agrobacteria taxonomy**

The *Agrobacterium* taxonomy was historically based upon pathogenicity traits that were later found to be determined by dispensable and highly exchangeable plasmids. It is now well known that crown gall and hairy root diseases —that are respectively characterized by tumors and root proliferations on infected plants— are due to the presence of infectious agrobacteria plasmids (i.e. Ti and Ri plasmids for tumor or root inducing plasmids, respectively) (Van Larebeke et al., 1974). However, agrobacteria were isolated and described very early—evidently before the discovery of the plasmidic nature of their pathogenicity. This is why first bacteria isolated from neoformed tissues, which had the ability to reproduce the symptoms, were named *Bacterium tumefaciens* and *Phytomonas rhizogenes*, respectively (Smith & Townsend, 1907; Riker, 1930). For their part, non pathogenic agrobacteria had been isolated earlier from soil and named *Bacillus radiobacter* because they displayed a particular star shape in certain growth conditions (Beijerinck & van Velden, 1902). The genus name *Agrobacterium* was created in 1942 by Conn as a genus very similar but different to the genus *Rhizobium* (Conn, 1942). Hooykaas et al. (1977) and Genetello et al. (1977) showed that Ti plasmid is transferable by conjugation in plasmid free agrobacteria that consecutively become able to incite crown gall. As a result, it was definitively evident that pathogenicity traits led to artificial classification of agrobacteria and could no longer be used to delineate species. In parallel, enzymatic abilities determined with biochemical galleries led to the distinction of several clusters among agrobacteria that were named biovars (Keane et al., 1970; Panagopoulos et al., 1978). Biovars are determined by chromosomal genes and not plasmids, thus tumorigenic, rhizogenic and non-pathogenic strains can be found within the same biovar.

In modern bacterial taxonomy, the criteria to delineate taxa now include genomic information. This has brought considerable modifications to the nomenclature. According to Wayne et al. (1987) and Stackebrandt et al. (2002), homogenous genomospecies is the ultimate criterion to validly delineate a *bona fide* species. Amongst agrobacteria *lato sensu*, genomospecies generally encompass the same set of strains than biovars. Upon this criterion, *Agrobacterium vitis* (i.e. biovar 3)*, A. rubi* and *A. larrymoorei* have been validated as *bona fide* species (Ophel & Kerr, 1990; Holmes & Roberts, 1981; Bouzar & Jones, 2001). For its part biovar 1 is heterogeneous, including ten genomospecies (De Ley, 1973, 1974; Popoff et al., 1984) currently called genomovar G1 to G9 (Mougel et al., 2002) and G13 (Portier et al., 2006 ). Biovar 1 is thus a species complex and not a single bacterial species. Noticeably, G4 contains both type-strains of the formerly described species "*A. tumefaciens*" and "*A. radiobacter*" that should be both renamed *A. radiobacter* for antecedence reasons (Young et al., 2006). However, even though the name "*A. tumefaciens*" is not valid to designate a *bona fide* genomic species, we proposed that the group of closely related species corresponding to biovar 1 should be collectively called "*A. tumefaciens* complex" in order to avoid confusion with the genomic species G4, which must be validly named *A. radiobacter* (Costechareyre et al., 2010). For the ICSP subcommittee on the taxonomy of *Rhizobium* and *Agrobacterium*, this seems to be a good interim solution until genomovars can be formally named (Lindström & Young, 2011). In addition to the epithet *radiobacter* for genomovar G4, *fabrum* and *pusense* have recently been proposed for homogenous species corresponding to genomovar G8 and to genomovar G2, respectively (Lassalle et al., 2011; Panday et al., 2011).

The *Agrobacterium* taxonomy was historically based upon pathogenicity traits that were later found to be determined by dispensable and highly exchangeable plasmids. It is now well known that crown gall and hairy root diseases —that are respectively characterized by tumors and root proliferations on infected plants— are due to the presence of infectious agrobacteria plasmids (i.e. Ti and Ri plasmids for tumor or root inducing plasmids, respectively) (Van Larebeke et al., 1974). However, agrobacteria were isolated and described very early—evidently before the discovery of the plasmidic nature of their pathogenicity. This is why first bacteria isolated from neoformed tissues, which had the ability to reproduce the symptoms, were named *Bacterium tumefaciens* and *Phytomonas rhizogenes*, respectively (Smith & Townsend, 1907; Riker, 1930). For their part, non pathogenic agrobacteria had been isolated earlier from soil and named *Bacillus radiobacter* because they displayed a particular star shape in certain growth conditions (Beijerinck & van Velden, 1902). The genus name *Agrobacterium* was created in 1942 by Conn as a genus very similar but different to the genus *Rhizobium* (Conn, 1942). Hooykaas et al. (1977) and Genetello et al. (1977) showed that Ti plasmid is transferable by conjugation in plasmid free agrobacteria that consecutively become able to incite crown gall. As a result, it was definitively evident that pathogenicity traits led to artificial classification of agrobacteria and could no longer be used to delineate species. In parallel, enzymatic abilities determined with biochemical galleries led to the distinction of several clusters among agrobacteria that were named biovars (Keane et al., 1970; Panagopoulos et al., 1978). Biovars are determined by chromosomal genes and not plasmids, thus tumorigenic, rhizogenic and non-pathogenic

In modern bacterial taxonomy, the criteria to delineate taxa now include genomic information. This has brought considerable modifications to the nomenclature. According to Wayne et al. (1987) and Stackebrandt et al. (2002), homogenous genomospecies is the ultimate criterion to validly delineate a *bona fide* species. Amongst agrobacteria *lato sensu*, genomospecies generally encompass the same set of strains than biovars. Upon this criterion, *Agrobacterium vitis* (i.e. biovar 3)*, A. rubi* and *A. larrymoorei* have been validated as *bona fide* species (Ophel & Kerr, 1990; Holmes & Roberts, 1981; Bouzar & Jones, 2001). For its part biovar 1 is heterogeneous, including ten genomospecies (De Ley, 1973, 1974; Popoff et al., 1984) currently called genomovar G1 to G9 (Mougel et al., 2002) and G13 (Portier et al., 2006 ). Biovar 1 is thus a species complex and not a single bacterial species. Noticeably, G4 contains both type-strains of the formerly described species "*A. tumefaciens*" and "*A. radiobacter*" that should be both renamed *A. radiobacter* for antecedence reasons (Young et al., 2006). However, even though the name "*A. tumefaciens*" is not valid to designate a *bona fide* genomic species, we proposed that the group of closely related species corresponding to biovar 1 should be collectively called "*A. tumefaciens* complex" in order to avoid confusion with the genomic species G4, which must be validly named *A. radiobacter* (Costechareyre et al., 2010). For the ICSP subcommittee on the taxonomy of *Rhizobium* and *Agrobacterium*, this seems to be a good interim solution until genomovars can be formally named (Lindström & Young, 2011). In addition to the epithet *radiobacter* for genomovar G4, *fabrum* and *pusense* have recently been proposed for homogenous species corresponding to genomovar G8 and

to genomovar G2, respectively (Lassalle et al., 2011; Panday et al., 2011).

**2. Current state of agrobacteria taxonomy** 

strains can be found within the same biovar.

Modifications of the nomenclature also happen at the genus level. Although, biovar 2 is a homogenous genomospecies (Mougel et al., 2002; Popoff et al., 1984), this species clearly appears phylogenetically more related to *Rhizobium* species such as *R. etli* or *R. tropici,* than to other *Agrobacterium* species. Therefore, biovar 2 was proposed to be classified in the genus *Rhizobium*, while all other species were kept in the genus *Agrobacterium* (Costechareyre et al., 2010). This proposal had the great advantage to solve the paraphyletic problem of the old classification pointed out by Young et al. (2001)*.* In agreement with taxonomy rule of name antecedence, biovar 2 should be named *Rhizobium rhizogenes*. The epithet for this species is *rhizogenes* because the type-strain ATCC 113225T received this epithet at the time of its first description by Riker (1930). However, most members of this taxon are nonpathogenic or induce crown gall but not hairy root symptoms. This is particularly the case for the famous crown gall biocontrol agent K84 (Kerr, 1974). Frequently named "*A. radiobacter*" , it is actually a true member of the species *Rhizobium rhizogenes* in its present definition.

Fig. 1. Phylogeny of the *recA* gene of type-strains of all *bona fide* genomic species of *Agrobacterium* spp. known to date using the revised nomenclature proposed by Costechareyre et al. (2010). The maximum likelihood method was used. Only significant ML values (>0.95) are given. The branch length unit is the number of substitutions per nucleotidic site. *B. Bradyrhizobium, Rh. Rhodopseudomonas, Az. Azorhizobium, M. Mesorhizobium, E. Ensifer, R. Rhizobium, A. Agrobacterium.*

There are likely numerous other species of *Agrobacterium* either within or out the *A. tumefaciens* complex, such as the unnamed species that includes strain NCPPB 1650, or the novel species that got the epithet *skierniewicense* (Puławska et al., 2011) and—at least provisionally— *nepotum* for the novel *A. tumefaciens* genomovar G14 found by Puławska et al. (unpublished). In addition, it is also likely that bacteria able to induce the formation of nodules with some plants are true members of the genus *Agrobacterium* in its novel definition. It could be the case of the nitrogen fixing bacteria initially called *Allorhizobium undicola* (Costechareyre et al., 2010). A reasonably good view of the phylogeny of the *Agrobacterium* genus is given by the *recA* gene phylogeny (Figure 1) adapted from Costechareyre et al. (2010), completed with sequences from type-strains of novel species *A. skierniewicense* and *A. nepotum* (i.e. genomovar G14). The recent denomination *A. fabrum* and *A. pusense* are used for genomovars G8 and G2, respectively. Table 1 lists type-strains and *recA* accession numbers used to construct the phylogeny.


Table 1. Representative strains and infraspecific diversity of *Agrobacterium* species and related species. T, type-strains. PT, proposed type-strain. LMG, Laboratorium voor Microbiologie, Ghent University, Belgium. *recA*, accession number of *recA* allele of representative strain. (HE610312) is the *recA* accession number of strain C3.4.1. Allele nb, number of strains displaying different MLSA patterns. Genome nb, number of completely sequenced genome. nd, not deposited.

#### **3. Isolation of agrobacteria from complex environments**

Agrobacteria usually form ecological guilds in soils consisting of several genomovars with several strains within genomovars. There are also several strains even within a single tumor (Nesme et al., 1987; Vogel et al., 2003; Costechareyre et al., 2010). As a consequence, it is necessary to take extreme care during strain isolation to be sure of the genotypic purity of isolates. Efficient purification of agrobacteria requires both selective media and the strict respect of the procedure described below.

There are likely numerous other species of *Agrobacterium* either within or out the *A. tumefaciens* complex, such as the unnamed species that includes strain NCPPB 1650, or the novel species that got the epithet *skierniewicense* (Puławska et al., 2011) and—at least provisionally— *nepotum* for the novel *A. tumefaciens* genomovar G14 found by Puławska et al. (unpublished). In addition, it is also likely that bacteria able to induce the formation of nodules with some plants are true members of the genus *Agrobacterium* in its novel definition. It could be the case of the nitrogen fixing bacteria initially called *Allorhizobium undicola* (Costechareyre et al., 2010). A reasonably good view of the phylogeny of the *Agrobacterium* genus is given by the *recA* gene phylogeny (Figure 1) adapted from Costechareyre et al. (2010), completed with sequences from type-strains of novel species *A. skierniewicense* and *A. nepotum* (i.e. genomovar G14). The recent denomination *A. fabrum* and *A. pusense* are used for genomovars G8 and G2, respectively. Table 1 lists type-strains and

Table 1. Representative strains and infraspecific diversity of *Agrobacterium* species and related species. T, type-strains. PT, proposed type-strain. LMG, Laboratorium voor Microbiologie, Ghent University, Belgium. *recA*, accession number of *recA* allele of representative strain. (HE610312) is the *recA* accession number of strain C3.4.1. Allele nb, number of strains displaying different MLSA patterns. Genome nb, number of completely

Agrobacteria usually form ecological guilds in soils consisting of several genomovars with several strains within genomovars. There are also several strains even within a single tumor (Nesme et al., 1987; Vogel et al., 2003; Costechareyre et al., 2010). As a consequence, it is necessary to take extreme care during strain isolation to be sure of the genotypic purity of isolates. Efficient purification of agrobacteria requires both selective media and the strict

**3. Isolation of agrobacteria from complex environments** 

*recA* accession numbers used to construct the phylogeny.

sequenced genome. nd, not deposited.

respect of the procedure described below.

Fig. 2. Typical crown gall symptom (A) and *Agrobacterium* colony morphology on tellurite amended medium (B).

Elective media 1A and 2E, proposed by Brisbane and Kerr (1983) to isolate different biovars, are based on the particular ability of agrobacteria to use certain compounds, such as carbon and energy sources. In addition, these media contain selenite salt (Na2SeO3) because agrobacteria *lato sensu* has the general ability to reduce this salt. However, as the ability to reduce selenite also determines a general ability to resist to toxic amounts of tellurite (K2TeO3), this latter compound was substituted for selenite to improve the medium selectivity, especially when isolating bacteria from complex environments such as soil and decaying tumors (Mougel et al., 2001). Members of the *A. tumefaciens* species complex (i.e. biovar 1) and *A. rubi* can be isolated with 1A-Te containing arabitol as an elective carbon source and 80 mg/l K2TeO3. *R. Rhizogenes* (i.e. biovar 2) can be isolated using 2E-Te containing erythritol and 320 mg/l K2TeO3. Agrobacteria *lato sensu* (i.e. *Agrobacterium* spp. and *Rhizobium rhizogenes*) as well as most *Rhizobiaceae* can be isolated using MG-Te containing mannitol and 200 mg/l K2TeO3 (Table 2). On tellurite-amended media, colonies have the typical circular glistening morphologies of agrobacteria plus a characteristic black color with a metallic shine (Figure 2B).

*Agrobacterium vitis* (i.e. biovar 3) and *Agrobacterium larrymoorei* can be isolated on 3DG medium (Brisbane and Kerr, 1983) or Roy and Sasser medium (Roy and Sasser, 1983). On this latter medium, colonies have a typical pinkish-white (red centre) color. Both media are based on the utilization of sodium L-tartrate as the carbon source. It is likely that the selectivity of these media could be improved by addition of tellurite. This is because *A. vitis* members are resistant to this compound as well as most *Rhizobiaceae* (unpublished results).

For the first purification step, ca. 0.5 g of soil, root, shoot or tumor tissues are crushed with a micropestle in microtube containing 500 µl of sterile distilled water and then indispensably let to macerate for at least 30 min. This maceration is thought to be necessary to allow efficient cell separation from polysaccharides produced by agrobacteria. Next, ca. 25 µl of macerate (100 µl for soil samples) are streaked on appropriate selective media and then incubated at 28°C. Agrobacteria usually require between 3 and 4 days to form colonies on tellurite amended media because this compound tends to slow down bacterial growth. For the second purification step, clear individual colonies with the typical morphology (Figure 2B) are picked and suspended separately in 100 µl of sterile distilled water. These


Table 2. Improved selective media for agrobacteria members (adapted from Mougel et al., 2001).

individual colony suspensions are left to rehydrate overnight with shaking at 28°C before they are streaked onto appropriate media that must NOT contain tellurite. A third purification step following the same procedure is absolutely necessary before introduction of the novel isolate into the lab collection.

#### **4. Presumptive genus determination by minimal biochemical tests**

The INCO-DC European program ERBIC18CT970198, "Integrated Control of Crown Gall in Mediterranean Countries" has delivered a simple and efficient identification scheme for agrobacteria. By a simple and fast urease and/or esculinase biochemical test, any strains isolated from soil, tumor, or root that posseses the typical circulate glistening colony morphology on tellurite amended media can be conviently and confidently classified as part of the genus *Agrobacterium* (Table 3). To test the presence of these activities, fresh cells are suspended in urease solution or esculin solution (Table 3). After incubation at 28°C for 1 h, a colorimetric change is observed for agrobacteria strains: pink and black coloration, respectively, for urease and esculinase tests.

The *A. tumefaciens* species complex (biovar 1) has the enzymatic ability to aerobically convert lactose to 3-ketolactose. This is tested by streaking on medium containing lactose (Bernaerts and De Ley, 1963). After 2 days of growth at 28°C, the plates are flooded with a layer of Benedict's reagents. The presence of 3-ketolactose in the medium is indicated by the formation of a yellow ring around the growth of a positive strain (Table 3 and Figure 3).

*A. fabrum* (G8 genomovar of *A. tumefaciens* species complex), has the biovar 1 characteristics, plus a specific ability to degrade ferulic acid and caffeic acid. These properties are due to a gene cluster present only in the *A. fabrum* strains (Lassalle et al., 2011). A minimal medium supplemented with caffeic acid turns the color brown in five days. As caffeic acid degradation by *A. fabrum* inhibits this browning, inoculation of an isolate of agrobacteria in a minimal medium supplemented with caffeic acid is an easy test to detect if a strain belong to the *A. fabrum* species (Figure 3).

Table 2. Improved selective media for agrobacteria members (adapted from Mougel et al.,

individual colony suspensions are left to rehydrate overnight with shaking at 28°C before they are streaked onto appropriate media that must NOT contain tellurite. A third purification step following the same procedure is absolutely necessary before introduction

The INCO-DC European program ERBIC18CT970198, "Integrated Control of Crown Gall in Mediterranean Countries" has delivered a simple and efficient identification scheme for agrobacteria. By a simple and fast urease and/or esculinase biochemical test, any strains isolated from soil, tumor, or root that posseses the typical circulate glistening colony morphology on tellurite amended media can be conviently and confidently classified as part of the genus *Agrobacterium* (Table 3). To test the presence of these activities, fresh cells are suspended in urease solution or esculin solution (Table 3). After incubation at 28°C for 1 h, a colorimetric change is observed for agrobacteria strains: pink and black coloration,

The *A. tumefaciens* species complex (biovar 1) has the enzymatic ability to aerobically convert lactose to 3-ketolactose. This is tested by streaking on medium containing lactose (Bernaerts and De Ley, 1963). After 2 days of growth at 28°C, the plates are flooded with a layer of Benedict's reagents. The presence of 3-ketolactose in the medium is indicated by the formation of a yellow ring around the growth of a positive strain (Table 3 and Figure 3).

*A. fabrum* (G8 genomovar of *A. tumefaciens* species complex), has the biovar 1 characteristics, plus a specific ability to degrade ferulic acid and caffeic acid. These properties are due to a gene cluster present only in the *A. fabrum* strains (Lassalle et al., 2011). A minimal medium supplemented with caffeic acid turns the color brown in five days. As caffeic acid degradation by *A. fabrum* inhibits this browning, inoculation of an isolate of agrobacteria in a minimal medium supplemented with caffeic acid is an easy test to detect if a strain belong

**4. Presumptive genus determination by minimal biochemical tests** 

2001).

of the novel isolate into the lab collection.

respectively, for urease and esculinase tests.

to the *A. fabrum* species (Figure 3).

Fig. 3. Positive and negative reactions to the 3-keto-lactose test of Bernaert and De Ley (1963) of various *A. tumefaciens* complex and *R. rhizogenes* members (A) and identification of *A. fabrum* by caffeic acid test (B). AT minimal medium (Petit et al., 1978) supplemented with caffeic acid 0.1 mg.ml-1 is browning after five days of incubation except when the medium is inoculated with *A. fabrum* (here *A. fabrum* C58 or J07).


Table 3. Recipe for esculin, urease, and 3-ketolactose tests (adapted from Bernaerts and De Ley, 1963).

#### **5. Species determination and strain typing using marker genes**

#### **5.1 Infraspecific diversity of** *Agrobacterium* **spp. at the genome level**

Multi-locus sequence analysis (MLSA) performed with housekeeping or ecologically relevant genes *recA, mutS, gyrB, glgC, chvA, ampC,* reveals the occurrence of several strains with different alleles in most species (Table 1). As already showed with *chvA* and *recA* (Costechareyre et al., 2009, 2010), these genes allow the identification of novel isolates of *Agrobacterium* spp. at both species and strain levels. Nevertheless, even if MLSA is better than single gene analysis, we found that *recA* alone is definitively a good proxy for the identification of *Agrobacterium* species, since we have so far not found any indication that this gene has been laterally transferred between different species. Identification can be easily done by a nucleotide Blast in databases. Thus, in order to facilitate the identification of *A. tumefaciens* genomovars, we have clearly indicated the genomovar and allele variant status of sequences deposited at EMBL in the description section, as shown below.


Extensive analysis of the infraspecific diversity is now conducted at the whole genome level by comparative genomics. The first analyses were done with DNA microarrays containing probes covering the whole genome of C58. This procedure permitted us to find species specific genes of *A. tumefaciens* genomovar G8 that were used to find typical phenotypic traits of this species (such as the ability to degrade the caffeic acid, Fig. 3). It was then possible to propose this genomovar as a novel species: *A. fabrum* (Lassalle et al., 2011). Comparative genomics of *Agrobacterium* spp. is now done with 22 complete genomes using the Agrobacterscope plateform at Genoscope:

#### https://www.genoscope.cns.fr/agc/microscope/about/collabprojects.php?P\_id=51

However, while MLSA and genome sequencing are suited to type a limited number of strains for taxonomy purposes, these methods are not easily applicable to analyze large agrobacterial populations, as is required for epidemiological investigations. For this reason, we developed a PCR-RFLP approach consisting of the restriction of PCR products obtained from selected genome regions (Figure 4). The intergenic region spanning between the 16S and 23S rRNA genes (16S+ITS) was found to allow the distinction of strains within both the *R. rhizogenes* and *A. tumefaciens* complex (Ponsonnet & Nesme, 1994), with an accuracy we now know to be equivalent to those of *recA* (unpublished results).

Fig. 4. Location of primers used for PCR-RLFP typing of Ti plasmid and chromosome. A, B and C indicate primer locations in the T-DNA region (regions between the *tmr* and *nos* genes), the *virA* and *virB2* genes, and the ribosomal operon (16S and 23S rRNA genes), respectively.

#### **5.2 Ti plasmid diversity**

10 Biochemical Testing

done by a nucleotide Blast in databases. Thus, in order to facilitate the identification of *A. tumefaciens* genomovars, we have clearly indicated the genomovar and allele variant status

Extensive analysis of the infraspecific diversity is now conducted at the whole genome level by comparative genomics. The first analyses were done with DNA microarrays containing probes covering the whole genome of C58. This procedure permitted us to find species specific genes of *A. tumefaciens* genomovar G8 that were used to find typical phenotypic traits of this species (such as the ability to degrade the caffeic acid, Fig. 3). It was then possible to propose this genomovar as a novel species: *A. fabrum* (Lassalle et al., 2011). Comparative genomics of *Agrobacterium* spp. is now done with 22 complete genomes using

https://www.genoscope.cns.fr/agc/microscope/about/collabprojects.php?P\_id=51 However, while MLSA and genome sequencing are suited to type a limited number of strains for taxonomy purposes, these methods are not easily applicable to analyze large agrobacterial populations, as is required for epidemiological investigations. For this reason, we developed a PCR-RFLP approach consisting of the restriction of PCR products obtained from selected genome regions (Figure 4). The intergenic region spanning between the 16S and 23S rRNA genes (16S+ITS) was found to allow the distinction of strains within both the *R. rhizogenes* and *A. tumefaciens* complex (Ponsonnet & Nesme, 1994), with an accuracy we

Fig. 4. Location of primers used for PCR-RLFP typing of Ti plasmid and chromosome. A, B and C indicate primer locations in the T-DNA region (regions between the *tmr* and *nos* genes), the *virA* and *virB2* genes, and the ribosomal operon (16S and 23S rRNA genes),

of sequences deposited at EMBL in the description section, as shown below.

genomovar G1, strain TT111, allele recA-G1-1

now know to be equivalent to those of *recA* (unpublished results).

the Agrobacterscope plateform at Genoscope:

Description: Agrobacterium tumefaciens partial recA gene for recombinase A,

Accession#: FM164286

respectively.

Although agrobacteria are primarily natural soil inhabitants and also commensal rhizospheric bacteria, they become a plant pathogen when they acquire a Ti (Tumor inducing) plasmid (size ~200 kb). Thus it becomes important to characterize agrobacterium plasmid content. Generally agrobacteria of the *A. tumefaciens* complex harbor, besides their chromosome and chromid, another large plasmid called the At plasmid (~400 kb). Large Ti and At plasmids can be visualized on an agarose gel using a method modified from Eckhardt (1978) by Wheatcroft et al. (1990), which is based on the action of gentle bacterium lysis directly in agarose gel wells in order to avoid DNA breakages. Briefly, agrobacterial strains are grown in standard medium to an optical density of 0.2 at 600 nm. After which a total of 1 ml of cell culture is centrifuged at 13,000g for 1 min at 4°C. The pellet is then washed once in 1 ml Na N-lauroyl sarcosinate 0.3% and resuspended in Ficoll buffer (10 mM Tris, pH 7.5, and 0.1 mM EDTA, pH 8.0*,* 20% Ficoll® 400; Sigma) and incubated on ice for 15 min. Then lysis solution consisting of 10 mM Tris, 10 mM EDTA, RNase A (0.4 mg.ml-1), bromophenol red (1mg.ml-1; Sigma), and lyzosyme (1mg.ml-1; Sigma) is added to the cell suspension. Immediately after addition of the lysis solution to cells, 25 µl of this solution is loaded on to a 0.75% agarose gel. Migration is run for 3 h at 100V. Characteristic plasmid size of different *Agrobacterium* and *Rhizobium* strains are given figure 5. In addition, the presence of a linear chromosome (i.e. chromid) can be easily visualized in the *A. tumefaciens* complex, *A. rubi* and *A. larrymoorei* by pulse field electrophoresis (Ramirez-Bahena et al., 2011).

While pathogenic agrobacteria differ from the non-pathogenic strains by the presence of Ti or Ri plasmids, there is also a large diversity within this plasmid class that must be considered in epidemiological investigations. Briefly, the eco-pathology of crown gall and hairy root diseases is centered upon the fact that Ti and Ri plasmids both incite the diseased plant to produce a particular class of compounds called 'opines', which give the agrobacteria a unique ability to use these particular compounds for their growth. There are several kind of opines produced in tumors and consumed by agrobacteria. The most common types of Ti plasmids produce nopaline, octopine or agropine and mannopine (for a review about opines and opine concept see Dessaux et al., 1992). While genes encoding the biosynthesis and the consumption of opines are very different according to opine types, genes involved in the pathogenic process are conserved amongst Ti and Ri plasmids and can be used to define the primers to amplify Ti-plasmid specific regions. Conserved regions are the region of virulence or *vir* region involved in the processing of the DNA transferred from agrobacterium to plant, (i.e. the T-DNA) and T-DNA genes determining the tumoral morphology such as *tmr* or *tms*. Thus, the PCR-RFLP approach is also applicable to study the Ti-plasmid diversity using T-DNA and *vir* regions (Figure 4 and table 4).

There are however a diversity of Ti plasmid conserved genes that can be used to rapidly assess the opine-type of pathogenic agrobacteria. With this idea we developed a set of nopaline-type specific primers in one of the pioneering works using PCR to detect plant pathogenic bacteria (Nesme et al., 1989). Primers F14 and F44 (Table 4) allowed the amplification of a 247 bp-long fragment only with nopaline-type Ti plasmids such as pTiC58 or pTiT37, while primer set F14-F750 permits us to specifically amplify a 217 bp-long amplicon of octopine-type Ti plasmids, such as pTiB6 or pTi15955. The 'universal' primer set F14-F749 was designed based on alignments of *vir* regions of the most common Ti-plasmid opine type (Figure 6). As these primers match different genes separated by intergenic regions of different lengths, amplicon sizes can be used to determine Ti-plasmid opine types.

Fig. 5. Plasmids in *Agrobacterium sp.* and *Rhizobium rhizogenes.* A and B plasmid content determined by the Wheatcroft's method using gentle in well lysis of bacteria. A , *A. fabrum*  C58T*, Rhizobium rhizogenes* K84 and CFBP2408T, *Agrobacterium vitis* LMG8750T. B, *A. fabrum*  C58 derivatives showing At and Ti plasmids of ca. 210 and 450 kbp, respectively. Molecular weight in kb. C, PCR detection of pathogenic *Agrobacterium* in environmental DNAs with primers VCF3 and VCR3. Lane 1, marker 1kb+; 2, negative control with DNA extracted from healthy plant tissue; 3 and 4, positive detections of a *virC1-virC2* region using DNAs extracted from tumors of tomatoes infected three weeks earlier with strain C58.


Table 4. PCR primers used to type agrobacteria by PCR-RLFP or for the detection of Ti and Ri plasmids (adapted from Bruce et al., 1992; Ponsonnet et al., 1994 and Kawaguchi et al., 2005).

#### **5.3 Strain and Ti plasmid diversities in crown gall outbreaks**

In the case study presented below, the PCR-RFLP method was used to type agrobacteria at both chromosomal and Ti plasmid levels. Agrobacteria were isolated from tumors of hybrid poplars (*Populus alba* x *P. termuloides*) in different tree nurseries (Orléans, Loiret, France and Peyrat-le-Château, Haute-Vienne, France) supposedly affected by the same crown gall epidemic. However, results summarized in figure 7 show that the pathogenic isolates belonged to different strains (as indicated by chromosomal ribotypes) of *Rhizobium rhizogenes* (28%) or *Agrobacterium tumefaciens* (72%). For the latter taxon, strains essentially belonged to genomovar G1 (96%). Results also showed a notable diversity of Ti-plasmids that were all of the nopaline-type (data not shown), despite the fact that they were dispatched in four different PCR-RFLP patterns: pTi2516 (50%), pTiM80 (8%), pTi1903 (23%), pTi2177 (9%) and pTi292 (10%).

Fig. 5. Plasmids in *Agrobacterium sp.* and *Rhizobium rhizogenes.* A and B plasmid content determined by the Wheatcroft's method using gentle in well lysis of bacteria. A , *A. fabrum*  C58T*, Rhizobium rhizogenes* K84 and CFBP2408T, *Agrobacterium vitis* LMG8750T. B, *A. fabrum*  C58 derivatives showing At and Ti plasmids of ca. 210 and 450 kbp, respectively. Molecular weight in kb. C, PCR detection of pathogenic *Agrobacterium* in environmental DNAs with primers VCF3 and VCR3. Lane 1, marker 1kb+; 2, negative control with DNA extracted from

healthy plant tissue; 3 and 4, positive detections of a *virC1-virC2* region using DNAs extracted from tumors of tomatoes infected three weeks earlier with strain C58.

Table 4. PCR primers used to type agrobacteria by PCR-RLFP or for the detection of Ti and Ri plasmids (adapted from Bruce et al., 1992; Ponsonnet et al., 1994 and Kawaguchi et al.,

In the case study presented below, the PCR-RFLP method was used to type agrobacteria at both chromosomal and Ti plasmid levels. Agrobacteria were isolated from tumors of hybrid poplars (*Populus alba* x *P. termuloides*) in different tree nurseries (Orléans, Loiret, France and Peyrat-le-Château, Haute-Vienne, France) supposedly affected by the same crown gall epidemic. However, results summarized in figure 7 show that the pathogenic isolates belonged to different strains (as indicated by chromosomal ribotypes) of *Rhizobium rhizogenes* (28%) or *Agrobacterium tumefaciens* (72%). For the latter taxon, strains essentially belonged to genomovar G1 (96%). Results also showed a notable diversity of Ti-plasmids that were all of the nopaline-type (data not shown), despite the fact that they were dispatched in four different PCR-RFLP patterns: pTi2516 (50%), pTiM80 (8%), pTi1903

**5.3 Strain and Ti plasmid diversities in crown gall outbreaks** 

(23%), pTi2177 (9%) and pTi292 (10%).

2005).


Fig. 6. Aligment of *vir*G15-*vir*B11 regions amplifiable with Ti-plasmid-universal primers F14-F749 in Ti plasmids of different opine types. The first box corresponds to F14. The second box corresponds to reverse complement of F749. oct, octopine-type Ti plasmids such as pTi15955 or pTiB6. agr, agropine-mannopine-type Ti plasmid pTiBo542. nop, nopalinetype Ti plasmids such as pTiC58 or pTiT37. Amplicon lengths are 384 bp, 387 bp and 372 bp for octopine-, agropine-mannopine- and nopaline-type Ti plasmids, respectively.

Remarkably, a given Ti plasmid type could be found in different strains while a given strains could harbor different Ti plasmids, likely as the results of Ti plasmid conjugal transfers. In addition, none of the indigenous agrobacteria isolated from a non-contaminated plot harbored a Ti plasmid. Indigenous agrobacteria were found to be different of pathogenic strains isolated from tumors, except for some isolates of ribotype 2520 that harbored a pTi2516. This strongly suggests that indigenous soil agrobacteria —that were initially Ti plasmid free and consequently not pathogenic, such as 2520— were able to receive a Ti plasmid from introduced pathogenic strains, and then in turn become pathogenic. This may facilitate the establishment of Ti plasmids in indigenous soil agrobacteria and could be at the basis of perennial soil contaminations recorded for decades in several instances (Krimi et al., 2002; Costechareyre et al., 2010).

From an epidemiological point of view, the present chromosome and Ti plasmid diversities were found even in single tumors, illustrating the fact that a single isolate is not enough to characterize pathogenic populations involved in crown gall epidemics. However, by studying numerous isolates, we found that pathogenic agrobacteria were almost the same in Orléans and Peyrat-le-Château (data not shown), demonstrating that the crown gall outbreak of the latter nursery was caused by the introduction of apparently healthy (i.e. tumor free) —but in fact contaminated— plant material from Orléans. The same procedure involving both chromosome and Ti plasmid typing of numerous isolates was used to discover the source of crown gall contamination of rose bushes (Pionnat et al., 1999) and is currently used for routine epidemiological investigations.

#### **6. Detection in complex environments**

The detection of pathogenic *Agrobacterium* in complex environments without prior strain isolation can be done easily by PCR using primers designed in the conserved *vir* region (Table 4). Kawaguchi et al. (2005) also reported primers VCF3 and VCR3 amplifying DNA fragments of 414 bp in the *virC1*-*virC2* regions of both Ti and Ri plasmids. Ti plasmid detection can be conducted with metagenomic DNAs extracted from complex environments such as bulk soil, rhizosphere and tumors (Figure 5C). To date, in terms of accuracy and reproducibility the best results are obtained with DNA extracted with the PowerSoil® DNA isolation kit (http://www.mobio.com/soil-dna-isolation/powersoil-dna-isolation-kit.html).

In a similar manner, it is also possible to detect *Agrobacterium* and *Rhizobium* spp. in complex environments by using PCR probes designed in the 16S rDNA gene (Mougel et al., 2001). However, the 16S is not diverse enough to allow the distinction of closely related species such as *A. tumefaciens* genomovars. Instead, Nucleotide sequences of protein encoding genes have a much more resolutive power at both species and infraspecies levels. Nevertheless, the present challenge is to find gene regions well conserved within a species but with marked differences between species in order to design species specific primers. Only a small number of genes fulfill this requirement in *A. tumefaciens*, because genomovars are very closely related but also have a huge infraspecies diversity. Primary results suggest however that *recA* could be a convenient gene to find species specific primers suited for in situ detection of *Agrobacterium* species and genomovars (unpublished results).

Remarkably, a given Ti plasmid type could be found in different strains while a given strains could harbor different Ti plasmids, likely as the results of Ti plasmid conjugal transfers. In addition, none of the indigenous agrobacteria isolated from a non-contaminated plot harbored a Ti plasmid. Indigenous agrobacteria were found to be different of pathogenic strains isolated from tumors, except for some isolates of ribotype 2520 that harbored a pTi2516. This strongly suggests that indigenous soil agrobacteria —that were initially Ti plasmid free and consequently not pathogenic, such as 2520— were able to receive a Ti plasmid from introduced pathogenic strains, and then in turn become pathogenic. This may facilitate the establishment of Ti plasmids in indigenous soil agrobacteria and could be at the basis of perennial soil contaminations recorded for decades

From an epidemiological point of view, the present chromosome and Ti plasmid diversities were found even in single tumors, illustrating the fact that a single isolate is not enough to characterize pathogenic populations involved in crown gall epidemics. However, by studying numerous isolates, we found that pathogenic agrobacteria were almost the same in Orléans and Peyrat-le-Château (data not shown), demonstrating that the crown gall outbreak of the latter nursery was caused by the introduction of apparently healthy (i.e. tumor free) —but in fact contaminated— plant material from Orléans. The same procedure involving both chromosome and Ti plasmid typing of numerous isolates was used to discover the source of crown gall contamination of rose bushes (Pionnat et al., 1999) and is

The detection of pathogenic *Agrobacterium* in complex environments without prior strain isolation can be done easily by PCR using primers designed in the conserved *vir* region (Table 4). Kawaguchi et al. (2005) also reported primers VCF3 and VCR3 amplifying DNA fragments of 414 bp in the *virC1*-*virC2* regions of both Ti and Ri plasmids. Ti plasmid detection can be conducted with metagenomic DNAs extracted from complex environments such as bulk soil, rhizosphere and tumors (Figure 5C). To date, in terms of accuracy and reproducibility the best results are obtained with DNA extracted with the PowerSoil® DNA isolation kit (http://www.mobio.com/soil-dna-isolation/powersoil-dna-isolation-kit.html). In a similar manner, it is also possible to detect *Agrobacterium* and *Rhizobium* spp. in complex environments by using PCR probes designed in the 16S rDNA gene (Mougel et al., 2001). However, the 16S is not diverse enough to allow the distinction of closely related species such as *A. tumefaciens* genomovars. Instead, Nucleotide sequences of protein encoding genes have a much more resolutive power at both species and infraspecies levels. Nevertheless, the present challenge is to find gene regions well conserved within a species but with marked differences between species in order to design species specific primers. Only a small number of genes fulfill this requirement in *A. tumefaciens*, because genomovars are very closely related but also have a huge infraspecies diversity. Primary results suggest however that *recA* could be a convenient gene to find species specific primers suited for in situ

detection of *Agrobacterium* species and genomovars (unpublished results).

in several instances (Krimi et al., 2002; Costechareyre et al., 2010).

currently used for routine epidemiological investigations.

**6. Detection in complex environments** 

Fig. 7. Chromosome and Ti-plasmid diversity of agrobacteria isolated from poplar tumors in a single crown gall epidemic involving two distant nurseries. Ribotypes and Ti-plasmid types were determined by PCR-RFLP of the 16S+ITS region using primers F667-PA and F38 (Table 4), and digestion with *Cfo*I, *Hae*III, *Nde*II and *Taq*I; and primers F9095 and F140, and F9096 and F9097 (Table 4) and digestion with *Taq*I, *Dde*I, *Msp*I and *Rsa*I, respectively as described in Ponsonnet & Nesme (1994). 2520 and 2178 are ribotypes identical to those of *R. rhizogenes* strains CFBP 2520 and CFBP 2178, respectively. 2517, 2177 and 2516: ribotypes identical to those of *A. tumefaciens* genomovar-G1 strains CFBP 2517, CFBP 2177 and CFBP 2516, respectively. pTi2516, pTiM80, pTi1903, pTi2177: nopaline-type Ti-plasmids with PCR-RFLP patterns identical to those of strains M80, CFBP 1903 (i.e. C58) and CFBP 2177, respectively. pTi292: pTi2177 with the insertion element IS292 (Ponsonnet et al., 1995). Tumor indicates total numbers of isolates for each conjugated chromosome and Ti-plasmid patterns obtained in four different plots in Orléans and Peyrat-le-Château. Soil indicates agrobacteria isolated directly from a non-contaminated soil plot in the vicinity of

contaminated plots planted with poplars at Peyrat-le-Château; none of which harboring a Ti plasmid. The dendrogram was built with UPGMA using number of substitutions per nucleotidic sites calculated from RFLP patterns as genetic distance.

#### **7. Conclusion**

The genus *Agrobacterium* encompasses several species and several strains within species that are now clearly named using a novel nomenclature. A set of efficient procedures is available to study either pure culture isolates or agrobacteria directly in soil microbiomes. These methodologies are necessary to unmask the ecologies of *Agrobacterium* species that usually live sympatrically in soils. By this knowledge we expect to differentially manage agrobacterial species directly in soils to improve the crown gall as well as to stimulate their putative PGPR effects.

#### **8. Acknowledgements**

The author wishes to thank J. Puławska that kindly provided strains of novel species before the definitive publication of their results and Cyrus Mallon for reading and correcting the English. This review prepared within the framework of COST Action 873 was supported by the EcoGenome project of Agence Nationale de la Recherche (grant number ANR-08-BLAN-0090).

#### **9. References**


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The genus *Agrobacterium* encompasses several species and several strains within species that are now clearly named using a novel nomenclature. A set of efficient procedures is available to study either pure culture isolates or agrobacteria directly in soil microbiomes. These methodologies are necessary to unmask the ecologies of *Agrobacterium* species that usually live sympatrically in soils. By this knowledge we expect to differentially manage agrobacterial species directly in soils to improve the crown gall as well as to stimulate their

The author wishes to thank J. Puławska that kindly provided strains of novel species before the definitive publication of their results and Cyrus Mallon for reading and correcting the English. This review prepared within the framework of COST Action 873 was supported by the EcoGenome project of Agence Nationale de la Recherche (grant

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### **Biochemical Isolation and Identification of Mycobacteria**

### Wellman Ribón

*Universidad Industrial de Santander Bucaramanga, Colombia* 

#### **1. Introduction**

20 Biochemical Testing

Young, J.M.; Pennycook, S.R. & Watson, D.R.W. (2006). Proposal that *Agrobacterium* 

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de Lajudie et al. 1998 as new combinations: *Rhizobium radiobacter*, *R. rhizogenes*, *R. rubi*, *R. undicola* and *R. vitis*. *International Journal of Systematic and Evolutionary* 

*radiobacter* has priority over *Agrobacterium tumefaciens*. Request for an Opinion. *International Journal of Systematic and Evolutionary Microbiology*, Vol. 56, No. 2, pp.

> The identification of the species that comprise the *Mycobacterium* genus is one of the best documented examples of scientific development and technology. Approximately 150 species are currently recognized that, besides comprising a genus because of structural and biochemical similarities, exhibit a great variety of characteristics that permit the establishment of differential patterns in order to identify and characterize them, or to establish clusters or complexes. At the present time it is insufficient for the performance of tests that would determine if a given specie belongs to the *Mycobacterium* genus. There is clearly a pressing need to establish its membership in a group or complex, and to determine its specie and genetic characterization so as to permit a comparison of characteristics such as frequency and distribution at a global level.

> Within this bacterial genus appear species that, for their public health impact, have been extensively studied; this is in contrast with those that have been recently identified, or of limited frequency, and of which only general characteristics are known.

> These species share conventional microbiological methodologies that are based on the classification made by Dr. Runyon in designating them as photochromogens, scotochromogens, and non-chromogens, depending on their capacity, or inability, to synthesize pigments with or without light stimulus. Other characteristics, such as the speed of growth, have been used in differentiation through a wide variety of biochemical tests. The development of molecular biology contributes to the identification and characterization of species in only one test, thus requiring less time, and promotes the process of biosecurity as it does not necessitate a live microorganism beyond the initial steps of each experimental protocol. The advance of the science is evidenced through the adoption of recent methodologies that quantify volatile compounds through the odors of a patient or from certain clinical specimens; the use of nanoparticles and mass spectrometry appear to be the most promising developments for the very near future.

> In conclusion, the analysis and documentation demonstrate that we do not yet possess the ideal methodology for the identification of mycobacteria. The most promising outlook for the identification of the species that comprise the *Mycobacterium* genus can be found in the recognition of its biochemical characteristics; the use of conventional methodologies; an

appreciation of the techniques of molecular biology in its contribution to science and the mitigation of human suffering, while being reasonable in its use; and an unceasing advancement toward the development and implementation of new methodologies produced through scientific, technical, and social advancement.

#### **2. Phenotypic identification**

The phenotypic identification of mycobacteria is based on the characteristics of the culture and biochemical features, and comprises a large variety of tests, some of which are conducted in order to locate the microorganisms into two large groups:

1. Species that comprise the *Mycobacterium tuberculosis* complex: to date, it has been reported that 9 species are included in this complex and, as the species are closelyrelated genetically, occasionally the phenotypic methodologies are unable to discriminate among some of the species, being able to determine only membership in the *M. tuberculosis* complex (Brosch et al 2002).

*M. tuberculosis*, as a causal agent of tuberculosis, is the species with the greatest worldwide distribution. The other species that comprise this complex are: *M. bovis, M. africanum, Bacille Calmette- Guerín (BCG)* vaccine strain, *M. microti, M. cannetti, M. caprae, M. pinnipedii, and M. mungi.*

The following tests and phenotypic observations identify this complex: velocity of growth; colony morphology; pigment production; niacin test; nitrate reduction; catalase activity; formation of the cord factor; urease test; pyrazinamidase test; growth in the presence of p-nitrobenzoic acid; and, growth in the presence of hydrazide of thiophene-2-carboxylic acid.


The fundamentals of each test used in the identification of mycobacteria are described as follows:

#### **2.1 Characteristics deduced from the direct observation of a culture medium, or growth inhibition, and microscopic observation**

#### **2.1.1 Velocity of growth**

Mycobacteria are grouped according to velocity of growth as either slow or rapid growth. mycobacteria that develop colonies, visible by eyesight, in a culture medium in less than 7 days are classified as rapid growth; those requiring more than 7 days in which to form visible colonies are designated as slow growth. The rRNA comprises 80% of the RNA of mycobacteria and one unit of its DNA. The RNA produced depends upon the number of *rrn*

operons and the efficiency of their transcription. One or two *rrn* operons are found in mycobacteria, regulated by P1 and P2 promoters. The *rrnA operon*, located in the *murA* gene, is found in all species, including *M. tuberculosis* and *M. leprae*; but, in some species, like those of rapid growth, an additional *rrnB* operon is found, located in the *tyrS* gene (Arnvig et al. 2005, Verma et al 1999).

#### **2.1.2 Colony morphology**

Mycobacteria in culture generally appear as two types of colonies: rough or smooth, and with a shiny or opaque aspect (Tasso et al 2003).

#### **2.1.3 Pigment**

22 Biochemical Testing

appreciation of the techniques of molecular biology in its contribution to science and the mitigation of human suffering, while being reasonable in its use; and an unceasing advancement toward the development and implementation of new methodologies

The phenotypic identification of mycobacteria is based on the characteristics of the culture and biochemical features, and comprises a large variety of tests, some of which are

1. Species that comprise the *Mycobacterium tuberculosis* complex: to date, it has been reported that 9 species are included in this complex and, as the species are closelyrelated genetically, occasionally the phenotypic methodologies are unable to discriminate among some of the species, being able to determine only membership in

*M. tuberculosis*, as a causal agent of tuberculosis, is the species with the greatest worldwide distribution. The other species that comprise this complex are: *M. bovis, M. africanum, Bacille Calmette- Guerín (BCG)* vaccine strain, *M. microti, M. cannetti, M. caprae,* 

The following tests and phenotypic observations identify this complex: velocity of growth; colony morphology; pigment production; niacin test; nitrate reduction; catalase activity; formation of the cord factor; urease test; pyrazinamidase test; growth in the presence of p-nitrobenzoic acid; and, growth in the presence of hydrazide of thiophene-

2. Non-Tuberculous Mycobacteria (NTM): reports indicate that about 140 species have been documented, with similar phenotypic characteristics; a large number of biochemical tests, in different culture media, are needed for identification and observation of the growth of the microorganism; various molecules are added to the culture to highlight the differential characteristics among the species that comprise the *Mycobacterium* genus. These tests should be conducted in conjunction with tests that identify the *M. tuberculosis* complex in order to guarantee an adequate analysis of

The fundamentals of each test used in the identification of mycobacteria are described as

Mycobacteria are grouped according to velocity of growth as either slow or rapid growth. mycobacteria that develop colonies, visible by eyesight, in a culture medium in less than 7 days are classified as rapid growth; those requiring more than 7 days in which to form visible colonies are designated as slow growth. The rRNA comprises 80% of the RNA of mycobacteria and one unit of its DNA. The RNA produced depends upon the number of *rrn*

**2.1 Characteristics deduced from the direct observation of a culture medium, or** 

produced through scientific, technical, and social advancement.

the *M. tuberculosis* complex (Brosch et al 2002).

conducted in order to locate the microorganisms into two large groups:

**2. Phenotypic identification** 

*M. pinnipedii, and M. mungi.*

2-carboxylic acid.

identification.

**2.1.1 Velocity of growth** 

follows:

3. *M. leprae*: is a non-cultivable species.

**growth inhibition, and microscopic observation** 

Some species of mycobacteria, classified as photochromogens, produce carotenoid pigment in the presence of light; the group designated as scotochromogenic develops yellow colonies independent of light stimulus; the non-chromogenic group does not produce pigment; their colonies appear with shades of pale yellow or cream, in the presence of light or in darkness, and the color does not intensify when exposed to light (Bernardelli et al 2007, Belén et al 2007).

#### **2.1.4 Growth in presence of P-nitrobenzoic acid**

This test contributes to the differentiation of species of the *M. tuberculosis* complex. Pnitrobenzoic acid inhibits the growth of: *M. tuberculosis*, *M. bovis*, *M. africanum,* and *M. microti (Palomino et al 2007).*

#### **2.1.5 Growth in the presence of hydrazide of thiophene-thiophene-2-carboxilic acid**

Permits the differentiation of *M. tuberculosis* that grows in the presence of the compound as opposed to other species such as *M. bovis*; it is sensitive to the compound when added to the Lowenstein Jensen (LJ) medium. Some species of NMT are positive for this test.

#### **2.1.6 Growth in McConkey agar without violet crystal**

Mycobacteria such as *M. fortuitum*, *M. chelonae, and M. abscessus* have the ability to grow in McConkey agar without violet crystal, as opposed to other mycobacteria such as *M. tuberculosis* and *M. bovis* that cannot grow in this medium.

#### **2.1.7 Growth in 5% sodium chloride**

Species of mycobacteria such as *M. fortuitum* show the capacity to grow in a LJ culture medium to which has been added 5% sodium chloride.

#### **2.1.8 Growth in the presence of hydroxylamine**

This test plays a very important role in determining the difference in capacity of growth of species such as *M. bovis* in LJ media supplemented with a concentration of 250 mg of hydroxylamine. NTM does not have this capability.

#### **2.1.9 Growth in Sauton picric medium and Sauton agar with 0.2% picric acid**

This test is fundamental to differentiate mycobacteria of slow and rapid growth. Mycobacteria of rapid growth, with the exception of *M. chelonae*, have the capacity to grow in this medium. Among mycobacteria of slow growth, *M. simie* is unable to grow in this medium.

#### **2.1.10 Cord formation in acid-alcohol resistant bacilli**

This involves demonstrating the capacity to form cords from aggregates of acid-alcohol resistant bacilli in which the longitudinal axis of the bacteria is parallel to the longitudinal axis of the cord (Tasso et al 2003). This characteristic is attributed to glycolipid trehalose 6,6 dimycolate or cord factor, which is composed of molecules of mycolic acids (Palomino et al 2007). The presence of the cord in Ziehl Neelsen (ZN) coloration from rough, nonchromogenic colonies is indicative of the presence of *M. tuberculosis*; on the contrary, the absence of a cord, or the uniform distribution of bacilli, is consistent with NTM (Tasso et al 2003).

#### **2.2 Biochemical test of identification**

#### **2.2.1 Niacin test**

Niacin is part of the energy metabolism of mycobacteria in redox reactions. All mycobacteria produce niacin, but *M. tuberculosis* accumulates it as a result of the major activity of Nicotinamide Adenine Dinucleotide and the inability to process the resulting Niacine (Palomino et al 2007, Cardoso et al 2004)). The test demonstrates the presence of cyanogen chloride formed through the reaction of chloramine T and potassium thiocyanate in the presence of citric acid. The cyanogen chloride breaks the pyridine ring of niacin, forming the aldehyde gammacarboxyglutamate that binds with the aromatic amine producing a yellow color.

#### **2.2.2 Nitrate reduction test**

Nitrate reductase, an enzyme capable of reducing nitrates to nitrites, appears in the cellular membranes of mycobacteria; the bacteria can utilize this enzyme as a source of nitrogen (Palomino et al 2007). The test detects the presence of nitrate reductase in a medium that contains sodium nitrate. The enzyme reduces nitrate to nitrite that appears through the addition of sulfalinamide and dihydrochloride –N- naphtyl ethylendiamine, forming a complex of diazonium chloride with a fuchsia color ( Bernardelli et al 2007).

#### **2.2.3 Catalase test**

This is an antioxidant enzyme responsible for eliminating molecules of hydrogen peroxide from the cells that are produced during respiration. The reaction results in the release of water and free oxygen (Palomino et al 2007). Two classes of catalase, thermolabile and thermostable, appear in mycobacteria. In *M. tuberculosis* and *M. bovis* the enzymatic activity is inhibited at 68°C; this contrasts with the remaining species of mycobacteria that maintain enzymatic activity following the increase in temperature (Organización Panamericana de la Salud 2008). Oxygen in the form of bubbles, caused by the activity of the enzyme in a solution of perhydrol 30% and Tween 80 at 10%, appears during the test. The height of the column of bubbles can be measured, thus quantifying the enzymatic activity (Bernardelli et al 2007).

#### **2.2.4 Urease test**

24 Biochemical Testing

This test is fundamental to differentiate mycobacteria of slow and rapid growth. Mycobacteria of rapid growth, with the exception of *M. chelonae*, have the capacity to grow in this medium. Among mycobacteria of slow growth, *M. simie* is unable to grow in this

This involves demonstrating the capacity to form cords from aggregates of acid-alcohol resistant bacilli in which the longitudinal axis of the bacteria is parallel to the longitudinal axis of the cord (Tasso et al 2003). This characteristic is attributed to glycolipid trehalose 6,6 dimycolate or cord factor, which is composed of molecules of mycolic acids (Palomino et al 2007). The presence of the cord in Ziehl Neelsen (ZN) coloration from rough, nonchromogenic colonies is indicative of the presence of *M. tuberculosis*; on the contrary, the absence of a cord, or the uniform distribution of bacilli, is consistent with NTM (Tasso et al

Niacin is part of the energy metabolism of mycobacteria in redox reactions. All mycobacteria produce niacin, but *M. tuberculosis* accumulates it as a result of the major activity of Nicotinamide Adenine Dinucleotide and the inability to process the resulting Niacine (Palomino et al 2007, Cardoso et al 2004)). The test demonstrates the presence of cyanogen chloride formed through the reaction of chloramine T and potassium thiocyanate in the presence of citric acid. The cyanogen chloride breaks the pyridine ring of niacin, forming the aldehyde gamma-

Nitrate reductase, an enzyme capable of reducing nitrates to nitrites, appears in the cellular membranes of mycobacteria; the bacteria can utilize this enzyme as a source of nitrogen (Palomino et al 2007). The test detects the presence of nitrate reductase in a medium that contains sodium nitrate. The enzyme reduces nitrate to nitrite that appears through the addition of sulfalinamide and dihydrochloride –N- naphtyl ethylendiamine, forming a

This is an antioxidant enzyme responsible for eliminating molecules of hydrogen peroxide from the cells that are produced during respiration. The reaction results in the release of water and free oxygen (Palomino et al 2007). Two classes of catalase, thermolabile and thermostable, appear in mycobacteria. In *M. tuberculosis* and *M. bovis* the enzymatic activity is inhibited at 68°C; this contrasts with the remaining species of mycobacteria that maintain enzymatic activity following the increase in temperature (Organización Panamericana de la Salud 2008). Oxygen in the form of bubbles, caused by the activity of the enzyme in a

carboxyglutamate that binds with the aromatic amine producing a yellow color.

complex of diazonium chloride with a fuchsia color ( Bernardelli et al 2007).

**2.1.9 Growth in Sauton picric medium and Sauton agar with 0.2% picric acid** 

**2.1.10 Cord formation in acid-alcohol resistant bacilli** 

**2.2 Biochemical test of identification** 

medium.

2003).

**2.2.1 Niacin test** 

**2.2.2 Nitrate reduction test** 

**2.2.3 Catalase test** 

The urease enzyme is coded by the *ureABC* genes and is able to hydrolyze urea, forming two molecules of ammonium; this is used by the mycobacteria in the process of biosynthesis (Palomino et al 2007). The determination is made by using a medium containing red phenol; production of ammonium results in alkalization of the medium with a change in color to fuchsia.

#### **2.2.5 Pyrazinamidase test**

This is an intracellular enzyme codified by the *pncA* gene which is able to hydrolyze pyrazinamide (PZA) in pyrazinoic acid. Some strains present a mutation in the *pncA* gene that generates resistance to PZA, the principal mechanism of resistance in *M. tuberculosis* to this drug. The method of transport of PZA into *M. tuberculosis* is by passive diffusion, where it is converted into pyrazinoic acid through the action of the pyrazinamidase enzyme. Its usefulness as a test of identification is based on the differentiation of *M. tuberculosis* (positive pyrazinamidase) from other species of the *M. tuberculosis* complex (negative pyrazinamidase), with the exception of *M. canetti* which is also positive (Palomino et al 2007, Zhang et al 2003).

#### **2.2.6 Acid phophatase**

The acid phosphatase of some mycobacteria separates the free phenolphthalein from phenolphthalein diphosphate using as a substrate of the reaction the magnesium salt thymophthalein monophosphate. The appearance of a red color is positive for acid phosphatase. The *M. tuberculosis* species is negative in this test.

#### **2.2.7 Hydrolysis of polyoxyethylene mono-oleate**

This shows the capability of some species of mycobacteria to separate the oleic acid which is esterified in polyoxyethylene monoleate; it is better known commercially and in the laboratory environment as "Tween 80".

#### **2.2.8 Arylsulfatase test**

The test is based on the capability of some species of mycobacteria, through the activity of arylsulfatase which acts on sulfate esters, to release its aryl radical. To confirm the activity of the enzyme a color development system should be attached, using phenolphthalein disulfate potassium in the culture medium, that is hydrolyzed by the enzyme producing free phenolphthalein; in the presence of an alkali, a red color appears when the test is positive. Examples of species positive for this test, after three days, are: *M. fortuitum* and *M. abcesuss*; *M*. *gastri* presents a positive reaction only after two weeks.

#### **2.2.9 Other phenotypic test**

Tellurite reduction, oxygen preference, utilization of carbon sources, iron uptake, *B* galactosidase. (See figure 1).

#### **3. Molecular identification of mycobacteria**

The amazing development of biological molecular methods, in the last years, for identification of mycobacteria were implemented for clinical and research uses. Different molecular approaches developed in research laboratories became speedily in diagnostic test. The reference molecular method for identification of mycobateria is the determination of sequences of 16S ribosomal DNA, due to this molecule is highly conserved. (Kirschner et al 1993). The 16S-23S internal transcribed spacer (ITS) sequencing is a supplement to 16S rRNA gene sequencing for identification of closely related species (Roth et al 1998). Others examples of DNA sequencing assays are, *gyrB*, and *rpoB* sequences. The DNA probes used in conjunction with culture methods was employed in many countries for identification of mycobacteria, examples of this methodologies are, AccuProbe, Line probe Assays, INNO LiPA Mycobactera, GenoType Mycobacterium, and GenoType MTBC. Currently, the most reported methodology for mycobacterial identification is the polymerase chain reaction (PCR) restriction-enzyme analysis, this methods is based on the amplification of a 441.bp fragment of the hsp65 gene by PCR and discriminate between all mycobacterial species (*M, tuberculosis* complex species presents the same patrons) (Telenty 1993, Castro et al 2007, 2010, Torres et al 2010).

Different molecular typing methods developed and implemented as a result of the accelerated development of molecular biology have been useful in various contexts of public health and scientific research, among which we can cite: the monitoring of individual clinical isolates of species belonging to the *M. tuberculosis* complex; studies of the transmission of tuberculosis (TB) in urban areas with low, medium, and high rates of transmission; analysis of recent cases of transmission, or TB cases caused by reactivation, reinfection, or mixed diseases; establishment of relationships between remote geographical areas; the influence of transcontinental movements of large numbers of tourists; determination of the geographical origin of isolates; epidemiological analysis of TB outbreaks in closed areas such as prisons, child-care centers, geriatric homes, and indigenous communities; the establishment of the epidemiological nexus of cases that are the product of contacts with a specific patient; and the identification of the index case through a combination of molecular typing techniques.

As typing techniques, both the spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU) methodologies are the most commonly used tools; they provide a high degree of reproducibility of results and every day are reported in more publications that attest to their versatility and wide use throughout the world; but it cannot be denied that other methodologies exist, which are described below, that have made great contributions in typing processes.

#### **3.1 Typing methods**

The *M. tuberculosis* genome is markedly homogeneous with 99.9% similarity and identity to the 16s rRNA sequence, with *M. africanum* and *M. mungi* being species genetically related to

Tellurite reduction, oxygen preference, utilization of carbon sources, iron uptake, *B*

The amazing development of biological molecular methods, in the last years, for identification of mycobacteria were implemented for clinical and research uses. Different molecular approaches developed in research laboratories became speedily in diagnostic test. The reference molecular method for identification of mycobateria is the determination of sequences of 16S ribosomal DNA, due to this molecule is highly conserved. (Kirschner et al 1993). The 16S-23S internal transcribed spacer (ITS) sequencing is a supplement to 16S rRNA gene sequencing for identification of closely related species (Roth et al 1998). Others examples of DNA sequencing assays are, *gyrB*, and *rpoB* sequences. The DNA probes used in conjunction with culture methods was employed in many countries for identification of mycobacteria, examples of this methodologies are, AccuProbe, Line probe Assays, INNO LiPA Mycobactera, GenoType Mycobacterium, and GenoType MTBC. Currently, the most reported methodology for mycobacterial identification is the polymerase chain reaction (PCR) restriction-enzyme analysis, this methods is based on the amplification of a 441.bp fragment of the hsp65 gene by PCR and discriminate between all mycobacterial species (*M, tuberculosis* complex species presents the same patrons) (Telenty 1993, Castro et al 2007,

Different molecular typing methods developed and implemented as a result of the accelerated development of molecular biology have been useful in various contexts of public health and scientific research, among which we can cite: the monitoring of individual clinical isolates of species belonging to the *M. tuberculosis* complex; studies of the transmission of tuberculosis (TB) in urban areas with low, medium, and high rates of transmission; analysis of recent cases of transmission, or TB cases caused by reactivation, reinfection, or mixed diseases; establishment of relationships between remote geographical areas; the influence of transcontinental movements of large numbers of tourists; determination of the geographical origin of isolates; epidemiological analysis of TB outbreaks in closed areas such as prisons, child-care centers, geriatric homes, and indigenous communities; the establishment of the epidemiological nexus of cases that are the product of contacts with a specific patient; and the identification of the index case

As typing techniques, both the spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU) methodologies are the most commonly used tools; they provide a high degree of reproducibility of results and every day are reported in more publications that attest to their versatility and wide use throughout the world; but it cannot be denied that other methodologies exist, which are described below, that have made great contributions in

The *M. tuberculosis* genome is markedly homogeneous with 99.9% similarity and identity to the 16s rRNA sequence, with *M. africanum* and *M. mungi* being species genetically related to

**2.2.9 Other phenotypic test** 

galactosidase. (See figure 1).

2010, Torres et al 2010).

typing processes.

**3.1 Typing methods** 

**3. Molecular identification of mycobacteria** 

through a combination of molecular typing techniques.

the *M. tuberculosis* complex and presenting few mutations (Niobe-Eyangoh et al 2004, Brosh et al 2002). This characteristic has been employed in the development of methodologies that permit the differentiation of species of this complex. As an example of the contribution of this characteristic in the differential identification of *M. tuberculosis* isolates, the following techniques were initially employed: determination of unusual resistance to drugs; serotyping; multilocus enzyme electrophoresis; biochemical heterogeneity, and typing through the use of phages, with the last being the standard technique in use until the end of the 1980's, but presenting problems such as the low number of phage types identified and the labor intensity associated with this experimental protocol. (Jones et al 1975, 1978, Kalndtri et al 2005, Dïaz et al 2003). The genome of the species that comprise the *M. tuberculosis* complex presents occasional recombinations caused by mobile fragments or sequences of DNA known as transposons; these are unstable elements potentially capable of causing rearrangements through transposition, deletion, inversion, and duplication, since they are insertion sequences. These mobile insertion elements are responsible for directing the generation of genetic polymorphisms which are commonly employed for discriminating among different isolates. In the studies of *M. tuberculosis* molecular epidemiology the most frequently used insertion sequence is *IS6110*, which is found exclusively in members of the *M. tuberculosis* complex, presenting between 0 – 25 copies which differ in position and number. The study of this element soon became an epidemiological milestone and a methodology of great clinical contribution (Coros et al 2008, Mostrom et al 2002).

In this context the clinical isolates of the *M. tuberculosis* complex that present the same genetic pattern are defined as groupings and it is believed that these cases could have been caused by a recent infection, being part of the same chain of transmission, as opposed to cases of family origin, nosocomial, reinfections, or reactivations. More recently, with genomic sequencing of various members of the *M. tuberculosis* complex, techniques have been employed such as: Sequencing of single nucleotide polymorphism (SNP) spoligotyping, Fluorescent amplified fragment length polymorphism (FAFLP), and, among others. (National TB Controllers Association 2004, Centers for Diseases Control and Prevention (CDC) 2006 and 2007, Caminero et al 2001, Shamputa et al 2007, Small et al 1994, Kamerbeek et al 1997, Kassama et al 2006, Gibson et al 2008, Kremer et al 2005, Orjuela et al 2010, Hernandez et al 2010).

Currently, the methods of molecular typing of members of the *M. tuberculosis* complex can be grouped as (Mostrom 2002, Narayabab et al 2004, Parra et al 2003, and Diaz 2003):

#### **3.1.1 Genomic methods for DNA studies**

Restriction Fragment Length Polymorphism IS6110 (RFLP IS6110) Polymorphic GC rich sequence (PGRS) Analysis and pulsed-field gel electrophoresis (PFGE)

#### **3.1.2 Methods based on the amplification of specific sequences of DNA using the PCR**

Spoligotyping, Fast ligation mediated PCR (Flip), Double-Repetitive Element (DRE-PCR), MIRU, Ligation Mediated PCR (LM-PCR), Fluorescent Amplified Fragment Length Polymorphism (FAFLP), and Single Nucleotide Polymorphism ( SNP). (Steinlein et al 2001,

Burgos et al 2004, Varela et al 2005, Reisig et al 2005, Kremer et al 2005, Kasama et al 2006, Ashworth et al 2008, Rozo et al 2010, Collins 2011, Li et al 2011, Blaschitz et al 2011).

#### **3.1.3 Typing methods based on genomic DNA**

#### **3.1.3.1 RFLP IS6110**

28 Biochemical Testing

Given the current knowledge of the *M. tuberculosis* genome H37Rv, the use of *IS6110* was, until recently, the most frequently reported method for discriminating among bacteriological clinical isolates of members of the *M. tuberculosis* complex; a reduction in its frequency of use is now observed given new methods such as those based on PCR. The RFLP *IS6110* technique has certain disadvantages such as: being labor-intensive; slow; requiring a large quantity of good-quality DNA (1-2 µg); limited ability to discriminate isolates with less than 6 copies of *IS6110* (< 6 bands in the RFLP pattern); and difficulty in comparing results obtained in different laboratories since an international database does not yet exist due to results being reported in patterns and not codes. This molecular marker possesses stability of approximately one to three years in patterns of RFLP *IS6110,* which is related to the number of copies present, since a larger number of copies imply a greater possibility of transposition, especially among isolates of 8 to 12 copies. In general, studies show a high stability in patterns of RFLP *IS6110* (Van et al 1991, Gordon et al 1999, Diel et al 2002, Mostrom et al 2002, Gopaul 2006).

#### **3.1.3.2 PGRS**

This method has been of great usefulness in typing clinical isolates of members of the *M. tuberculosis* complex that present a low number of copies of the insertion sequence *IS6110*. These repetitive sequences are present in multiple copies through the mycobacterial genome; approximately 26 to 30 copies of these repetitive sequences are found per chromosome in members of the *M. tuberculosis* complex. It has a discriminating power very similar to that of RFLP, requiring DNA of good quality extracted from cultures of approximately 15 days growth in order to conduct the experimental protocol; several weeks are needed in order to obtain the pattern of each bacterial isolate in electrophoresis gel similar to that obtained through analysis of the *IS6110* sequence. It has a low discrimination in isolates that possess multiple copies of this genetic marker. (Burgos et al 2004, Rozo et al 2010).

#### **3.1.3.3 PFGE**

Fig. 1. Flowchart for phenotypic identification of mycobacteria.

This method possesses a high power of discrimination, requiring large concentrations of DNA with purity and quality; for its implementation it is necessary to make various adaptations in the experimental protocol according to the unique requirements of each laboratory. The introduction of PFGE has had great impact in the study and investigation of the mycobacterial genome, above all in the creation of genomic maps of closely related species such as those that comprise the *M. tuberculosis* complex, thus allowing for the establishment of differences through the identification of multiple rearrangements and of the nonrandom location of insertion elements (Wolfgang et al 1998).

#### **3.1.4 Typing methods based on PCR**

#### **3.1.4.1 Spoligotyping**

The direct repetitive regions of the "Direct Repeat" sequence in members of the *M. tuberculosis* complex are composed of multiple direct variant repetitive sequences, each of which is comprised of direct repetitions of approximately 36 pairs of bases separated by unique spacer sequences of 35 to 41 pb, generating a large polymorphism which can be used in molecular epidemiological studies for the differentiation of species of the *M. tuberculosis* complex. The methodology of spoligotyping is based on PCR which is targeted on a small DR sequence sandwiched within a spacer region; a total of 94 sequences have been identified, of which 43 are generally used in the genotyping of isolates. All of these can be simultaneously amplified using a single primer set. The presence or absence of these sequences is determined by hybridization with a set of 43 oligonucleotides derived from *M. tuberculosis* H37Rv. This technique has been shown to be useful in the typing of clinical isolates of species of the *M. tuberculosis* complex, especially in those with less than 6 copies of *IS6110*, and in different clinical samples (Bauer 1999, Doroudchi et al 2000, Parra et al 2003).

#### **3.1.4.2 Flip**

A rapid method with high reproducibility and great power of discrimination, based on the study of the *IS6110* sequence. In comparison with the RFLP *IS6110* methodology, this technique requires small quantities of DNA (1ng) or crude cell lysates. This methodology has limited discriminating power in isolates with less than six copies of the *IS6110* sequence; it gives results in less time than LM-PCR (Reisig et al 2005).

#### **3.1.4.3 DRE – PCR technique**

A methodology with great power of discrimination that consists in the amplification of DNA segments located between the *IS6110* sequence or the polymorphic region rich in guanine cytosine (PGRS). The DRE-PCR method is based on the number of copies and the distance between the repetitive sequences of *IS6110* and PGRS; these distances vary among the different clinical isolates analyzed, and the variations allow a differentiation with respect to the size and number of the amplified fragments of DNA, producing a single pattern band for the different strains of *M. tuberculosis*. It is a method that can be completed using the primary culture of the microorganism and the results can be interpreted after eight hours of work. One of the limitations of this methodology has been the poor power of resolution of the bands procured (Mostron et al 2002).

#### **3.1.4.4 MIRU**

A typing methodology based on the varible number tandem repeat (VNTR) or MIRU, with a power of resolution similar to that produced by RFLP *IS6110*, has been useful for typing of members of the *M. tuberculosis* complex. The MIRU are short elements of DNA (40 to 100 pb) found in tandem repetitions and dispersed in intergenic regions of the genome within members of the complex. Given its discriminating power, among the 41 sequences of MIRU that have been described, only 12 or 16 of them are frequently reported as genetic markers. The clinical isolates that are characterized by this genetic marker are designated with a 12 digit code corresponding to the number of repetitions in each MIRU locus, thereby forming the basis of a system that facilitates global comparison among laboratories (Supply et al 2000, 2001, Evans et al 2004, Narayanan et al 2004, Gibson et al 2005).

#### **3.1.4.5 LM-PCR**

30 Biochemical Testing

The direct repetitive regions of the "Direct Repeat" sequence in members of the *M. tuberculosis* complex are composed of multiple direct variant repetitive sequences, each of which is comprised of direct repetitions of approximately 36 pairs of bases separated by unique spacer sequences of 35 to 41 pb, generating a large polymorphism which can be used in molecular epidemiological studies for the differentiation of species of the *M. tuberculosis* complex. The methodology of spoligotyping is based on PCR which is targeted on a small DR sequence sandwiched within a spacer region; a total of 94 sequences have been identified, of which 43 are generally used in the genotyping of isolates. All of these can be simultaneously amplified using a single primer set. The presence or absence of these sequences is determined by hybridization with a set of 43 oligonucleotides derived from *M. tuberculosis* H37Rv. This technique has been shown to be useful in the typing of clinical isolates of species of the *M. tuberculosis* complex, especially in those with less than 6 copies of *IS6110*,

and in different clinical samples (Bauer 1999, Doroudchi et al 2000, Parra et al 2003).

it gives results in less time than LM-PCR (Reisig et al 2005).

A rapid method with high reproducibility and great power of discrimination, based on the study of the *IS6110* sequence. In comparison with the RFLP *IS6110* methodology, this technique requires small quantities of DNA (1ng) or crude cell lysates. This methodology has limited discriminating power in isolates with less than six copies of the *IS6110* sequence;

A methodology with great power of discrimination that consists in the amplification of DNA segments located between the *IS6110* sequence or the polymorphic region rich in guanine cytosine (PGRS). The DRE-PCR method is based on the number of copies and the distance between the repetitive sequences of *IS6110* and PGRS; these distances vary among the different clinical isolates analyzed, and the variations allow a differentiation with respect to the size and number of the amplified fragments of DNA, producing a single pattern band for the different strains of *M. tuberculosis*. It is a method that can be completed using the primary culture of the microorganism and the results can be interpreted after eight hours of work. One of the limitations of this methodology has been the poor power of resolution of

A typing methodology based on the varible number tandem repeat (VNTR) or MIRU, with a power of resolution similar to that produced by RFLP *IS6110*, has been useful for typing of members of the *M. tuberculosis* complex. The MIRU are short elements of DNA (40 to 100 pb) found in tandem repetitions and dispersed in intergenic regions of the genome within members of the complex. Given its discriminating power, among the 41 sequences of MIRU that have been described, only 12 or 16 of them are frequently reported as genetic markers. The clinical isolates that are characterized by this genetic marker are designated with a 12 digit code corresponding to the number of repetitions in each MIRU locus, thereby forming the basis of a system that facilitates global comparison among laboratories (Supply et al

2000, 2001, Evans et al 2004, Narayanan et al 2004, Gibson et al 2005).

**3.1.4 Typing methods based on PCR** 

**3.1.4.1 Spoligotyping** 

**3.1.4.2 Flip** 

**3.1.4.4 MIRU** 

**3.1.4.3 DRE – PCR technique** 

the bands procured (Mostron et al 2002).

This is a highly reproducible technique that uses a primer directed toward a specific region of insertion sequence *IS6110*, and a second that is targeted to a linker ligated to restricted genomic DNA. This methodology requires small quantities of DNA and has a high power of discrimination (Prodhom et al 1997, Kremer et al 2005).

#### **3.1.4.6 FAFLP**

 The FAFLP is a complementary technique for the typing of *M. tuberculosis*, principally in clinical isolates with less than 6 copies of *IS6110*. It has the limitation of requiring the extraction of DNA from the microorganism in growth; subsequently, this is digested with EcoRI and Msel restriction enzymes, although some studies report using BamHI and Mspl (Mortimer 2001, Sims et al 2002).

#### **3.1.4.7 SNP**

This methodology is based on the premise that MicroRNAs (miRNA) are thought to play important roles in the pathogenesis of diseases. SNPs within miRNAs can change their characteristics via altering their target selection and/or expression, resulting in functional and/or phenotypic changes. This methodology has produced results similar to those obtained from RFLP I*S6110* and spoligotyping. This technique generates a binary code that permits the creation of a comparative database of different studies (Li et al 2011).

#### **3.1.4.8 Other methodologies**

Other methodologies have been developed for the identification and study of mycobacteria such as "in house" PCR (Puerto et al 2007), but statistics have not been reported that would permit an extensive evaluation of these methodologies. There is a large variation among the reported studies which precludes any conclusions regarding their contributions.

### **4. Recent promising technologies for the detection of** *M. tuberculosis*

#### **4.1 Urinary antigen detection**

This method is based on the direct detection of lipoarabinomannan (LAM) in urine, when the mycobacteria are metabolically active; accordingly, this glycolipid is found in the urine of patients with active turberculosis. This antigen can be detected through an ELISA test and the determination presents high ranges of sensitivity (from 38 to 51%) and of specificity, close to 89% (Daley et al 2009, Lawn et al 2009, Mutetwa et al 2009, Reither et al 2009, Deng et al 2011).

#### **4.2 Volatile markers**

Through chromatographic techniques, various molecules or organic compounds have been identified in human clinical samples with the objective of showing their potential use as markers in the surveillance of illnesses. The volatile organic compounds (VOCs) patterns identified in the *M. tuberculosis* specie through sputum samples allow, through the use of an electronic nose based 14-sensor conducting polymer array, the performance of in vitro and in situ studies. Reports indicate that this methodology identifies 100% of positive *M. tuberculosis* cultures from among others and were able to discriminate between sputum containing either *M. tuberculosis* alone, or a mixed infection. The technique is commonly referred to as "breath testing," with the great advantage of not being an invasive procedure; in a small study it distinguished between patients with a positive sputum culture from those with a negative sputum culture (Pavlou et al 2000, 2004, Buszewski et al 2007, Phillips et al 2007).

#### **4.3 Bead-based methods**

This method is based on the application of monoclonal antibodies to nano magnetic beads in order to identify the bacillus. Microsciences Medtech Ltd. (London, United Kingdom) developed a kit of beads coated with a chemical ligand that binds to mycobacteria present in the sputum of patients; the linkage is evident after coloring the preparation and observing it through a fluorescent microscope. Alternative diagnostic techniques have been developed using nanoparticles coupled to PCR, while other methodologies employ monoclonal anti-BCG antibodies. This methodology can be used for detection and identification of *M. tuberculosis* (Kluge et al 2008, Lee et al 2009).

#### **4.4 Simplified smart flow cytometry (S-FC)**

Following the use by many researchers of flow cytometry to count CD4 T cells, the S-FC has been recently proposed for arriving at a rapid diagnosis of active TB among Human Immunodeficiency Virus (HIV) negative patients and those with the TB/HIV coinfection. The S-FC is considered to be of great use when a sputum sample cannot be obtained, or among patients with negative sputum, with the recommendation that it be combined with microbiological procedures (Castiblanco et al 2006, Breen et al 2007, Streitz et al 2007, Janossy et al 2008).

#### **4.5 Broad nucleic acid amplification-mass spectrometry**

The integration of advances in various technologies such as the technique of analysis and separation of masses, bioinformatics, and ionization techniques linked to mass spectrometry, has facilitated their application in the identification and characterization of pathogens. The successful identification of *M. tuberculosis* complex through the pattern of its mycolic acids has been achieved through the use of Electrospray ionization-tandem mass spectrometry (ESI-MS). Other similar methodologies developed by Abbott Laboratories include the IBIS T5000 which is based upon the amplification of genome sequences of the pathogen linked to mass spectrometry (Ecker et al 2008, 2009, Ho et al 2010, Eshoo et al 2010, Grant et al 2010).

#### **5. Detection of drug susceptibility testing**

The increase in TB cases has been accompanied by a growth in the number of patients with isolates of resistant *M. tuberculosis*, thereby confirming TB as a grave, world-wide public health problem. The situation becomes even more complex with the association of *M. tuberculosis* resistant bacilli with HIV, making control more difficult (Bentwich et al 2000, Castiblanco et al 2006, Lemus et al 2004, and World Health Organization (WHO) 2009).

Multidrug-resistant TB (MDR TB), defined as a combined resistance to two first-line medications, isoniazid (INH) and rifampicin (RIF), attained a global level of 500,000 cases in

containing either *M. tuberculosis* alone, or a mixed infection. The technique is commonly referred to as "breath testing," with the great advantage of not being an invasive procedure; in a small study it distinguished between patients with a positive sputum culture from those with a negative sputum culture (Pavlou et al 2000, 2004, Buszewski et al 2007, Phillips et al

This method is based on the application of monoclonal antibodies to nano magnetic beads in order to identify the bacillus. Microsciences Medtech Ltd. (London, United Kingdom) developed a kit of beads coated with a chemical ligand that binds to mycobacteria present in the sputum of patients; the linkage is evident after coloring the preparation and observing it through a fluorescent microscope. Alternative diagnostic techniques have been developed using nanoparticles coupled to PCR, while other methodologies employ monoclonal anti-BCG antibodies. This methodology can be used for detection and identification of *M.* 

Following the use by many researchers of flow cytometry to count CD4 T cells, the S-FC has been recently proposed for arriving at a rapid diagnosis of active TB among Human Immunodeficiency Virus (HIV) negative patients and those with the TB/HIV coinfection. The S-FC is considered to be of great use when a sputum sample cannot be obtained, or among patients with negative sputum, with the recommendation that it be combined with microbiological procedures (Castiblanco et al 2006, Breen et al 2007, Streitz et al 2007,

The integration of advances in various technologies such as the technique of analysis and separation of masses, bioinformatics, and ionization techniques linked to mass spectrometry, has facilitated their application in the identification and characterization of pathogens. The successful identification of *M. tuberculosis* complex through the pattern of its mycolic acids has been achieved through the use of Electrospray ionization-tandem mass spectrometry (ESI-MS). Other similar methodologies developed by Abbott Laboratories include the IBIS T5000 which is based upon the amplification of genome sequences of the pathogen linked to mass spectrometry (Ecker et al 2008, 2009, Ho et al 2010, Eshoo et al 2010,

The increase in TB cases has been accompanied by a growth in the number of patients with isolates of resistant *M. tuberculosis*, thereby confirming TB as a grave, world-wide public health problem. The situation becomes even more complex with the association of *M. tuberculosis* resistant bacilli with HIV, making control more difficult (Bentwich et al 2000, Castiblanco et al 2006, Lemus et al 2004, and World Health Organization (WHO) 2009).

Multidrug-resistant TB (MDR TB), defined as a combined resistance to two first-line medications, isoniazid (INH) and rifampicin (RIF), attained a global level of 500,000 cases in

2007).

**4.3 Bead-based methods** 

Janossy et al 2008).

Grant et al 2010).

*tuberculosis* (Kluge et al 2008, Lee et al 2009).

**4.4 Simplified smart flow cytometry (S-FC)** 

**4.5 Broad nucleic acid amplification-mass spectrometry** 

**5. Detection of drug susceptibility testing** 

the year 2007 and is expected to continue to increase. According to WHO, 85% of these cases are concentrated in 27 countries The clinical significance of resistance has been defined as the in vitro growth of the microorganism in the presence of the critical concentration of the drug used in treatment, which should be equal or superior to 1% of the growth of the microorganism in the absence of the drug. The critical concentration of a drug is the concentration that inhibits the growth of most wild type within a population without affecting the growth of resistant cells that might be present (Kent et al 1985, Woods et al 2007).

The problem of *M. tuberculosis* resistance appears when the resistant mutants that naturally occur in the microbial population are selected as result of inadequate administration of treatment (irregular administration of dosages, poor absorption, or an inadequate treatment plan) (Parson et al 2004).

When a diagnosis is made of a type of resistance to first-line medications such as INH, RIF, ethambutol (EMB), streptomycin (SM), and PZA, the WHO recommends treatment plans that should be adjusted based upon the pattern of resistance identified in the microorganism and each patient's particular state of health. Second-line medications include: aminoglycosides, cycloserine, capreomycin, fluoroquinolones, and para aminosalicylic acid. These treatment plans are prolonged in comparison with those of the first-line, with increased costs and toxicity, resulting in reduced patient compliance with treatment. When second-line treatment plans are not adequately administered, extensively drug-resistant TB (TB XDR) will probably appear, which is defined as TB MDR with greater resistance to any fluoroquinolone and to at least one of the three injectable medications (amikacin, kanamycin, or capreomycin) (Zumia et al 2001,Gillespie 2002, Raviglione et al 2007, 2004, 2003, 2006, 2007, 2008).

The treatment of TB, compared with that used to cure other infectious diseases, has some differences due to the particular physiological characteristics of *M. tuberculosis,* such as generation time and the capacity to enter into long periods of latency with minimal metabolic activity. In the process that arises from the interaction between the bacteria and the host's defenses, populations of bacilli are identified that are differentiated based upon location, metabolic activity, or velocity of multiplication. Characteristics, therefore, vary depending upon location. For example, when located within pulmonary cavities, they actively multiply; as opposed to being situated in the interior of macrophages where reduced pH and oxygen induce a physiology approximating a state of latency; or, if found within the site of necrosis, they are occasionally able to replicate. Due to the characteristics of the bacillus and the interaction with the host, various effective drugs are used, as appropriate, in the treatment of TB for each of the bacterial populations. Another fundamental aspect in the selection and study of drugs used in the treatment of TB involves the study of the cell structure of the microorganism, in which the most studied has been the bacterial cell wall complex, being hydrophobic, with little permeability, thereby precluding the entry of many therapeutic alternatives (Wayne 1974, Guillespi 2002, Coll 2003, Parson et al 2004, De Rossi et al 2006).

INH,RIF, SM, EMB and PZA are first-line medications for their efficacy in bactericidal or bacteriostatic activity, having less adverse effects than other antimycobacterial medications, as opposed to second-line drugs such as aminoglycosides, polypeptides, fluoroquinolones, ethionamide, cycloserine, and para aminosalicylic acid.

Some characteristics of first- and second-line medications used in the treatment of TB are described below in table 1:


References: Svensson et al 1982,Pfyffer et al 1999, Caminero 2003, Coll 2003, Cohenet al 2003, Zhan 2005, CDC 2006, Abate et al 2007, Boldú et al 2007, Loether et al 2010,Cremades et al 2011, Pholwat et al 2011, pholwat et al 2011, Table 1.

*M. tuberculosis* acquires resistance to anti-TB agents through mutations in the genome due to such causes as previous exposure to some drug or rearrangements in restricted regions of the DNA of the bacillus; this generally produces resistance of the microorganism to only one medication. No plasmids or transposable elements are involved in this process. (Musser 1995, Quros et al 2001).

*M. tuberculosis* acquires resistance to anti-TB agents through mutations in the genome due to such causes as previous exposure to some drug or rearrangements in restricted regions of the DNA of the bacillus; this generally produces resistance of the microorganism to only one medication. No plasmids or transposable elements are involved in this process. (Musser

Ciprofloxacin

Ethionamide

Kanamycin

Levofloxacin

**Second line**

Cycloserine

Ofloxacin

Para-aminosalicylic

Synthetic molecule

derive from

Oral

Antagonist in the synthesis of folate from all mycobacteria

200mg/kg

 Bactericidal Active bacilli References: Svensson et al 1982,Pfyffer et al 1999, Caminero 2003, Coll 2003, Cohenet al 2003, Zhan 2005, CDC 2006, Abate

et al 2007, Boldú et al 2007, Loether et al 2010,Cremades et al 2011, Pholwat et al 2011, pholwat et al 2011,

Table 1.

ND

Gastrointestinal disorder; hepatotoxic

2.0

1.0

ND

ND

4.0

salicilic acid

acid

Synthetic molecule

derived of carbonic

Oral and parenteral

Direct inhibition of DNA gyrase

 500mg

 Bactericidal

Active and dormant bacilli

Cross-resistance with other quinolones

Tendinitis, neurotoxicity, intestinal disorders, rash, photosensitivity.

2.0

2.0

1.5-2.0

ND

ND

acid

Natural

(*Streptomyces* 

Oral

Inhibition of essential pyridoxal 5′-

phosphate (PLP)-dependent

10-20mg/kg until

Bacteriostatic

Active and

Alcohol

Neurotoxicity, psychosis, seizures, depression

25-30

40

ND

ND

≤50

dormant bacilli

and bactericidal

1g

enzymes.

*garyphalus* ) Synthetic molecule

derived of carbonic

Oral

Direct inhibition of DNA gyrase

 600-800mg

Bactericidal Active bacilli

Cross-resistance with other quinolones

Gastrointestinal disorder; CNS; hypersensitivity

2.0

2.0

2.0

ND

2.0

acid

 Syntetic Natural

(*Streptomyces* 

Parenteral

Inhibition of protein synthesis

15-30mg/kg until 1g

Bactericidal Active bacilli

Neuromuscular bloking drugs

Lesion of cranial pair VIII, nefrotoxicity

5.0

30

ND

ND

5.0

*kanamyceticus* )

Oral

Inhibition of mycolic acid synthesis

15-30mg/kg until 1g

Bacteriostatic

Active and dormant bacilli

ND

Gastrointestinal disorder; hepatotoxic

5.0

40

5.0

ND

1.25

1995, Quros et al 2001).

**Nature of compound**

**Drug**

INH RIF EMB

**First line** PZA SM

Amikacin

Capreomycin

 Semisynthetic

Natural

(*Streptomyces* 

Parenteral

Inhibition of protein synthesis

15-30mg/kg until 1g

Bacteriostatic Active bacilli

Neuromuscular bloking drugs

Lesion of cranial pair VIII, nefrotoxicity

10.0

40

2.5-3.0

ND

1.25

*capreolus* ) Synthetic molecule

derived of carbonic

Oral and parenteral

Direct inhibition of DNA gyrase

150mg/kg until 12g

Bactericidal Active bacilli

Cross-resistance with other quinolones

Gastrointestinal disorder; CNS; hypersensitivity

2.0

2.0

1.0

ND

2.0

acid

 Parenteral

Inhibition of protein synthesis

 15mg/kg

 Bactericidal Active bacilli

Natural

*(Streptomyces* 

Parenteral

 Blocking RNA translation

 15mg/kg

 Bactericidal Active bacilli

*griseus)*

Synthetic

Oral

Affects the membrane potential for bioaccumulation

25mg/kg

 Bactericidal

Persistant and dormant bacilli

ND Neuromuscular bloking

Nephrotoxicity,

2; 10

4.0

1.0; 4.0

1.0

2.0; 6.0

ototoxicity

drugs, cross resistance

with capreomycin

Neuromuscular bloking drugs

Lesion of cranial pair VIII, nefrotoxicity

4.0

ND

1.5

ND

1.0

Arthritis, dermatitis, hepatotoxicity, gastrointestinal disorders NA

NA

100

ND

100

Synthetic

Oral

Affects cell wall biosynthesis

 20mg/kg Bacteriostatic Active bacilli

Semisynthetic

Oral

Inhibition of transcription by interacting with RNA polymerase

 10mg/kg

 Bactericidal All population

Inhibe anticonceptivos orales, Quinidina.

ND

Optic neuritis, alopecia, hypersensitivity

5.0; 10.0

2.0

5.0; 7.5

2.5

2.5; 7.5

Hepatotoxic, gastric intolerance, hypersensitivity

1.0

40.0

1.0

1.0

2.0

Synthetic

Oral

Inhibition of mycolic acid synthesis

5mg/kg

 Bactericidal

Active and dormant bacilli

Fenitoína

Hepatotoxic, peripheral neuropathy, seizures

0.2; 1.0

0.2

0.1; 0.4

1.0

0.1; 0.4

**Administration way**

**Mechanism of action**

**Daily dose Actividad microbiana**

**activity** 

**Microbial** 

**Susceptible population**

**Pharmacological interactions**

**Side effects**

**7H10 solid medium**

**LJ solid medium**

**MGIT MB/BacT Alert BACTEC 460**

Determining the probability that *M. tuberculosis* will develop spontaneous resistance to two drugs used in treatment can be established through the sum of probabilities; for example, a probability exists of 1x106-1x108 of a combined resistance to INH and RIF, being equal to a total probability of 1x1014. In the process of the development of TB, the total number of possible bacilli in the patient, including cases of chronic cavitary disease, seldom results in this quantity of bacilli (1014). Consequently, the natural development of bacilli resistant to two medications is infrequent; therefore, it is assumed that in the majority of cases, this type of resistance is due to previous exposure to the medications (Long 2000).

Some data suggest that about 17% of new TB cases in a zone of high incidence evidence multiple infections, causing discordance and difficulty in the interpretation of tests and in patient management. The expansion of isolates of resistant *M. tuberculosis* is a concern and creates urgency in the implementation of effective diagnostic measures and control, focusing hopes on the development of a new vaccine, diagnostic methods, and effective alternative therapies (Zumia et al 2001, Richardson et al 2002, Gandhi et al 2006, Palomino et al 2007, WHO 2006, Raizada et al 2009).

#### **5.1 Diagnostic methods of susceptibility**

A great variety of methodologies exist for determining the susceptibility of *M. tuberculosis* to medications, among which include standard tests showing the growth of bacillus, such as the method of multiple proportions and BACTEC MIGT 960. Other methods of great impact, and experiencing growing implementation at a global level, are those based on the amplification of nucleic acids through PCR, designated as genotypic methods (Wilson M. et al 2011). Some of these include microarrays of DNA, and solid- phase hybridization, among others. The aforementioned methodologies possess excellent percentages of sensitivity and specificity; however, they vary in the time required to obtain results, cost, and technical complexity, thus complicating their large-scale use, especially in countries with limited resources that employ conventional methods. Rapid phenotypic techniques have recently been endorsed by WHO, and they constitute good solutions for a fast and timely diagnosis of susceptibility compared with standard methodologies, and with the cost and infrastructure required for molecular techniques (Canettii et al 1963, 1969, Siddiqi et al 1981, Telenti et al 1993, Abate et al 1998, Palomino et al 2002, 2005, Mitchison 2005,Lakshmi et al 2006).

#### **5.1.1 Phenotypic methods**

Phenotypic methods are extensively used and a wide variety of studies are available that establish their principal advantages and disadvantages. They begin with the cultivation of isolates of *M. tuberculosis* obtained from clinical samples processed in an egg- or liquidbased culture medium, some of which can include antibiotics, which are used for direct determination (observation of the microorganism) or indirect (for example, through color reactions) of the growth of the microorganism and subsequent identification of the resistance of the microorganism through its growth in the presence of anti-TB drugs.

#### **5.1.1.1 Mycobacteria Growth indicator Tube (MGIT BACTEC)**

Although various conventional phenotypic methods have been employed in the study of the susceptibility of *M. tuberculosis*, the most frequently reported, using solid media, are: the multiple proportions method; the ratio of resistance and absolute concentrations; and the standard, automated radiometric BACTEC method that, as opposed to the others, uses a culture in liquid medium and the tube growth indicator method. Modifications to the BACTEC system have been adopted in order to avoid the use of radioactive material; these have consisted of using the principle of detecting oxygen consumption and recording the emission of fluorescent signals as a product of the reaction in each test, which is detected and controlled from the interior of the equipment coupled to software algorithms. The system interprets as resistant all cultures with growth equal or greater than that of the tube used as a control in the test (Pffyer et al 1997, 1999, Heifets et al 2000, Lin et al 2009, Yu et al 2011).

#### **5.1.1.2 The Trek Diagnostic System Inc**

This method measures the growth of mycobacteria through changes of pressure that occur in the space between the liquid medium and the cap of the tube where the reaction occurs. Results are obtained in approximately three days (Woods et al 2007).

#### **5.1.1.3 The MB/BacT Alert instrument (bioMérieux)**

This system emits results from an isolate of *M. tuberculosis* when the bottle that contains the microorganism and the drug is positive before the control tube. The result is obtained through measurement of carbon dioxide released to the medium, detected by a sensor located at the bottom of the bottle used in the test (Siddiqi et al 2006, Woods et al 2007, Lin et al 2009).

#### **5.1.1.4 The multiple proportions method**

This method determines the number of mutants resistant to each drug, establishing a relation between the numbers of bacilli that grow in a solid medium with antibiotic as compared with the number of bacilli that grow in a medium without antibiotic. The methodology used with solid medium evaluates the susceptibility of *M. tuberculosis* to firstand second-line medications. Frequently, the method employed uses the LJ culture medium and results are obtained in 21 days, or before, if there are indications of growth (Cannetti et al 1963, 1969, Heifets 2000, WHO 2004, Fisher 2002, Clinical and Laboratory Standards Institute 2003, Woods et al 2007, Pfyffer 1997, 2007).

Currently, BACTEC MGIT (version 960 or 320) and the multiple proportions method are considered as "gold standard" for the diagnosis of TB susceptibility to first- and second-line medications (Pino et al 1998).

As mentioned previously, phenotypic methods of relatively recent implementation now exist, and are noted for having succeeded in establishing a group of non-conventional phenotypic techniques that have been postulated as good diagnostic alternatives for the susceptibility of *M. tuberculosis* depending upon their performance given the particular situations in which they are used.

#### **5.1.1.5 The diagnosis of TB and drug resistance with mycobacteriophages**

This group of methodologies offers rapid results, low cost, and uses only viable cells, thereby differing from molecular methodologies. Two types of tests that use mycobacteriophages have been documented: the phage amplified biological assay (phaB) and the luciferase reported phages (LRPs). Both methodologies study the capacity of a clinical isolate of *M. tuberculosis* to harbor an infectious mycobacteriophage and to permit its replication (Albert 2001, Butt et al 2004, Pai et al 2005, and Kalantri et al 2005).

#### *5.1.1.5.1 The PhaB*

36 Biochemical Testing

multiple proportions method; the ratio of resistance and absolute concentrations; and the standard, automated radiometric BACTEC method that, as opposed to the others, uses a culture in liquid medium and the tube growth indicator method. Modifications to the BACTEC system have been adopted in order to avoid the use of radioactive material; these have consisted of using the principle of detecting oxygen consumption and recording the emission of fluorescent signals as a product of the reaction in each test, which is detected and controlled from the interior of the equipment coupled to software algorithms. The system interprets as resistant all cultures with growth equal or greater than that of the tube used as a control in the test (Pffyer et al 1997, 1999, Heifets et al 2000, Lin et al 2009, Yu et al

This method measures the growth of mycobacteria through changes of pressure that occur in the space between the liquid medium and the cap of the tube where the reaction occurs.

This system emits results from an isolate of *M. tuberculosis* when the bottle that contains the microorganism and the drug is positive before the control tube. The result is obtained through measurement of carbon dioxide released to the medium, detected by a sensor located at the bottom of the bottle used in the test (Siddiqi et al 2006, Woods et al 2007, Lin et

This method determines the number of mutants resistant to each drug, establishing a relation between the numbers of bacilli that grow in a solid medium with antibiotic as compared with the number of bacilli that grow in a medium without antibiotic. The methodology used with solid medium evaluates the susceptibility of *M. tuberculosis* to firstand second-line medications. Frequently, the method employed uses the LJ culture medium and results are obtained in 21 days, or before, if there are indications of growth (Cannetti et al 1963, 1969, Heifets 2000, WHO 2004, Fisher 2002, Clinical and Laboratory Standards

Currently, BACTEC MGIT (version 960 or 320) and the multiple proportions method are considered as "gold standard" for the diagnosis of TB susceptibility to first- and second-line

As mentioned previously, phenotypic methods of relatively recent implementation now exist, and are noted for having succeeded in establishing a group of non-conventional phenotypic techniques that have been postulated as good diagnostic alternatives for the susceptibility of *M. tuberculosis* depending upon their performance given the particular

This group of methodologies offers rapid results, low cost, and uses only viable cells, thereby differing from molecular methodologies. Two types of tests that use

**5.1.1.5 The diagnosis of TB and drug resistance with mycobacteriophages** 

Results are obtained in approximately three days (Woods et al 2007).

**5.1.1.3 The MB/BacT Alert instrument (bioMérieux)** 

Institute 2003, Woods et al 2007, Pfyffer 1997, 2007).

medications (Pino et al 1998).

situations in which they are used.

2011).

al 2009).

**5.1.1.2 The Trek Diagnostic System Inc** 

**5.1.1.4 The multiple proportions method** 

The PhaB determines the protection and amplification of a phage by mycobacteria in clinical specimens (Eltringham et al 1999). The methodology consists of:


#### *5.1.1.5.2 LRPs*

The LRPs are phages harboring the firefly luciferase (*flux*) gene, which produces visible light when expressed in the presence of lucifering and cellular ATP (Hazbon 2004). The LRPs behave as molecular vectors of the *flux* gene that encodes for firefly luciferase in the interior of *M. tuberculosis*. Once this genetic information has been incorporated within the bacterial chromosome, it is expressed, and there is availability of ATP and luciferin, photon emissions are produced that are detected by a photographic film in a luminometer. The registering of light emitted indicates the viability of the bacilli (Riska et al 1997, 1998, 1999, Carriere et al 1997, 2003, Bardarov et al 2003).

#### **5.1.1.6 The E-Test**

The E-Test determines the pattern of susceptibility of *M. tuberculosis* through growth inhibition halos around strips impregnated with known concentrations of different drugs. Solid medium is used in Middlebrook 7H10 agar plates; the microorganism to be studied should be viable and concentrated in the solution that will be used for inoculation of the medium (Heifets et al 1999, Djiba et al 2004).

#### **5.1.1.7 The colorimetric methods**

These methodologies are simple and coupled to oxidation-reduction reactions that employ different indicators. Alamar blue salt is used in the case of REMA and MABA, and bromide 3 (4,5-dimethylthiazol-2-i)-2,5-diphenyltetrazolium, or MTT in the case of TEMA. Fundamentally, the result is obtained through observing a change of color, proportional to the number of viable mycobacteria in the test. TEMA was implemented in 1983 for the study of cellular proliferation and was subsequently utilized to evaluate the viability of microorganisms such as *Staphylococcus aureus* and *Listeria monocytogenes*; it has been extensively used in the study of *M. tuberculosis* resistance. The resulting color in this methodology changes from yellow to purple from the production of insoluble crystals of formazan that can be quantified by spectrophotometry, or a qualitative interpretation can be made by observing the change of color. The methodology provides results within 7 to 9 days in the model proposed by Abate and Mishana for the rapid detection of RIF resistance. The advances of this methodology have included the study of clinical samples applied directly to the test, reporting good values of sensitivity and specificity, close to 100%, for sputum samples. This test permits the determination of the susceptibility of *M. tuberculosis* to firstand second-line drugs in establishing the diagnosis of MDR and XDR TB (Mossmann 1983, Abate et al 1998, Palomino et al 1999, 2002, Morcillo et al 2004, WoldeMeskel et al 2005, Montoro et al 2005, Palomino et al 2007, CDC 2007). The REMA test employs resazurin as an indicator, with an increase in the production of NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD during multiplication of the bacilli to be studied. The viability of the microorganism is evidenced by the change from blue to pink in the indicator color. This test shows values of sensitivity and specificity exceeding 90% in the evaluation of drugs such as RIF and INH, as well as for second-line drugs. Some reports indicate difficulty in the interpretation of results due to intermediate tones in the change of indicator color (BioSource International, O´brien et al 2000, Maeda et al 2001,Palomino et al 2002, Martin et al 2003, Montoro et al 2005, Rivoire et al 2007).

#### **5.1.1.8 Microscopic-observation drug-susceptibility (MODS)**

The MODS are used in various countries with TB MDR problems and permit the rapid observation of the growth of *M. tuberculosis* in culture media with the different first- and second-line drugs employed in the treatment of TB. One of those methods is MODS, that uses an inverted microscope to view the growth of the microorganism. Other methods include a thin layer of agar (TLA) and the HSTB agar method, that use special, transparent, solid culture media, permitting the early observation of the growth of the microorganism with the aid of a conventional microscope (Caviedes et al 2000, Heifets et al 2003, Mejia et al 2004, Robledo et al 2008).

#### **5.1.1.9 The Nitrate Reductase Assay (NRA) or Griess Method**

The NRA is based upon the characteristic expressed by mycobacteria to reduce nitrate to nitrite, is a rapid method used in the TB control programs of various countries for the evaluation of the susceptibility of *M. tuberculosis*; this is coupled to processes that permit observation of color change. The test employs the LJ solid medium (Angeby et al 2002, lemus et al 2006, Palomino et al 2007).

#### **5.1.2 Genotypic methods**

The genotypic molecular methods for the diagnosis of the susceptibility of *M*. *tuberculosis* to drugs used in the treatment of TB are based on genetic determinants of resistance, requiring for its development the amplification of specific segments of DNA, and the identification of point mutations in the amplified products, thereby permitting the detection of resistance to medications (Cockerill et al 1999, Telenti et al 1993, Palomino 2005, Fluit et al 2011). Compared to phenotypic methods, these methodologies have the advantage of not requiring the previous growth of the microorganism, with results being obtained in a short time (24 to 48 hours). The following is a description of some methods:

#### **5.1.2.1 DNA sequencing**

This methodology is the best and most accurate method for the detection of previously known mutations and for the identification of new mutations. It is the "gold standard" method. It is very effective for detecting mutations to RIF in *M. tuberculosis* by amplification of a single target of study in contrast to studies of various genes involved in resistance.

#### **5.1.2.2 DNA microarrays**

38 Biochemical Testing

in the model proposed by Abate and Mishana for the rapid detection of RIF resistance. The advances of this methodology have included the study of clinical samples applied directly to the test, reporting good values of sensitivity and specificity, close to 100%, for sputum samples. This test permits the determination of the susceptibility of *M. tuberculosis* to firstand second-line drugs in establishing the diagnosis of MDR and XDR TB (Mossmann 1983, Abate et al 1998, Palomino et al 1999, 2002, Morcillo et al 2004, WoldeMeskel et al 2005, Montoro et al 2005, Palomino et al 2007, CDC 2007). The REMA test employs resazurin as an indicator, with an increase in the production of NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD during multiplication of the bacilli to be studied. The viability of the microorganism is evidenced by the change from blue to pink in the indicator color. This test shows values of sensitivity and specificity exceeding 90% in the evaluation of drugs such as RIF and INH, as well as for second-line drugs. Some reports indicate difficulty in the interpretation of results due to intermediate tones in the change of indicator color (BioSource International, O´brien et al 2000, Maeda et al 2001,Palomino et al 2002, Martin et

The MODS are used in various countries with TB MDR problems and permit the rapid observation of the growth of *M. tuberculosis* in culture media with the different first- and second-line drugs employed in the treatment of TB. One of those methods is MODS, that uses an inverted microscope to view the growth of the microorganism. Other methods include a thin layer of agar (TLA) and the HSTB agar method, that use special, transparent, solid culture media, permitting the early observation of the growth of the microorganism with the aid of a conventional microscope (Caviedes et al 2000, Heifets et al 2003, Mejia et al

The NRA is based upon the characteristic expressed by mycobacteria to reduce nitrate to nitrite, is a rapid method used in the TB control programs of various countries for the evaluation of the susceptibility of *M. tuberculosis*; this is coupled to processes that permit observation of color change. The test employs the LJ solid medium (Angeby et al 2002,

The genotypic molecular methods for the diagnosis of the susceptibility of *M*. *tuberculosis* to drugs used in the treatment of TB are based on genetic determinants of resistance, requiring for its development the amplification of specific segments of DNA, and the identification of point mutations in the amplified products, thereby permitting the detection of resistance to medications (Cockerill et al 1999, Telenti et al 1993, Palomino 2005, Fluit et al 2011). Compared to phenotypic methods, these methodologies have the advantage of not requiring the previous growth of the microorganism, with results being obtained in a short time (24 to

This methodology is the best and most accurate method for the detection of previously known mutations and for the identification of new mutations. It is the "gold standard"

al 2003, Montoro et al 2005, Rivoire et al 2007).

2004, Robledo et al 2008).

**5.1.2 Genotypic methods** 

**5.1.2.1 DNA sequencing** 

lemus et al 2006, Palomino et al 2007).

**5.1.1.8 Microscopic-observation drug-susceptibility (MODS)** 

**5.1.1.9 The Nitrate Reductase Assay (NRA) or Griess Method** 

48 hours). The following is a description of some methods:

This methodology is based on hybridization and allow for the simultaneous analysis of a large quantity of gene segments involved in resistance. The segments amplified by PCR are marked with a fluorescent substance and hybridized with probes attached to a solid phase. Consistent results have been reported for this methodology in the study of resistance to RIF (*rpoB* gene) and genes implicated in resistance to other drugs such as: *KatG*, *inhA*, *rpsL*, and *gyrA*. The wide-scale implementation of this methodology has been limited due to the required technical expertise of the operator and the sophisticated equipment. The QIAplex test (Qiagen) system has been recently developed which detects 24 mutations in the *katG*, *inhA*, *rpoB*, *rrs*, *rpsL*, and *embB* genes (Gegia et al 2008).

#### **5.1.2.3 The Line Probes Assay (LPA)**

This method is employed to identify *M. tuberculosis* and determine resistance to RIF, identifying mutations in a specific region of the *rpoB* gene through amplification of this segment using PCR-based reverse hybridization, and using biotin in the test. These labeled products of PCR hybridize with immobilized probes coupling to a color development system that permits the subsequent analysis of resulting color patterns. This test detects four of the most frequent mutations in the *rpoB* gene arguing that 75% of the clinical isolates resistant to RIF have one of these four mutations. The method can be developed from clinical samples with results obtained in 48 hours or less (De Beenhouwer et al 1995, Somoskovi et al 2003, Ramaswamy et al 1998, WHO 2007, Ando et al 2011).

#### **5.1.2.4 Single-strand conformation polymorphism (SSCP)**

This methodology detects specific mutations in the region of DNA under study, through the difference in patterns of movement of polyacrylamide gels. The region of the gene involved in resistance should be amplified by PCR and then denatured until two completely separated DNA strands are obtained. These denatured strands migrate by electrophoresis permitting the detection of mutated sequences by the migration pattern, as compared with that which has been established for the wild strain. This methodology has been employed in studies of resistance to RIF; it has not been practical for the investigation of other drugs as it requires the study of various genes involved in resistance to those medications (Kim et al 1997).

#### **5.1.2.5 Real – time PCR or molecular beacons**

This methodology is reported as the most rapid methodology among those most frequently used. This technique includes the use of a probe that is designed in the form of a loop that possesses a sequence that is complementary with the sequence of the gene under study, and with the appearance of a fluorescent moiety in one of its extremities. When the probe locates the complementary sequence, hybridization occurs with the emission of fluorescence; the signal is detected and monitored by the associated system. This methodology is highly sensitive and specific; only one change in the sequence under study prevents hybridization, allowing the identification of point mutations. This is a closed system; therefore, minimizing the possibilities of amplification of contaminants. For the study of resistance to RIF, a set of five beacons has been designed, which includes the study of the entire *rpoB* region in only one test. Results are provided in three hours using this methodology, detecting a minimum of two bacilli present in the sample. The test includes the study of genes such as *katG*, *inhA*, *oxyR-ahpC*, and *kasA* (Cockeril 1999, Fluid et al 2001, Piatek et al 1998, 2000, Torres et al 2000, De Viedman 2003).

#### **5.1.2.6 Fluorescence resonance energy transfer (FRET) probes**

The FRET is a widely-studied, well-established methodology referred to as "light cycler probes." It is highly specific in identifying mutations in real-time PCR tests. The test permits the detection of RIF and INH resistance in less than two hours with the advantage of facilitating the study of large gene segments involved in resistance (Garcia et al 2002, Torres et al 2003).

#### **5.1.2.7 Other methodologies**

Other methodologies have been developed for the study of resistance to drugs used in the treatment of TB such as "in house" PCR, but statistics have not been reported that would permit an extensive evaluation of these methodologies. There is a large variation among the reported studies which precludes any conclusions regarding their contributions.

#### **5.1.2.8 Amplification refractory mutation system (ARMS)**

This methodology uses a set of primers, one of which should hybridize with the mutation site; in the absence of this, there is not a place for the amplification of the gene involved in resistance; therefore, the band of the expected product is not observed in the electrophoresis gel. This methodology is used for the study of mutations in the *rpo*B, *katG* y, and *embB* genes with good results (Mokrousov et al 2002, Fan et al 2003).

#### **5.1.2.9 Branch migration inhibition (BMI)**

The BMI requires two reactions from PCR, one using DNA from a known strain, and the other using DNA which is the object of study, using a primer labeled with digoxigenin or biotin. This test has been used to study resistance to RIF and PZA; a disadvantage is the identification of silent mutations that conflict with findings reported by phenotypic methods (Lishanski et al 2000, Lyiu et al 2000).

#### **6. Conclusion**

The implications of the processes in which mycobacteria participate are diverse and documented but there is a lack of knowledge concerning many of their characteristics. Health, environment, industry, and research are involved with these mycobacterial species in order to advance or improve their processes, verify their findings, or confirm their suspicions. In each of these fields the principal objectives are the detection, identification, and characterization of the species of the *Mycobacterium* genus; only the knowledge of its biological characteristics together with the availability of methods for its study can help us to achieve a complete documentation. The disciplined, periodic and timely revision of the biology, tendencies, innovations, and discoveries regarding this microbial genus should prove to be the best allies of academics, researchers and the general community in addressing any situation that involves mycobacteria in its processes.

#### **7. References**

40 Biochemical Testing

five beacons has been designed, which includes the study of the entire *rpoB* region in only one test. Results are provided in three hours using this methodology, detecting a minimum of two bacilli present in the sample. The test includes the study of genes such as *katG*, *inhA*, *oxyR-ahpC*, and *kasA* (Cockeril 1999, Fluid et al 2001, Piatek et al 1998, 2000, Torres et al 2000,

The FRET is a widely-studied, well-established methodology referred to as "light cycler probes." It is highly specific in identifying mutations in real-time PCR tests. The test permits the detection of RIF and INH resistance in less than two hours with the advantage of facilitating the study of large gene segments involved in resistance (Garcia et al 2002, Torres

Other methodologies have been developed for the study of resistance to drugs used in the treatment of TB such as "in house" PCR, but statistics have not been reported that would permit an extensive evaluation of these methodologies. There is a large variation among the

This methodology uses a set of primers, one of which should hybridize with the mutation site; in the absence of this, there is not a place for the amplification of the gene involved in resistance; therefore, the band of the expected product is not observed in the electrophoresis gel. This methodology is used for the study of mutations in the *rpo*B, *katG* y, and *embB* genes

The BMI requires two reactions from PCR, one using DNA from a known strain, and the other using DNA which is the object of study, using a primer labeled with digoxigenin or biotin. This test has been used to study resistance to RIF and PZA; a disadvantage is the identification of silent mutations that conflict with findings reported by phenotypic methods

The implications of the processes in which mycobacteria participate are diverse and documented but there is a lack of knowledge concerning many of their characteristics. Health, environment, industry, and research are involved with these mycobacterial species in order to advance or improve their processes, verify their findings, or confirm their suspicions. In each of these fields the principal objectives are the detection, identification, and characterization of the species of the *Mycobacterium* genus; only the knowledge of its biological characteristics together with the availability of methods for its study can help us to achieve a complete documentation. The disciplined, periodic and timely revision of the biology, tendencies, innovations, and discoveries regarding this microbial genus should prove to be the best allies of academics, researchers and the general community in

addressing any situation that involves mycobacteria in its processes.

reported studies which precludes any conclusions regarding their contributions.

**5.1.2.6 Fluorescence resonance energy transfer (FRET) probes** 

**5.1.2.8 Amplification refractory mutation system (ARMS)** 

with good results (Mokrousov et al 2002, Fan et al 2003).

**5.1.2.9 Branch migration inhibition (BMI)** 

(Lishanski et al 2000, Lyiu et al 2000).

**6. Conclusion** 

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