**2.1.1 Repetitive element sequence-based PCR (rep-PCR)**

A variety of repetitive DNA sequence elements have been identified in bacterial pathogens, which have been exploited to develop strategies for bacterial typing. Rep-PCR is a simple PCR-based technique that targets multiple copies of repetitive elements in the bacterial genome to generate DNA fingerprints (Versalovic et al. 1991). Primers designed to anneal in the outward direction, near the end of these repetitive elements bind to multiple noncoding, repetitive sequences interspersed throughout the bacterial genome. Multiple DNA

The results of bacterial strain typing have many different applications including outbreak investigation and surveillance in clinical care settings and public health investigations and also within other contexts such as food and pharmaceutical industries and

The aim of this chapter is to provide an overview of the methods available for analyzing bacterial isolates, focusing on those methods employed for typing *Streptococcus pneumoniae* and *Staphylococcus aureus*. Different molecular approaches have been used to better understand the epidemiology of these medically relevant gram-positive cocci (Willems.

The application of molecular biology tools to infectious disease epidemiology is perhaps just as revolutionary in advancing knowledge and concepts in epidemiology. Genotypic typing

The advantages of nucleic acid-based typing systems lie in that they are less likely to be affected by growth conditions or the laboratory manipulations to which organisms are subjected. Undoubtedly, genetic materials undergo changes due to natural or artificial

Compared to the classical phenotypic typing techniques, genotypic typing techniques have

A molecular technique must take into consideration the relative accumulation of variation (short or long term) of a targeted set of genes in a pathogen. Nearly all the typing systems can be grouped into variants of just three basic analytical procedures: (i) PCR, (ii) the use of restriction enzymes, and (iii) nucleic acid sequencing. These procedures allow for the use of common equipment and standard reagents to analyze many different types of infectious agents. In addition, genotypic characterization of pathogens facilitates standardization of information storage and data analyses, interpretation, and communication, which are all

In the last years, a number of PCR-based strategies have been developed for use as typing tools. PCR can be readily performed with commercially available supplies and there is little variation in the reagents and equipment needed to perform PCR assays from different microorganisms. The major advantages of PCR-based techniques are speed and simplicity.

A variety of repetitive DNA sequence elements have been identified in bacterial pathogens, which have been exploited to develop strategies for bacterial typing. Rep-PCR is a simple PCR-based technique that targets multiple copies of repetitive elements in the bacterial genome to generate DNA fingerprints (Versalovic et al. 1991). Primers designed to anneal in the outward direction, near the end of these repetitive elements bind to multiple noncoding, repetitive sequences interspersed throughout the bacterial genome. Multiple DNA

selective pressures, but this mechanism is exactly the basis for their typeability.

several advantages such as general applicability and a high discriminatory power.

environmental analysis.

**2. Genotypic methods** 

methods assess genome variation in bacterial isolates.

amenable to computer-assisted manipulations.

**2.1.1 Repetitive element sequence-based PCR (rep-PCR)** 

**2.1 PCR-based typing methods** 

et al. 2011).

fragments between those sites (interrepeat fragments) are amplified. Since the number and location of the repetitive elements are variable, the sizes and number of effectively amplified fragments vary depending on the strain (Figure 1).

Fig. 1. Schematic representation of REP-PCR assay. On the right BOX-PCR patterns of *S. pneumoniae* isolates using BOXAR1 primer.

Two different Rep-PCR have been used for typing enteric bacteria: a 38-bp repetitive extragenic palindromic element (REP) and a 126-bp enterobacterial repetitive intergenic consensus (ERIC) sequence (Versalovic et al. 1991), whose function has not yet been elucidated.

A BOX repetitive element is a highly conserved repeated DNA element that has been identified in the *Streptococcus pneumoniae (*pneumococcus) chromosome. Although the function of this element has not yet been completely understood, it has been demonstrated that the presence of a BOX element is associated with variation in colony opacity of the pneumococcus (Saluja & Weiser 1995). BOX-PCR has been effectively used for typing *S. pneumoniae* as well as other bacterial species (van Belkum et al. 1996).

Several genetic elements have been used for developing Rep-PCR to type *Staphylococcus aureus*. The element IS*256* occurs in the genome either independently or as part of the composite transposon Tn*4001*, IS*256* insertion position is strain-specific and spaced close enough to allow amplification of polymorphic inter-IS*256* element sequences (Deplano et al. 1997). Another element used for this methodology is RW3A, a repetitive sequence initially found in *Mycoplasma pneumoniae*, which also generates strain-specific DNA fragments when *S. aureus* DNA is used as template (van der Zee et al. 1999).

#### **2.1.2 Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR) or Arbitrarily-Primed PCR (AP-PCR)**

Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR), also referred to as Arbitrarily-Primed PCR (AP-PCR), is a variation of the PCR technique employing a single, generally short primer, that is not targeted to amplify a specific bacterial DNA sequence. Low annealing temperatures are used during amplification, allowing imperfect hybridization at multiple chromosomal locations. When the primer binds in two sites on opposite strands, at the proper orientation and with sufficient affinity to allow the initiation of polymerization,

Application of Molecular Typing Methods to the

*IS*256 and *mecA* gene.

band patterns.

fragments.

(Goering 2010; Halpin et al. 2010).

Study of Medically Relevant Gram-Positive Cocci 117

of this technique has declined, mainly due to its limited discriminatory power compared to other techniques. Several DNA probes have been employed for the study of methicillinresistant *S. aureus* (MRSA) outbreaks, including various insertion sequences, such as *IS*431,

Fig. 3. Schematic representation of restriction endonuclease analysis by Southern-blotting assay. On the right *IS*6110-restriction fragment length of *Mycobacterium tuberculosis* isolates.

Pulsed-field gel electrophoresis is based on the digestion of bacterial DNA with restriction endonucleases that recognize few sites along the chromosome, generating large DNA fragments (30-800 Kb) that cannot be effectively separated by conventional electrophoresis. The basis for PFGE separation is the size-dependent time-associated reorientation of DNA migration achieved by periodic switching of the electric field in different directions. The DNA fragments will form a distinctive pattern of bands in the gel, which can be analyzed visually and electronically (Figure 4 A). Bacterial isolates with identical or very similar band patterns are more likely to be related genetically than bacterial isolates with more divergent

This technique is laborious and includes several steps, requires good standardization and takes at least two days for obtention of results. Procedures will differ to some extent

Regarding DNA preparation, PFGE requires intact DNA for restriction endonuclease treatment. The risk of mechanical breakage to DNA molecules during the extraction procedure is avoided by embedding intact organisms into agarose plugs where cells are enzymatically lysed and cellular proteins digested. After endonuclease treatment, the agarose plugs containing the digested DNA are then submitted to PFGE (Figure 4B). The choice of the restriction enzyme for DNA digestion and pulse-time switching parameters for PFGE are critical variables for the obtention of restriction profiles to show well- resolved

Recent protocols can be completed in as little as two days through shortcuts such as the direct addition of lytic enzymes to the agarose mixture before the blocks are cast and also high temperature short-term washes which facilitate the extraction of unwanted compounds

Isolates with identical PFGE patterns were considered to represent the same epidemiological type. Isolates differing by one genetic event were considered epidemiologically-related subtypes, expecting that a single genetic event could occur in the chromosome of an

**2.2.2 Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA** 

depending on the organism that is being analyzed.

the amplification of the fragment between those sites will occur. The amplified products will be various different-sized fragments that can be resolved by conventional agarose gel electrophoresis (Figure 2).

Although the method is much faster than many other typing methods, it is much more susceptible to technical variation. Slight variations in the reaction conditions or the reagents can lead to difficulty in result reproducibility and in the band patterns generated. Therefore, trying to make comparisons among potential outbreak strains can be very problematic (van Belkum et al. 1995). When RAPD-PCR is tightly controlled, it can provide a high level of discrimination, especially when multiple amplifications with different primers are performed.

PCR-based typing methods are simplest and rapid genotyping methods, but is remarkable for its susceptibility to minor variations in experimental conditions.

Fig. 2. Schematic representation of RAPD-PCR assay. On the right RAPD-PCR patterns of *Enterococcus faecalis* isolates using D8635 primer.

#### **2.2 Based on enzymatic restriction of chromosomal DNA**

#### **2.2.1 Restriction endonuclease analysis of chromosomal DNA by hybridization with a nucleic acid probe (Southern blotting)**

Following digestion with high frequency restriction endonucleases, chromosomal DNA is separated into different-sized fragments by conventional agarose gel electrophoresis, but this type of polymorphism is difficult to interpret due to the high number of fragments generated. However, interpretation of these polymorphisms can be facilitated by a Southern blot hybridization technique. By this methodology, fragments are separated by electrophoresis and transferred to a nitrocellulose or nylon membrane and hybridized using specific chemically or radioactively-labeled probes (Figure 3). DNA probes are designed for specific sequences that are found in multiple copies and in different positions of the chromosome. One of the most frequently used probes is ribosomal RNA (16s rRNA) because most species have more than one chromosomal rRNA operon distributed around the chromosome. This particular technique is denominated ribotyping. In recent years, the use

the amplification of the fragment between those sites will occur. The amplified products will be various different-sized fragments that can be resolved by conventional agarose gel

Although the method is much faster than many other typing methods, it is much more susceptible to technical variation. Slight variations in the reaction conditions or the reagents can lead to difficulty in result reproducibility and in the band patterns generated. Therefore, trying to make comparisons among potential outbreak strains can be very problematic (van Belkum et al. 1995). When RAPD-PCR is tightly controlled, it can provide a high level of discrimination, especially when multiple amplifications with different primers are performed. PCR-based typing methods are simplest and rapid genotyping methods, but is remarkable

Fig. 2. Schematic representation of RAPD-PCR assay. On the right RAPD-PCR patterns of

**2.2.1 Restriction endonuclease analysis of chromosomal DNA by hybridization with a** 

Following digestion with high frequency restriction endonucleases, chromosomal DNA is separated into different-sized fragments by conventional agarose gel electrophoresis, but this type of polymorphism is difficult to interpret due to the high number of fragments generated. However, interpretation of these polymorphisms can be facilitated by a Southern blot hybridization technique. By this methodology, fragments are separated by electrophoresis and transferred to a nitrocellulose or nylon membrane and hybridized using specific chemically or radioactively-labeled probes (Figure 3). DNA probes are designed for specific sequences that are found in multiple copies and in different positions of the chromosome. One of the most frequently used probes is ribosomal RNA (16s rRNA) because most species have more than one chromosomal rRNA operon distributed around the chromosome. This particular technique is denominated ribotyping. In recent years, the use

for its susceptibility to minor variations in experimental conditions.

*Enterococcus faecalis* isolates using D8635 primer.

**nucleic acid probe (Southern blotting)** 

**2.2 Based on enzymatic restriction of chromosomal DNA** 

electrophoresis (Figure 2).

of this technique has declined, mainly due to its limited discriminatory power compared to other techniques. Several DNA probes have been employed for the study of methicillinresistant *S. aureus* (MRSA) outbreaks, including various insertion sequences, such as *IS*431, *IS*256 and *mecA* gene.

Fig. 3. Schematic representation of restriction endonuclease analysis by Southern-blotting assay. On the right *IS*6110-restriction fragment length of *Mycobacterium tuberculosis* isolates.
