**6. Identification based on physiological and biochemical properties and sequence analysis of the 16S rRNA gene**

Identification of the SRB by morphological, physiological, and biochemical characteristics can be conducted according to Bergey's Manual of Determinative Bacteriology (ninth edition, 1994), where SRB belong to the seventh group and are called "dissimilatory sulfate- or sulfur-reducing bacteria" [11]. This group is divided into four subgroups (**Figure 5**).

However, more modern and complex classification of SRB is published in Bergey's Manual of Systematic Bacteriology (2005), where SRB are divided into different classes, for example, class IV, *Deltaproteobacteria*, including order II, *Desulfovibrionales*; family I, *Desulfovibrionaceae* (genus I. *Desulfovibrio*); or family II, *Desulfomicrobiaceae* (genus I. *Desulfomicrobium*) [10]. For details identification based on physiological and biochemical characteristics is necessary to use both Bergey's manuals.

As was mentioned above, the representatives of *Desulfovibrio* genus are often found in the animal and patients with IBD and healthy subjects, because it is necessary to pay attention to the more detailed steps for identification of the second subgroup where this genus belongs (see **Figure 4**).

#### **Figure 5.**

*The scheme of classification of dissimilatory sulfate- or sulfur-reducing bacteria group, according to Bergey's manual of determinative bacteriology (ninth edition, 1994) [11].*


#### **Table 1.**

*The differences between features of the second subgroup SRB genera according to Bergey's manual of determinative bacteriology [11].*

The second subgroup includes *Desulfovibrio* (Dvi), *Desulfomonas* (Dmo), *Desulfobulbus* (Dbu), *Desulfomicrobium* (Dmi), and *Thermodesulfobacterium* (Tdb) genera (**Table 1**).

Other SRB genera can be identified by Bergey's manuals [10, 11]. However, for complete identification based on morphological, physiological, and biochemical properties, the molecular methods, in particular the sequence analysis of 16S rRNA gene, are also necessary to be applied [50]. Except sequence analysis of 16S rRNA gene, it is important to confirm the SRB species by using primers of functional genes of dissimilatory sulfate-reduction, such as *DsrAB* and А*prBA* (**Table 2**).

Further on the example of one isolate of intestinal SRB, identification based on sequence analysis of 16S rRNA gene by using the universal primers will be described. The schema of this identification is presented in **Figure 6**.

**33**

**Table 3.**

PCR amplification.

[51] and Persing [52].

ized water, and 2.0 μl of DNA supernatant.

1492RPL 5'-GGTTACCTTGTTACGACTT-3' position 1510–1492

*Universal primers for amplification of 16S rRNA gene fragments.*

*Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*

DSR4R 5'-GTGTARCAGTTDCCRCA-3'

aprA-5-RV 5'-GCGCCAACYGGRCCRTA-3'

*The scheme of identification based on sequence analysis of 16S rRNA gene.*

**DsrAB gen**

А**prBA gen**

**Table 2.**

**Figure 6.**

*amplification.*

*DNA isolation*. Isolation and purification of DNA were carried out with a 72-h culture of SRB by using a "QIAmp DNA Mini Kit (QIAGEN), Cat. No 51304." One single SRB colony was taken from modified Postgate agar medium and suspended in 50 μl of deionized water in a screw cap micro-centrifuge tube. The samples were boiled at 98°C for 5 min prior to being centrifuged for 5 min/14,000 g to settle cell debris. In total 2 μl of supernatant, containing the genomic DNA, were used for

**Functional genes Primer sequence Amplicon length (pb)**

DSR1F <sup>5</sup>'-ACSCAYTGGAARCACG-3<sup>&#</sup>x27; <sup>1900</sup>

aprB-1-FW <sup>5</sup>'-TGCGTGTAYATHTGYCC-3<sup>&#</sup>x27; 1200–1350

*Primers designed based on functional gens of dissimilatory sulfate reduction, which can be used for* 

*Amplification of gene fragments*. Amplification of 16S rRNA gene fragments was carried out using the universal primers (**Table 3**) according to Weisburg et al.

*PCR procedure*. PCR was carried out on DNA isolated from SRB cells in a final volume of 20 μl consisting of 10.0 μl Taq PCR Master Mix Kit (Cat. No 201445), 0.1 μl of each primer, 0.1 μl uracil D-glycosylase (Cat. No. M0280 L), 7.7 μl deion-

The amplicons were amplified by a preliminary incubation at 94°C for 5 min (initial denaturation) and then 34 cycles of 94°C for 1 min (denaturation), 55°C for 1 min (annealing of primers), and 72°C for 2 min (polymerization), using a

**Primers Sequence Amplicon length (pb)** 8FPL <sup>5</sup>'-AGTTTGATCCTGGCTCAG-3' position 8–27 Approximately 1500

806R 5'-GGACTACCAGGGTATCTAAT-3' position 806–787 Approximately 800

*Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*


**Table 2.**

*Microorganisms*

**32**

genera (**Table 1**).

*determinative bacteriology [11].*

**Table 1.**

**Figure 5.**

Optimal temperature range

Ability of bacteria to grow in the presence of sulfate

*manual of determinative bacteriology (ninth edition, 1994) [11].*

**Notes***: the feature is presence "+" or absence "–".*

and А*prBA* (**Table 2**).

The second subgroup includes *Desulfovibrio* (Dvi), *Desulfomonas* (Dmo), *Desulfobulbus* (Dbu), *Desulfomicrobium* (Dmi), and *Thermodesulfobacterium* (Tdb)

*The differences between features of the second subgroup SRB genera according to Bergey's manual of* 

Other SRB genera can be identified by Bergey's manuals [10, 11]. However, for complete identification based on morphological, physiological, and biochemical properties, the molecular methods, in particular the sequence analysis of 16S rRNA gene, are also necessary to be applied [50]. Except sequence analysis of 16S rRNA gene, it is important to confirm the SRB species by using primers of functional genes of dissimilatory sulfate-reduction, such as *DsrAB*

**Features Dvi Dmo Dbu Dmi Tdb** Spiral or vibrio-shaped cells + — — — — Oval or rod-shaped cells — + + + + Movement with the polar flagella +/− +/− + +/−

*The scheme of classification of dissimilatory sulfate- or sulfur-reducing bacteria group, according to Bergey's* 

+25…+40°C + + + + — +65…+70°C — — — — +

H2 + CO2 + acetate as a carbon source + + + + + Lactate + + + + + Propionate — — + — — Desulfoviridin + + — — —

Further on the example of one isolate of intestinal SRB, identification based on sequence analysis of 16S rRNA gene by using the universal primers will be

described. The schema of this identification is presented in **Figure 6**.

*Primers designed based on functional gens of dissimilatory sulfate reduction, which can be used for amplification.*

**Figure 6.**

*The scheme of identification based on sequence analysis of 16S rRNA gene.*

*DNA isolation*. Isolation and purification of DNA were carried out with a 72-h culture of SRB by using a "QIAmp DNA Mini Kit (QIAGEN), Cat. No 51304." One single SRB colony was taken from modified Postgate agar medium and suspended in 50 μl of deionized water in a screw cap micro-centrifuge tube. The samples were boiled at 98°C for 5 min prior to being centrifuged for 5 min/14,000 g to settle cell debris. In total 2 μl of supernatant, containing the genomic DNA, were used for PCR amplification.

*Amplification of gene fragments*. Amplification of 16S rRNA gene fragments was carried out using the universal primers (**Table 3**) according to Weisburg et al. [51] and Persing [52].

*PCR procedure*. PCR was carried out on DNA isolated from SRB cells in a final volume of 20 μl consisting of 10.0 μl Taq PCR Master Mix Kit (Cat. No 201445), 0.1 μl of each primer, 0.1 μl uracil D-glycosylase (Cat. No. M0280 L), 7.7 μl deionized water, and 2.0 μl of DNA supernatant.

The amplicons were amplified by a preliminary incubation at 94°C for 5 min (initial denaturation) and then 34 cycles of 94°C for 1 min (denaturation), 55°C for 1 min (annealing of primers), and 72°C for 2 min (polymerization), using a


**Table 3.**

*Universal primers for amplification of 16S rRNA gene fragments.*

thermocycler (model MJ Research PTC-200, USA). After the last amplification cycle, the samples were incubated further at 72°C for 2 min for complete elongation of the final PCR products and cooled at 10°C.

*Analysis of PCR products*. Analysis of PCR products was carried out by electrophoresis in 1.5% agarose gel, with field strengths of 5 V/cm. Electrophoresis time was 40 min. The 100 bp ladder (Malamité, Czech Republic) was used as a size standard and molecular weight markers. Isolation and purification of fragments from agarose were performed by centrifugation of gel strips containing DNA through aerosol filters. For purification of SRB amplicons, the commercial kit from QIAGEN "MinElute Gel Extraction Kit" was used. The sequence was carried out using a "genetic analyzer" and reagents "BigDye Terminator v3.1 Cycle Sequencing Kit." Search homologous deposited in the GenBank nucleotide sequence encoding the 16S rRNA gene, was performed using BLASTN and Blast2 programs.

The 16S rRNA gene amplicons which were used for sequence analysis were obtained by using the PCR method. The PCR products were separated by electrophoresis (**Figure 7**). Before sequence analysis the absorbance of amplicons (8FPL/806R, amplicon I about 800 bp; 8FPL/1492RPL, amplicon I about 1500 bp; 8FPL/806R, amplicon II about 800 bp; 8FPL/1492RPL, amplicon II about 1500 bp) was determined [50].

By comparison of individual sequencing data from the amplicons 1–5, the following gene for 16S rRNA sequence of the total length 1370 bp was completed:

**Black nucleotides** are a summary from 1, 2, 3, and 5: the best sequence data (totally four sequencing). **Green nucleotides** are a summary from 3 and 4: good sequence data (two sequencing). **Red nucleotides** are the rest of 4: the worse sequence data because they were received from one sequencing only, but its quality was excellent. The obtained sequence BLASTN was analyzed. The highest homology of SRB colony was identified with *Desulfovibrio piger* ATCC 29098 from GenBank.

The obtained sequence results of SRB isolated colony were also compared by BLASTN analysis with the nucleotide sequences of 16S rRNA gene of other strains (**Table 4**).

Thus, the nucleotide sequence of the 16S rRNA gene of SRB has the highest homology (99%) compared to deposited nucleotide sequence *D. piger* ATCC 29098 (AF192152) in the GenBank database.

**35**

**Table 4.**

**Figure 7.**

pletely to acetate [10, 11].

The *D. piger* belongs to sulfate-reducing bacteria which are usually considered

*The results of electrophoresis 16S rRNA PCR products (amplicons): 8FPL/1492RPL, 1500 bp (1); 8FPL/806R,* 

**SRB strains Acc. No Identities Identity (%)** *Desulfovibrio piger* ATCC 29098 AF192152 1352/1368 99 *Desulfovibrio fairfieldensis* ATCC700045 U42221 1313/1374 96 *Desulfovibrio desulfuricans* Essex 6 AF192153 1299/1369 95 *Desulfovibrio intestinalis* DSM 11275 Y12254 1289/1373 94 *Desulfovibrio desulfuricans* MB AF192154 1293/1373 94 *Bilophila wadsworthia* ATCC49260 L35148 1242/1369 91 *Desulfovibrio vulgaris* subsp. Oxamicus DSM 1925 AJ295677 1089/1195 91 *Desulfovibrio longreachensis* ACM 3958 Z24450 1253/1374 91 *Desulfovibrio termitidis* DSM 5308 X87409 1237/1372 90 *Lawsonia intracellularis* NCTC 12656 U30147 1215/1374 88 *Desulfovibrio vulgaris* subsp. vulgaris DSM 644 M34399 726/834 87

*800 bp (2); 8FPL/1492RPL, 1500 bp (3); 8FPL/806R, 800 bp (4); M is marker (100 bp ladder).*

as a commensal bacterium in humans [5, 10, 50]. More recently, *D. piger* has attracted more interest as it was found to be the most prevalent species of SRB in feces of patients with inflammatory bowel disease [20, 21, 25, 31]. The obtained bacterial strains have such phenotypic features as the presence of desulfoviridin, cytochrome *c*3, and menaquinone MK-6. They oxidize organic compounds incom-

*Comparing the resulting sequence of 16S rRNA gene with other* Desulfovibrio *species.*

*Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*

#### **Figure 7.**

*Microorganisms*

was determined [50].

thermocycler (model MJ Research PTC-200, USA). After the last amplification cycle, the samples were incubated further at 72°C for 2 min for complete elongation

the 16S rRNA gene, was performed using BLASTN and Blast2 programs.

The 16S rRNA gene amplicons which were used for sequence analysis were obtained by using the PCR method. The PCR products were separated by electrophoresis (**Figure 7**). Before sequence analysis the absorbance of amplicons (8FPL/806R, amplicon I about 800 bp; 8FPL/1492RPL, amplicon I about 1500 bp; 8FPL/806R, amplicon II about 800 bp; 8FPL/1492RPL, amplicon II about 1500 bp)

By comparison of individual sequencing data from the amplicons 1–5, the following gene for 16S rRNA sequence of the total length 1370 bp was completed:

**Black nucleotides** are a summary from 1, 2, 3, and 5: the best sequence data (totally four sequencing). **Green nucleotides** are a summary from 3 and 4: good sequence data (two sequencing). **Red nucleotides** are the rest of 4: the worse sequence data because they were received from one sequencing only, but its quality was excellent. The obtained sequence BLASTN was analyzed. The highest homology of SRB colony was identified with *Desulfovibrio piger* ATCC 29098 from GenBank. The obtained sequence results of SRB isolated colony were also compared by BLASTN analysis with the nucleotide sequences of 16S rRNA gene of other

Thus, the nucleotide sequence of the 16S rRNA gene of SRB has the highest homology (99%) compared to deposited nucleotide sequence *D. piger* ATCC 29098

*Analysis of PCR products*. Analysis of PCR products was carried out by electrophoresis in 1.5% agarose gel, with field strengths of 5 V/cm. Electrophoresis time was 40 min. The 100 bp ladder (Malamité, Czech Republic) was used as a size standard and molecular weight markers. Isolation and purification of fragments from agarose were performed by centrifugation of gel strips containing DNA through aerosol filters. For purification of SRB amplicons, the commercial kit from QIAGEN "MinElute Gel Extraction Kit" was used. The sequence was carried out using a "genetic analyzer" and reagents "BigDye Terminator v3.1 Cycle Sequencing Kit." Search homologous deposited in the GenBank nucleotide sequence encoding

of the final PCR products and cooled at 10°C.

**34**

strains (**Table 4**).

(AF192152) in the GenBank database.

*The results of electrophoresis 16S rRNA PCR products (amplicons): 8FPL/1492RPL, 1500 bp (1); 8FPL/806R, 800 bp (2); 8FPL/1492RPL, 1500 bp (3); 8FPL/806R, 800 bp (4); M is marker (100 bp ladder).*


#### **Table 4.**

*Comparing the resulting sequence of 16S rRNA gene with other* Desulfovibrio *species.*

The *D. piger* belongs to sulfate-reducing bacteria which are usually considered as a commensal bacterium in humans [5, 10, 50]. More recently, *D. piger* has attracted more interest as it was found to be the most prevalent species of SRB in feces of patients with inflammatory bowel disease [20, 21, 25, 31]. The obtained bacterial strains have such phenotypic features as the presence of desulfoviridin, cytochrome *c*3, and menaquinone MK-6. They oxidize organic compounds incompletely to acetate [10, 11].

Moore W.E. found SRB for the first time in people's feces and identified it as *Desulfomonas pigra* [53], which subsequently is reclassified as *Desulfovibrio piger* [16]. The described bacterial strains are similar to that of Moore et al. [53] except for the G–C content of the DNA, which is 64 mol%. Obligate anaerobic, sulfate-reducing, non-saccharolytic, non-proteolytic, nonspore-forming, and Gram-negative bacteria that are straight and vibrio-like and have rounded ends (0.8–1.0 × 2.5–10.0 μm) [10]. These microorganisms use lactate, pyruvate, ethanol, and hydrogen as electron donors for sulfate reduction. They oxidize lactate and pyruvate incompletely to acetate. The optimum temperature for growth is +37°C. Growth is not affected by 20% bile. Colonies on anaerobic blood agar are translucent, 2 mm in diameter, circular, and non-hemolytic. Cells contain desulfoviridin and cytochrome *c*3. These bacteria isolated from human specimens (feces, peritoneal fluids, and intra-abdominal collections). The type strain, isolated from human feces, is ATCC 29098 [10].
