The normal gut biota represents a complex microbial ecosystem (the “enterome”) that plays a crucial role in homeostasis of the gastrointestinal tract (McCracken and Lorenz, 2001). The predominant anaerobic members of endogenous human gastrointestinal tract belong to the following anaerobic genera- Bacteroides, Bifidobacterium, Clostridium, Fusobacterium, Ruminococcus, Peptococcus and Peptostreptococcus  (Salminen et al. 1995). In comparison to less efficient and time consuming traditional culture based methods, PCR allows the rapid detection of a wide range of both cultivable and non-cultivable bacterial species (Carrino and Lee, 1995; Dutta et al. 2001). The 16S rRNA molecule consists of a mosaic of highly conserved, semi-conserved and highly variable regions (Woese, 1987). Among these regions conserved sequences in all species for a particular genus can be selected as primer sequence; flanking the variable regions. A rapidly accumulating 16S rRNA database facilitated designing of species or group-specific oligonucleotide probes that can be used for the rapid and culture independent enumeration of anaerobes (Suau et al. 1999).

The human gut microbiota composition may get disturbed during antibiotic associated diarrhoeal condition that may be either due to overgrowth of Clostridium difficile or due to loss of beneficial metabolic activities of intestinal microbes (Bartlett, 2002; Dunne, 2001). Global changes in the composition and quantity of the gut microbiota can result in perturbations of global colonic metabolism that lead to AAD (Högenauer et al. 1998).

In the present study, genus specific primers were designed and validated using faecal samples collected from one healthy and two diarrhoeal patients.

Designing of primers

The 16S rRNA sequences of maximum species of a specific genus encountered in the human gut were downloaded from the EMBL and Genbank database and subjected to CLUSTALX (Jeanmougin et al. 1998) programme. The blocks of hypervariable regions among different genera were picked up and subjected to second round of alignment where the maximum number of species belonging a genus was aligned and the conserved regions were selected as genus specific primers. Specificity of primers was checked using Genbank program BLAST (Altschul et al. 1990).

Six different primer sets were designed to differentiate following major anaerobic genera - Bifidobacterium (Bif), Ruminococcus (Rum), Lactobacillus (Lacb), Campylobacter (Camp), Peptococcus (Pep), and Clostridium (Clos) (Table 1). Primers for Bacteroides (Bacto) and Peptostreptococcus productus (PSP) were obtained from the published work (Menaja et al. 1996; Wang et al. 1996). For Staphylococcus aureus, primers (mecA-1 and mecA-2) based on methicillin resistant gene were used (Louie et al. 2002). Primer set used for the detection of E. coli was based on the malB promoter gene (Wang et al. 1996) (Table 1). All the primers were commercially synthesized from Microsynth (GMBH, Switzerland). 

Standard cultures used

Lyophilized cultures of following bacterial strains were procured from the Microbial Type Culture Collection Center (MTCC) located at the Institute of Microbial Technology (IMTECH) Chandigarh, India. They were, Bacillus subtilis (MTCC 121), Staphylococcus aureus (MTCC 740), Lactobacillus cassie (MTCC 1423), Enterococcus faecalis (MTCC 439), Escherichia coli (MTCC 1302), Salmonella infantis (MTCC 1107), Klebsiella pneumoniae (MTCC 432), Pseudomonas aeruginosa (MTCC 741) and Bacteroides fragilis (ATCC 25285D). Genomic DNA was isolated from the lyophilized cultures of bacterial strains using Sarkosyl- Proteinase K method (Hancock, 2002).

Clinical status of subjects

Samples were collected from one healthy and two diarrhoeal subjects. Sample was collected from healthy individual (an adult female; NF) who didn't consume any antibiotic for a period of six months, prior to sample collection. Sample D1 was collected from an old age bronchitis infected patient who was prescribed augmentin for 8 days (875mg/12h). On the third day of antibiotic intake, patient noted the onset of bulky, loose stools. The second diarrhoea patient; an adult male (D2) suffering from acute giardiasis, was on flagyl treatment for three days. Faecal samples collected in sterile vials were immediately frozen at -20ºC for DNA isolation purpose.

DNA isolation

Concentration of the DNA isolated from 180 mg of frozen faecal samples using Qiagen stool DNA mini kit (Qiagen), was determined by eye estimation on 0.6% agarose gel electrophoresis.

Testing and validation of primers for PCR amplification

A 20 μl PCR reaction containing 2.5 μl of template DNA (Faecal sample DNA) was performed in Tech gene, thermal cycler (Nugen Scientific, USA). Annealing temperatures are mentioned in Table 1. PCR products for expected sizes were checked on 1.5% agarose gel (Table 1). Approximately 80 ng genomic DNA from the above mentioned pure strains was used as control for the subsequent PCR experiments. Genus specific amplicons from faecal DNA from healthy individual were purified using Qiagen gel extraction kit and cloned into pGEMT^®- easy vector. Similarly Campylobacter specific amplicon from diseased individual was purified and cloned into pGEMT-easy vector. The sequences of cloned products were analyzed and submitted to EMBL nucleotide database (WEBIN). These cloned products were used as control DNA when PCR reactions were carried out with diseased individuals.

Findings

Analysis of PCR products generated using the genus specific primers

Amplicons were obtained specifically with targeted genera DNA (genomic DNA of pure strains).The accession numbers of amplicons were: AM042696 (Bacteroides vulgatus partial 16S rRNA gene), AM042697 (Uncultured Clostridium sp. partial 16S rRNA gene), AM042698 (Uncultured Bifidobacterium sp. partial 16S rRNA gene), AM042699 (Campylobacter coli partial 16S rRNA gene), AM042700 (Peptococcus sp. RR-2005 species partial 16S rRNA gene), AM042701 (Lactobacillus acidophilus partial 16S rRNA gene), AM117587 (Peptostreptococcus productus partial 16S rRNA gene) and AM117597 (Ruminococcus partial 16S rRNA gene). The species names, in given accession numbers have been mentioned on the basis of BLAST results showing highest hits with a particular species. 

PCR Results

Genera Lactobacillus, Bacteroides, Peptococcus, Bifidobacterium, and E. coli were invariably present in all subjects. Ruminococcus and Peptostreptococcus were absent in the diseased subjects (Figure 1c and Figure 1d). Clostridium was absent in the antibiotic associated diarrhoea patient (D1). Campylobacter was present only in the diseased individuals (Figure 1e). No amplicon was observed in any samples when methicillin resistant gene specific primers (mecA-1 and mecA-2) were used.

The microbiota of the gastrointestinal tract of humans needs to be studied extensively because of the role played by gut bacteria in the maintenance of gut homeostasis. Identification of bacteria based on 16S rRNA primers is a sensitive and specific technique. According to BLASTN analysis, Bacteroides specific primers showed partial homology with Streptomyces, Cytophagals that are not inhabitants of human intestine. No homology was observed with any non-targeted genera for Bifidobacterium specific primers. Similarly other genus specific primers showed partial homology with Marichromatium purpuratum, Roseospirillum parvum and Thiomicrospira species; not encountered in a healthy human gut. Specific primers are difficult to design from 16SrRNA region for the identification of closely related Shigella spp. and E. coli (Christense et al. 1998). Therefore, mal B promoter gene sequence based primer set was used for E. coli detection (Wang et al. 1996). It is advantageous to use genus specific primers since it encompasses maximum number of species of a genus. Primers used for the PCR reaction showed specificity for targeted genera.

The sequences obtained from the cloned PCR products were subjected to BLAST analysis for confirming the specific genus. These clones were further used as appropriate controls. Ruminococcus and Peptostreptococcus were absent in the diseased samples whereas they were present in healthy individuals. Both the organisms are well-known butyrate-producing bacteria (Schwiertz et al. 2002, Wilson et al. 2000), which is a preferred energy source for colonic epithelial cells (Barcenilla et al. 2000). Clostridium was absent in old age female diarrhoea patient. There are reports indicating loss of normal resident flora due to antibiotic intake in diarrhoea and other infectious diseases (Wang et al. 2002; Tanaka et al. 2005). Augmentin (taken by D1, old age AAD patient) contains amoxicillin; is a broad-spectrum drug used against bacterial infection (Bartlett, 2002). Our results for AAD patient also support the study by Young and Schmidt (2004) where loss of Clostridia cluster IV was observed due to intake of Amoxicillin. The drug Flagyl (metronidazole) used for giardiasis patient (D2) treatment is an effective drug against protozoa and anaerobic bacteria (Muller, 1983). This may be responsible for the absence of the above flora in the giardiasis patient.

Presence of the members of Enterobacteriaceae e.g. E. coli as observed by us both in healthy and in diseased conditions has been reported earlier by Young and Schmidt (2004). In order to check nosocomial infection due to Staphylococcus aureus, PCR was performed using mecA primer set. No methicillin resistant strains of Staphylococcus aureus was observed in any of the samples studied. Presence of Campylobacter in diarrhoeal subjects supported the earlier studies (Haque et al. 2003) probably indicating mixed infection in giardiasis patients.

Our results show that Lactobacillus, Bacteroides, Peptococcus and Bifidobacterium were more stable flora and did not alter in diarrhoea conditions. However, due to lack of quantitative PCR data, it is difficult to comment on any change in titer of a specific genus due to diseased condition.

Through this study we were able to design and validate genus specific primers and use them as molecular probes for PCR based detection of important gut bacteria in faecal samples. Whether the variation in flora is due to physiological status cannot be concluded due to limited number of the samples analyzed and more number of samples, in each category needs to be analyzed for comparing gut flora profile between healthy and diseased individuals.

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