Molecular charecterization of pathogenic bacterial isolates from khoa

Introduction

S. S aureus is one of the most dangerous and harmful organisms to human health due to its ability to produce enterotoxins associated with food poisoning. The source of contamination is probably human (Kumar and Prasad 2010) [9] . Presence of E. coli in khoa samples is an indicator of faecal contamination (Sohal et al. 1993) [15] . Salmonella enters food chain due to human contamination. Condensed milk product like peda prepared from khoa also gets contaminated by Salmonella (Tambekar and Bhutda 2006) [18] . In recent times, PCR method is being widely used for identification and characterization of pathogens due to its high sensitivity and specificity. 16S rRNA gene identification in S. aureus isolates has an edge over coagulase; catalase and DNase test (Abdel All et al., 2010) [1] . Shanehbandi et al. (2014) [14] successfully used PCR technique targeting 16S rRNA gene for identification of S. aureus isolates from milk products. E coli identification by PCR method is being done by targeting 16S rRNA and pal genes. However molecular characterization of E. coli isolates obtained from milk products is being done by targeting pal gene (Virpari et al., 2013) [19] . Identification of Salmonella isolates is a critical in microbial assay due to its morphological and staining characters similarities with other enteric pathogens. In recent times PCR method has been successfully exploited for Salmonella identification. A cent percent Salmonella detection could be achieved by targeting invA gene using PCR method (Das et al., 2012) [5] .

Materials and Methods Primary Isolation of S. aureus, E.coli and Salmonella spp.

Primary isolation of of S. aureus is done by using 0.1 ml inoculums of 10 -3 and 10 -4 dilutions of sample on Vogel Johnsen Agar (Hi-Media Laboratories, Mumbai).Characteristic colonies of staphylococci showing typical black colonies were selected. For E. coli isolation a quantity of 0.1 ml of inoculam from dilution of 10 -3 and 10 -4 were used by spread plate technique on Levine's Eosine Methylene Blue Agar (EMB) (Himedia Laboratories, Mumbai). Bluish purple coloured colonies with greenish metallic sheen were considered indicative of E. coli.

For Primary isolation of Salmonella spp. pre enrichment was done by using 1 percent Buffered Peptone Water (BPW) (Hi-Media Laboratories, Mumbai) at 37 o C for 24 hours. Preenriched sample was inoculated in tetrathionate broth (TTB) (Hi-Media Laboratories, Mumbai) and incubation was done for 24 hours at 42 o C. Streaking of TTB grown bacteria was done on Xylose Lysine Deoxycholate (XLD) (Hi-Media Laboratories, Mumbai) agar and incubated for 24 hours at 37 o C. Pink colonies with or without black centers were considered as positive for Salmonella. All the isolates are further processed for biochemical tests.

Molecular characterization of S. aureus, E. coli, Salmonella spp

Molecular characterization of S. aureus, E. coli and Salmonella spp. isolates was done by using PCR at Department of Animal Biotechnology, College of Veterinary and Animal Sciences Parbhani. Molecular characterization of S. aureus, E. coli and Salmonella spp. isolates for their confirmations as per the method described by Mason et al.

(2001) [10] , Kuhnert et al. (1995) [8] , Rahn et al. (1992) [12] respectively with suitable modifications. S. aureus, E. coli, Salmonella spp. isolates were screened for presence of 16S rRNA, pal, inv A genes respectively.

Isolation of genomic DNA of S. aureus, E. coli, Salmonella spp.

The DNA of each isolates of S. aureus, E. coli, Salmonella spp. was extracted by boiling method. Approximately a loopful of culture were mixed in 100 µl of autoclaved distilled water in micro centrifuge tube. Then it was vortexed and heated at 98 o C for 10 min, cell debris was removed by centrifugation and 5μl of supernatant was used as a DNA template in PCR reaction mixture.

PCR reaction

Enumeration of Master Mixture, Forward and reverse primers, template DNA nuclease free water used in PCR reaction are given in Table 1.

Identification of pal gene in E. coli isolates

The DNA templates were used for PCR assay performed for the detection of pal genes. Each reaction contained 12.5 μL of PCR Master Mix, 1 μl (500 picomole conc.) of each forward and reverse primer, 5.5 μL Nuclease-free water, and 5 μL template DNA. After this the above mixture was subjected to PCR under conditions given in table 2. The PCR products so obtained were further electrophoresed on 2 percent agarose gel at 80 V and 200 mA for 1 hr. As per cycling conditions mentioned in Table 2.

Identification of inv A gene in Salmonella isolates

The DNA templates were used PCR assay performed for the detection inv A gene for identification of Salmonella. Each reaction contained 12.5 μL of PCR Master Mix, 1 μl (500 picomole conc.) of each forward and reverse primer, 5.5 μL Nuclease-free water, and 5 μL template DNA. After this the above mixture was subjected to PCR under conditions given in table 2. The PCR products so obtained were further electrophoresed on 2 percent agarose gel at 80 V and 200 mA for 1 hr. As per cycling conditions mentioned in Table 2.

Agarose Gel Electrophoresis

To confirm the targeted PCR amplification, 5 μl of PCR product from each tube was mixed with 1μl 6 X gels loading buffer and electrophoresed on 2.0 percent agarose gel along with 100 bp DNA Ladder (GeneRuler-Fermentas) and stained with ethidium bromide at constant 80 V for 40 minutes and 1 hour as per conditions in 0.5X TBE buffer. The amplified product was visualized as a single compact band of expected size under UV trans illuminator. [13,6,7,14] .

In present study also 16S rRNA gene was successfully exploited for identification of S. aureus isolates obtained from primary isolation from khoa samples by using PCR technique.

References

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