Ethical approval The present study was conducted during the period from July to December 2013, in the Bacteriology Laboratory of the Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University (BAU), Mymensingh.
Collection and transportation of samples Guava samples were collected from KR market, Sheshmor, Kewatkhali, Meshubazar, and Nutunbazar. Samples were collected in sterile polythene bags separately. The samples were transported carefully to the Bacteriology Laboratory of the Department of Microbiology and Hygiene, BAU, Mymensingh, for bacteriological analysis.
Processing of guava samples Each guava sample was washed with 20 ml sterile PBS and transferred the washing sample into a separate poly bag. A ten-fold serial dilution of the samples was performed in the nutrient broth.
Determination of total viable count (TVC) 0.5 ml of each 10-fold diluted sample was transferred and spread on Plate Count Agar using a sterile pipette and a sterile glass spreader. Then, the plates were kept in an incubator at 37°C for 24-48 h. The number of colonies in a particular dilution was multiplied by the dilution factor to obtain TVC which was expressed as a mean log10 ± standard deviation colony-forming unit (cfu) per ml.
Isolation of bacteria in pure culture The isolation and identification of bacteria were performed according to the method described by Carter [6]. Samples were enriched in nutrient broth at 37°C for 24 h. The overnight cultures were streaked on SS agar (for Salmonella spp.), eosin methylene blue (EMB) (for Escherichia coli), BA (for Bacillus spp.), MS agar (for Staphylococcus spp.), and thiosulfate-citrate-bile salts-sucrose (TCBS) agar (for Vibrio spp.). The inoculated plates were incubated at 37°C for 24 h. A single colony was further subcultured until pure culture was obtained. Identification of bacteria was performed on the basis of colony morphology, Gram’s staining reaction, motility test, and biochemical tests.
Molecular detection of E. coli by polymerase chain reaction (PCR) DNA extraction A pure bacterial colony of E. coli was mixed with 100 µl of distilled water which was boiled for 10 min and then immediately kept on ice for cold shock. Finally, centrifugation was done at 10,000 rpm for 10 min. The supernatant was collected and used as a DNA template for PCR.
Primers used for PCR A genus-specific PCR was performed to amplify 16S rRNA of E. coli using previously published primers.
Antibiotic sensitivity test Five isolates randomly selected from five genera were tested for antimicrobial drug susceptibility against five commonly used antibiotics by disc diffusion or Kirby-Bauer method [8]. Selection of 3–5 well-isolated colonies from the SS, BA, EMB, MS, and TCBS agar plate. Touched the top of colony with a loop and the growth is transferred into the nutrient broth. The broths were streaked onto Mueller-Hinton agar plates using sterile glass spreader homogeneously. Then, the antibiotic disc was placed onto Muller-Hinton agar and incubated at 37°C for 24 h. The plates were examined, and the diameter of the zones of inhibition was measured in mm from the edge of the disc to the edge of the zone using a meter ruler.
Statistical analysis The results of TVC of the bacteria of guava sold at local markets were analyzed for statistical significance using [9] multiple range test (SPSS 11.5, US). A p≤0.01 was considered to be statistically significant.