Enrichment Broth Culture-duplex Pcr Assay

An enrichment broth culture-duplex PCR combination assay was devised to identify Clostridium perfringens directly from fecal samples. The method consists of a combination of short enrichment of samples in selective media, DNA isolation, and performing duplex PCR using two pairs of primers which identify C. perfringens strains that harbor the virulence enterotoxin gene. Comparison of two selective enrichment media and two incubation temperatures showed that the reinforced clostridial medium with neomycin was better than the fluid thioglycollate medium with neomycin (p<0.001); and incubation at 37oC vs 45oC showed no statistically significant difference (p=0.238). The optimal short time for pre-enrichment culture was 4 hours. The developed assay was applied to detect phospholipase C (plc) and enterotoxin (cpe) genes for C. perfringens in feces inoculated artificially with enterotoxigenic C. perfringens. The method could detect both gene products in samples inoculated with a minimum of 104 CFU per ml. When the method was applied to detect enterotoxigenic C. perfringens in 198 diarrhea patients, C. perfringens was found in 121 samples; 7 out of 121 samples were positive for both plc and cpe (prevalence of 5.8%). These results indicate that the developed assay was a suitable method for the rapid and specific detection of enterotoxigenic C. perfringens directly in fecal specimens of diarrhea patients, which will assist epidemiological investigations of food poisoning outbreaks and quality control of food products. for enterotoxin production, is limited in the usual culture media. Many strains of C. perfringens do not sporulate well in these media which can lead to false-negative results (Kokai-Kun et al, 1994). We previously developed a duplex PCR to identify enterotoxigenic strains of fecal isolates directly from primary culture plates (Tansuphasiri, 2001; Tansuphasiri et al, 2002) by using two sets of primers which amplify in the same reaction two different DNA fragments for C. perfringens simultaneously: alpha toxin (phospholipase C, plc) and enterotoxin (cpe) genes. However, this method has yet to be perfected because PCR is sensitive to inhibition by factors present in the crude clinical samples (Widjojoatmodjo et al, 1992). Recently, an enrichment culture-PCR combination assay was reported for the detection of Salmonella directly from fecal samples (Chiu and Ou, 1996). Prior to the PCR assay, a short enrichment procedure was used to circumvent the effect of inhibitory compounds as well as selecSOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 1230 Vol 36 No. 5 September 2005 tive cultivation of the bacteria. In this study we applied enrichment broth culture with a slight modification that is suitable for the enrichment of C. perfringens in fecal specimens of diarrhea patients. The assay included a short pre-enrichment period of the fecal specimen in selective enrichment media, DNA isolation, and then analysis by a duplex PCR. MATERIALS AND METHODS Bacterial strains and fecal samples Enterotoxigenic C. perfr ingens ATCC 12916, NCTC 8198, and NCTC 8239 were obtained from A Heikinheimo, Faculty of Veterinary Medicine, Universi ty of Hels inki , F in land; nonenterotoxigenic strains, ATCC 3624, 3629, 27324, 29348 and 43402, were obtained from Prof Dr O Suthienkul, Faculty of Public Health, Mahidol University. The non-C. perfringens which served as negative controls were E. coli ATCC 25922, clinical isolates of C. sporogenes, C. difficile, food isolates of C. botulinum and Bacillus cereus. A total of 193 fecal samples were obtained from diarrhea patients at two hospitals, Siriraj Hospital, Bangkok; and Nakhon Nayok Hospital, Nakhon Nayok. These patients were over 2 years old and all had acute diarrhea. Fresh fecal specimens were collected in sterile containers, held at 4oC, and then sent within 3 hours to a laboratory for culture and enterotoxin analysis by duplex PCR. Conventional culture and identification Fecal specimens were serially diluted in 0.1% (w/v) peptone-water and plated onto tryptose-sulfite-cycloserine (TSC)-egg yolk agar (Merck) overlaid with an additional 10 ml of TSC agar. The plates were incubated overnight at 37oC in an anaerobic jar with Gaspak (Difco), and the black colonies by sulfite reduction with a zone of turbidity due to lecithinase activity were counted and presumptively identified as C. perfringens. Up to five presumptive colonies were randomly selected and each colony was subcultured on duplicate blood agar. One plate was incubated overnight at 37oC in an anaerobic jar and another was incubated overnight at 37oC in aerobic conditions. The colonies grown only on blood agar with dual hemolytic zones under anaerobic conditions were confirmed as C. perfringens based on the following tests: Gram stain and morphology, motility, nitrate reduction, gelatin liquefaction and lactose fermentation (FDA, 1998). To test the enterotoxigenicity of C. perfringens, these colonies were used as templates for the duplex PCR assay (Tansuphasiri, 2001). Determination of optimal conditions for preenrichment before PCR analysis Selective enrichment broth cultivation. Two selective enrichment broth, including reinforced clostridial medium (RCM) (Scharlau, Spain) and fluid thioglycollate (FTG) (Difco), with neomycin (100 mg/l) and without neomycin, were used to compare the growth and recovery rate of C. perfringens and other bacteria after overnight incubation at either 37oC or 45oC. All 13 bacterial strains were serially diluted 10-fold in 0.1% peptone water, and 1 ml of each dilution (ranged from 108 to 103 /ml) was inoculated into 9 ml of the enrichment broth. Two sets of each selective medium (with and without neomycin) were prepared. After incubation, the first set at 37oC and the second set at 45oC, for 24 hours, the number of colony forming units (CFU) of the original suspension per ml was estimated by growing on TSC agar. Briefly, the broth cultures were 10-fold serially diluted in 0.1% peptone-water (from 10-1 to 10-6) and appropriate dilution (0.1 ml) was inoculated onto duplicate TSC agar by the spread plate. After overnight incubation in an anaerobic jar, the number of colonies were counted and reported as CFU/ml of enrichment culture. Three replicates were done for each experiment. Optimal time for preenrichment. Seeded fecal samples from healthy individuals were used to determine the optimal short time for pre-enrichment before PCR analysis. An enterotoxigenic strain (C. perfringens ATCC 12916) was used as the seed. One ml of bacteria (108 CFU/ml) was serially diluted 10-fold with 0.1% peptonewater, and 1 ml of each dilution (ranged from 108 to 103 CFU/ml) was seeded into 9 ml of fecal suspension (1:10 in 0.1% peptone-water). The 6 concentrations of C. perfringens in the spiked feces corresponded to approximately 102 ENRICHMENT BROTH CULTURE-DUPLEX PCR ASSAY FOR C. PERFRINGENS DETECTION Vol 36 No. 5 September 2005 1231 to 107 CFU/ml of fecal suspension, respectively. A same aliquot of fecal specimen without C. perfringens was used as an original control. One ml of spiked feces with 6 concentrations of C. perfringens that ranged from 102 to 107 CFU/ml was inoculated into 9 ml of predetermined suitable enrichment broth. Each concentration was subjected to 4 different pre-enrichment times: 0 hour, 4 hours, 8 hours, and 24 hours before PCR analysis. After aerobic incubation at 37oC for each time duration, all the fecal specimens (both spiked and nonspiked with C. perfringens) were taken for DNA isolation and PCR analysis. The number of CFU of the original suspension per ml was also estimated by growing onto duplicate TSC agar; and colonies were counted as mentioned before. DNA isolation from enrichment broth culture. The pre-enrichment broth culture of spiked feces was processed to remove particulate fecal substances by low-speed centrifugation at 300g for 3 minutes, then high-speed centrifugation at 17,000g for 3 minutes. Following three more cycles of washing with TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8), the final pellet was suspended in 400 μl of TE buffer and divided into 2 aliquots for DNA isolation by the boiling method and the QIAamp DNA Stool Mini Kit (QIAGEN Pty Ltd, Australia). For boiling, 200 μl of concentrated broth culture was boiled in a water bath at 95oC for 10 minutes, then placed on wet ice. The pellet was removed by centrifugation at 12,000g for 2 minutes, and 10 μl of the supernatant was used in the PCR reaction. The second aliquot was extracted using the QIAamp DNA Stool Mini Kit, following the manufacturer protocol. Ten microliters of the eluate was used in the PCR reaction.