Molekularna karakterizacija i otpornost na antibiotike bakterije Clostridium perfringens izolirane iz trupova goveda i ovaca u klaonicama Shiraza u južnom Iranu

Clostridium perfringens type A food-borne poisoning is often caused by C. perfringens enterotoxin (CPE) encoded by chromosomal cpe. Contamination of meat with C. perfringens usually leads to food poisoning outbreaks. To find more information regarding the causative agent, we focused on the identification of type A containing cpe and netB genes in cattle and sheep carcasses slaughtered at Shiraz slaughterhouse and investigated the prevalence of antibiotic-resistant plasmid in isolated C. perfringens. 200 specimens were randomly collected by swabbing the whole outer and inner surface of the carcasses, and processed for selective culture on sulfadiazine polymyxin sulphate agar (SPS). The suspected colonies were further identified using species-specific primers as to confirm the presence of the cpa, cpe, netB and tetracycline and enrofloxacin gene resistance patterns. Our results demonstrated that out of 90 and 70 colonies of the positive cultures from cattle and sheep samples, respectively, 40% and 35.7% of the suspected colonies were identified as C. perfringens type A by PCR assay. Moreover, from those type A isolates, only 1 (2.7%) isolate was positive for both cpe and netB genes in the cattle carcasses. The MIC values also showed high tetracycline resistance patterns for cattle (45.8%) and sheep (92.3%) while all of the PCR positive C. perfringens type A isolates were susceptible to enrofloxacin. The high prevalence of C. perfringens in slaughtered animals with a high rate of resistance to tetracycline implies the need for caution in the use of antibiotic in food animals.Trovanje hranom uzrokovano bakterijom Clostridium perfringens tipa A često uzrokuje C. perfringens enterotoksin (CPE), kodiran kromosomskim cpe. Kontaminacija mesa s C. perfringens obično uzrokuje otrovanje hranom. Da bismo doznali više informacija o uzročniku, iz trupova goveda i ovaca zaklanih u klaonicama Shiraza, identificirali smo tip A koji sadržava gene cpe i netB. Osim toga, u izoliranim bakterijama C. perfringens utvrdili smo prevalenciju plazmida rezistentnih na antibiotike. Obriskom cijele vanjske i unutarnje površine nasumično odabranih trupova, prikupljeno je 200 uzoraka koji su obrađeni selektivnom kulturom na sulfadiazin-polimiksin sulfatnom agaru (SPS). Sumnjive kolonije dodatno su identificirane primjenom specifičnih početnica kako bi se potvrdila prisutnost gena cpa, cpe, netB te gena za otpornost na tetraciklin i enrofloksacin. Naši su rezultati pokazali da je PCR analizom od 90, odnosno 70 kolonija pozitivnih kultura iz uzoraka goveda i ovaca, njih 40 %, odnosno 35,7 % identificirano kao C. perfringens tipa A. Štoviše, iz izolata tipa A dobivenih od goveđih trupova, samo je jedan izolat (2,7 %) bio pozitivan i za gene cpe i za netB. MIC vrijednosti također su pokazale visoku razinu otpornosti na tetracikline kod goveda (45,8 %) i ovaca (92,3 %), dok su svi PCR pozitivni na C. perfringens tipa A bili osjetljivi na enrofloksacin. Visoka prevalencija bakterije C. perfringens kod zaklanih životinja zajedno s visokom stopom otpornosti na tetraciklin upućuje na potrebu za oprezom u primjeni antibiotika kod životinja koje služe za ljudsku hranu

Determination of Tetracycline and Enrofloxacine Resistance in Salmonella Isolated From Poultry

Background: In recent years, an increase in antibiotic resistance has been observed in Salmonella in different countries. The aim of this study was to determine the tetracycline and enrofloxacine resistance in salmonella isolated from poultry. Methods: The pattern of antibiotic resistance to tetracycline and enrofloxacin in isolated Salmonella of fecal broiler chickens from Shiraz, southern Iran, was assessed using minimum inhibitory concentration (MIC) and PCR methods. Results: Of 100 fecal samples of broiler chickens, 5 samples (5%) were infected to Salmonella. The antimicrobial susceptibility showed that MIC90 of isolated Salmonella strains for enrofloxacin and tetracycline was less than 0.2 μg/mL and 180 μg/mL, respectively, indicating a high sensitivity to these antibiotics. In two samples the presence of tetracycline resistance plasmid was also found, while all the strains were susceptible to enrofloxacin. Conclusion: According to the results, the isolated Salmonella spp. showed higher resistance to tetracycline than enrofloxacin, which seems due to the excessive usage of this antibiotic in poultry industry

Growth Rates of Bacillus Species Probiotics using Various Enrichment Media

Background: Probiotics are well-known as valuable functional foods to promote specific health benefits to consumers. Some Bacillus bacteria have been recently considered as probiotic and food additives. We aimed to investigate the growing rate of probiotic B. subtilis and B. coagulans using several enrichment media incubated at 37 °C for 24 hours. Methods: Various enrichment media including nutrient broth (NB), tryptic soy broth (TSB), double strength TSB, Mueller Hinton broth (MH), brain-heart infusion broth (BHIB), de Man, Rogosa and Sharpe (MRS), and nutrient yeast extract salt medium (NYSM) were used to enrich the probiotics and they were subsequently incubated for 18 h at 37 °C. The bacteria were then enumerated on TSA medium. Results: The results showed that B. subtilis ATCC 6633, B. subtilis PY79, and B. coagulans developed in TSB, double strength TBS, TSB yeast extract, BHIB and NYSM, respectively. Moreover, the formulas were achieved based on the optical density curve and the number of bacteria. Conclusion: Considering that the probiotics are significantly employed as food supplements, it is essential to identify appropriate enrichment media to proliferate these beneficial bacteria

The effects of probiotic Bacillus subtilis on the cytotoxicity of Clostridium perfringens type a in Caco-2 cell culture

Abstract Background Some Bacillus strains have recently been identified for potential use as probiotics and food additives. The present study evaluated the antimicrobial effects of Bacillus subtilis ATCC 6633 and its metabolite on the enterotoxin and vegetative cells, spore and germinated spore of Clostridium perfringens type A in Caco-2 cells. Results We used flow cytometry and MTT assays to evaluate the cytotoxicity effect of treatments. According to the results, the most cell survival was found in the 4% crude antimicrobial substance (CAS) with the vegetative form of C. perfringens among co-cultured groups. Furthermore, the apoptosis and necrosis in co-cultured groups were significantly decreased (P < 0.05). Conclusion The present results suggested the crucial role of the current probiotic in the control of various forms of C. perfringens type A which was investigated for the first time. Also, the majority of treatments showed higher cell viability in flow cytometry compared to the MTT assay

Monitoring the Various Types of Clostridium botulinumin in Four Kinds of Food Stuffs Using Multiplex PCR

Background &Objective: Food poisoning (FP) caused by C. botulinum is the most serious feature of FP inpeople consuming the contaminated foodstuffs (Canned meat, vegetarian foods, dairy products and seafood products). Botulism is basically detected by the identification of live bacteria and/or its toxins. Among various types of microorganisms (i.e. A, B, C1, C2, D, E, F), serotypes A, B, E and F are considered as the main human pathogens. The present study was aimed at investigating the possible roles of various foodstuffs to induce the food intoxication and also to compare the culture and molecular assays for identifying the microorganism.Materials &Methods: Three Lab techniques including biochemical, culture (enriched in TPGY and cooked meat medium) and MPCR were used to detect C. botulinum in the samples. As the molecular based techniques have recently employed for the rapid and reliable identification of the bacteria and its toxins, the PCR assay, using three pairs of primers were designed and optimized to identify A, B and E strains in the contaminated specimens. The PCR was able to amplify 782, 205 and 389 bp genes specified for A, B and E types of the bacteria, respectively. Results: Total number of 290 specimens including fish, honey,&quot;kashk&quot;and&quot;Dough&quot; were tested, in which 5%, 4%, 2.5% and 1.25%, were found positive, respectively. Using selective culture of the specimens on the enriched samples, it was shown that just four samples were found positive.Conclusion: As a final conclusion, the molecular based techniques are recommended as a reliable tool to detect C. botulinum and, its toxins and spores in foodstuffs. Moreover, it is strongly advised to use it in food microbial Lab and also the epidemiological surveys

Additional file 5: of The effects of probiotic Bacillus subtilis on the cytotoxicity of Clostridium perfringens type a in Caco-2 cell culture

Percent of cell viability (MTT assay). (DOC 37 kb

Additional file 4: of The effects of probiotic Bacillus subtilis on the cytotoxicity of Clostridium perfringens type a in Caco-2 cell culture

Percent of cytotoxicity (MTT assay). (DOC 32 kb