Toxigenic Genes Research Articles

Prevalence of Bacillus cereus in dairy powders focusing on its toxigenic genes and antimicrobial resistance

Bacillus cereus is a common environmental foodborne microorganism that is mainly found to harbor toxigenic genes with multiple antibiotic resistances and is linked to threatening the safety of dried milk in concern to powdered infant milk formula. In the current investigation, the mean value of B. cereus in 140 samples of powdered milk was 0.57 × 102 ± 0.182 × 102, 0.15 × 102 ± 0.027 × 102, 0.21 × 102 ± 0.035 × 102, and 0.32 × 102 ± 0.072 × 102 CFU/g in a percentage of 64.0 samples of whole milk powder, 43.3 of skim milk powder, 26.7 of powdered infant milk formula and 36.7 milk–cereal-based infant formula, respectively. The results revealed that B. cereus isolates were found to harbor toxigenic genes in the following percentages: 77.8, 2.0, 72.7, 16.2, and 67.7 for nhe, hbl, cytK, ces, and bceT, respectively. Despite all evaluated B. cereus strains were originated from dairy powders, they showed a significant difference (P < 0.05) in their harbored toxigenic cytK gene between whole and skim milk powders with powdered infant formula and milk–cereal-based infant formula, as well as between powdered infant formula and milk–cereal-based infant formula. All isolated B. cereus strains were resistant to cefoxitin, colistin sulfate, neomycin, trimethoprim–sulfamethoxazole, oxacillin, and penicillin. Based on the antimicrobial resistance of B. cereus strains to cephalothin, chloramphenicol, nalidixic acid, and tetracycline, there was a significant difference (P < 0.05) between powdered infant milk formula and whole milk powder strains. This survey is one of few studies proceeded in Egypt to determine the prevalence of toxigenic B. cereus strains in milk–cereal-based infant formula and powdered infant formula as well as skim milk powder.

Occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in 'coalho' cheese produced in Brazil.

The experiments reported in this research communication analysed the presence of methicillin-resistant Staphylococcus aureus (MRSA) in 112 samples of 'coalho' cheese, from 56 dairy producing farms in 28 cities in all mesoregions of the State of Ceará, Brazil. To assess antimicrobial resistance we also examined the presence of genes encoding enterotoxins and toxic shock syndrome toxin, as well as the presence of the blaZ gene for β-lactamases, and resistance to oxacillin. The research found 69 isolates of S. aureus, of which 13.04% had the mecA gene encoding the penicillin-binding protein, which confers resistance to methicillin, in cheese samples from 6 different cities. This included the state capital, Fortaleza, which had the largest prevalence (23.19%) of mecA positive isolates. It was also found that 55.07% of the isolates of S. aureus had the blaZ gene, and 7.25% demonstrated resistance to oxacillin in the plate disc diffusion tests. We did not show the presence of isolates carrying toxigenic genes. The findings suggest that strict supervision of production processes in the dairy industry is necessary in all production scale processes, thus preventing contamination and possible problems for consumers.

Safety evaluation of Lactococcus lactis IDCC 2301 isolated from homemade cheese.

For applications of microorganisms as probiotics in the food industry, safety evaluation has increasingly become important to ensure the health of consumers. Although people have been using various lactic acid bacteria for different purposes, some studies have reported that certain lactic acid bacteria exhibit properties of virulence and produce toxic compounds. Thus, it is necessary to examine the characteristics associated with lactic acid bacteria that are safe for use as probiotics. This research aimed to assess the safety of Lactococcus lactis IDCC 2301 isolated from homemade cheese using in vitro and in vivo assays, including antibiotic resistance, hemolytic activity, toxin production, infectivity, and metabolic activity in immune‐compromised animal species. The results demonstrated that the strain was susceptible to nine antibiotics suggested by the European Food Safety Authority (EFSA). Whole‐genome analysis revealed that L. lactis IDCC 2301 neither has toxigenic genes nor harbors antibiotic resistance. Moreover, L. lactis IDCC 2301 showed neither hemolytic nor β‐glucuronidase activity. Furthermore, none of the D‐lactate and biogenic amines were produced by L. lactis IDCC 2301. Finally, it was demonstrated that there was no toxicity and mortality using single‐dose oral toxicity tests in rats. These results indicate that L. lactis IDCC 2301 can be safely used as probiotics for human consumption.

Evaluation of microbial quality and safety of selected dairy products with special focus on toxigenic genes of Bacillus cereus

Although dairy products play a significant role in human nutrition, they may cause a risk to the consumers. This study intends to determine the microbial contamination level in selected milk products concerning Bacillus cereus enterotoxigenic genes. Three hundred plant-origin flavoured cheese, flavoured yogurt, flavoured drinking yogurt, kareish (skim milk cheese), soft cheese, and Ras (Romy) cheese (50 samples for each) were randomly collected from retail markets at different districts in El Fayoum Governorate, Egypt. The microbiological analysis and the molecular identification of Bacillus cereus by multiplex PCR were applied and revealed potential risks associated with consumption of the tested milk products (white soft, Ras, and kareish cheeses). In all analysed cheeses (white soft, Ras and kareish) a high contamination level with coagulase positive staphylococci (20.0%, 22.0% and 20.0%) and coliform bacteria (76.0, 86.0, and 88.0%) was detected. The highest percent (78.0%) of fungi was found in kareish cheese samples. Bacillus licheniformis was the most aerobic spore forming bacteria detected in kareish cheese (23.7%) and flavoured drinking yogurt (17.0%) samples. The highest prevalence (8.0%) of Bacillus cereus was recorded in the flavoured drinking yogurt and white soft cheese samples. The (nhe) was the most detected gene (100.0%) in all of the examined samples. There was a significant difference (p&lt;0.05) between nhe and ces genes in all investigated samples except in flavoured drinking yogurt samples. Salmonella sp., Escherichia coli and Pseudomonas aeruginosa were not detected. This survey is one of the very few surveys that describe the safety and quality status of plant-origin flavoured cheeses and flavoured drinking yogurt, particularly in Egypt.

Wastewater based environmental surveillance of toxigenic Vibrio cholerae in Pakistan.

BackgroundPakistan has been experiencing intervals of sporadic cases and localized outbreaks in the last two decades. No proper study has been carried out in order to find out the environmental burden of toxigenic V. cholerae as well as how temporal and environmental factors associated in driving cholera across the country.MethodsWe tested waste water samples from designated national environment surveillance sites in Pakistan with RT-PCR assay. Multistage sampling technique were utilized for samples collection and for effective sample processing Bag-Mediated Filtration system, were employed. Results were analysed by district and month wise to understand the geographic distribution and identify the seasonal pattern of V. cholera detection in Pakistan.ResultsBetween May 2019, and February 2020, we obtained and screened 160 samples in 12 districts across Pakistan. Out of 16 sentinel environmental surveillance sites, 15 sites showed positive results against cholera toxigenic gene with mostly lower CT value (mean, 34±2) and have significant difference (p < 0.05). The highest number of positive samples were collected from Sindh in month of November, then in June it is circulating in different districts of Pakistan including four Provinces respectively.ConclusionV. cholera detection do not follow a clear seasonal pattern. However, the poor sanitation problems or temperature and rainfall may potentially influence the frequency and duration of cholera across the country. Occurrence of toxigenic V. cholerae in the environment samples showed that cholera is endemic, which is an alarming for a potential future cholera outbreaks in the country.

English

Verotoxin-producing E. coli are getting an emergence for human health and day by day, it is also becoming more alarming in the fact that, they are acquiring multidrug-resistant profiles. This study aimed at isolation and identification of verotoxigenic E. coli followed by the antibiogram of the species from salad vegetables at Jashore district in Bangladesh. A total of 119 raw vegetable salad samples were collected from different areas of the Jashore district. The collected samples were initially enriched in nutrient broth and then used for streak plating on MacConkey and followed by Eosin Methylene Blue (EMB) agar media. Escherichia coli species were isolated and identified following observation of the cultural, microscopic, and biochemical characteristics and further analyzed to determine the presence of verotoxin (VT) producing genes through polymerase chain reaction (PCR) using specific primers (VT1, VT2, and Eae). A panel of antibiotics were tested following the disc diffusion method for determining the antibiotic profile. Total 55 (46.22%) E. coli was isolated phenotypically. 6 (10.9%) isolated E. coli showed positive by amplifying verotoxin-producing gene vt1, but none of the samples were positive vt2 and eaeA gene. Antibiogram for 55 E. coli isolates showed the highest resistance to Erythromycin (83.64%), Streptomycin (78.18%), Ceftriaxone (70.91%). On the other hand, the most heightened susceptibility was observed for Nalidixic acid (76.36%). Moreover, six vt1 positive isolates showed a multidrug-resistant profile. Thus, the presence of toxigenic genes in the isolates suggests the proper maintenance and regular monitoring of raw salad items should be increase to ensure healthy life in this region. &nbsp; Key words: Vegetable salad, Escherichia coli, verotoxin, antimicrobial resistant, polymerase chain reaction (PCR).

Morphometric and Genomic Characterization of Toxigenic Aspergillus Isolates Associated with Poultry Feeds and Feed Ingredients from Five Agro-Ecological Zones of Nigeria

Introduction: Toxigenic aspergilli are storage contaminants of poultry feeds and feed ingredients which produce secondary metabolites known as mycotoxins in these products. This research is aimed at detecting and characterizing mycotoxin producing aspergillus species present in feeds given to poultry birds and in the ingredients used in the production of these feeds.&#x0D; Method: A total of 102 samples of feed (n=30) and feed ingredients (n=72) were collected across 5 agro-ecological zones of Nigeria and analyzed for toxigenic Aspergilli using morphometric and molecular genomic characterization techniques. Different mycological media was used for isolation, by deploying pour plate method. The Aspergilli isolates morphometric identity were characterized using macroscopic and microscopic observed features. The isolated Aspergillus species were further characterized molecularly using genomic characterization protocols.&#x0D; Results: The results of the morphometric characterization indicated that nine (9) Aspergillus species were detected in the feed and feed ingredients, with Aspergillus flavus having the highest rate of occurrence among the isolates. Furthermore, the genomic typing using internal transcribe spacer (ITS) as a barcode, as well as comparison of the fungal isolates with other Aspergillus species in the genebank showed 99-100% similarity. Also, toxigenic gene typing indicated that some of the A. flavus were toxigenic with the detection of the following genes Nor-1, ver-1, OmtB and AflR.&#x0D; Conclusion: The findings of this study showed that toxigenic Aspergilli isolates are present in both poultry feed and feed ingredients from 5 agro-ecological zones in Nigeria and poses great public health issues.

Control of Virulent Listeria monocytogenes Originating from Dairy Products and Cattle Environment Using Marine Algal Extracts, Silver Nanoparticles Thereof, and Quaternary Disinfectants.

IntroductionListeria monocytogenes is an important foodborne pathogen of public- and animal-health concern globally. The persistence of L. monocytogenes in the dairy-processing environment has multifactorial causes, including lack of hygiene, inefficient cleaning, and improper disinfection practices.Materials and MethodsA total of 300 dairy-product and environmental samples were collected from dairy-cattle facilities and local dairy shops and vendors in Qena, Egypt. Samples were screened for the incidence of Listeria spp. and to detect virulence determinants and disinfectant-resistance genes. Three marine algal species — Caulerpa racemosa, Jania rubens, and Padina pavonica — were collected from Hurghada on the Red Sea coast. Algal extracts were screened using gas chromatography–mass spectrometry. The antimicrobial activity of some marine algal extracts, nanoparticles derived therefrom, and some disinfectants against L. monocytogenes strains were assessed in vitro using agar-well diffusion and liquid-broth methods. The impact of P. pavonica extract on the growth and survival of virulent L. monocytogenes in cheese and whey were clarified.Results and DiscussionThe incidence of L. monocytogenes in dairy products and environmental samples was 15.5% and 19%, respectively. The most common toxigenic gene profile found among the isolates was hlyA+–inlA+–prfA+. The sensitivity pattern of L. monocytogenes strains to disinfectant containing alkyl (C12–16) dimethyl BAC was high compared to other tested quaternary ammonium compounds (QAC) disinfectants tested, which showed lower log reductions against resistant strains. The QAC disinfectant–resistance gene qacH was detected in 40% of the isolates. Potent bactericidal activity of a petroleum ether extract of P. pavonica and silver nanoparticles of P. pavonica were obtained against the virulent L. monocytogenes strain. The population of L. monocytogenes in cheese curd and whey after 14 days was reduced at a rate of 9 log CFU/g and 8 log CFU/mL, respectively due to the effect of P. pavonica extract. After 28 days of storage, L. monocytogenes was completely inactivated in those dairy products.ConclusionP. pavonica extract showed promising antimicrobial properties, calling for further comprehensive studies prior to it being applied in the food industry to enhance the safety, quality, and shelf life of products and protect public health.

Genomic-, phenotypic-, and toxicity-based safety assessment and probiotic potency of Bacillus coagulans IDCC 1201 isolated from green malt

Probiotics are beneficial microorganisms, and the evaluation of their safety for human use in the food industry has become critical. This study examines the safety of Bacillus coagulans IDCC 1201 isolated from green malt by analyzing its genomic and phenotypic characteristics and determining its toxicity. The presence of antibiotic resistance and toxigenic genes and gene transferability were investigated using whole-genome analysis. The strain's hemolytic and enzyme activities, minimum inhibitory concentrations of antibiotics, and biogenic amine and D-lactate production were also examined. Furthermore, the principal properties of B. coagulans IDCC 1201 as probiotics, such as resistance to abiotic stress and intestinal adhesion, were studied. The whole-genome analysis demonstrated that B. coagulans IDCC 1201 had no antibiotic resistance or toxigenic genes; the strain was susceptible to the nine antibiotics proposed by the European Food Safety Authority. Moreover, this strain lacked hemolytic and β-glucuronidase activities. Additionally, it was confirmed that B. coagulans IDCC 1201 produced undesirable metabolites, including biogenic amines or D-lactate, at a safe level. Finally, the strain exhibited functional potential as a probiotic in terms of abiotic tolerance, such as bile tolerance and intestinal adhesion in in vitro experiments. In conclusion, B. coagulans IDCC 1201 can be considered as a safe probiotic with regard to human health.

Faecal contamination and prevalence of pathogenic E. coli in shellfish growing areas along south-west coast of India

Seasonal prevalence of faecal coliforms in Indian black clam (Villorita cyprinoides), sediment and harvesting waters from shellfish harvesting areas along Cochin estuary was evaluated. Enumeration was done by 5 tube most probable number (MPN) method. High prevalence of faecal coliforms was observed in shellfish, sediment and harvesting waters during monsoon and post-monsoon seasons compared to pre-monsoon season. Escherichia coli was isolated and identified by biochemical methods and further confirmed by PCR based detection of uid A gene. A total 125 E. coli strains were isolated; out of which 99 (79.2%) could be serotyped and comprised of 17 diverse serotypes. Approximately 89.06% of the serotyped strains belonged to 5 different pathogenic serotypes. Enterotoxigenic strains were the most prevalent, followed by enterohaemorrhagic, uropathogenic, enteropathogenic and enteroinvasive strains. Enterohaemorrhagic E. coli O157 strains were isolated from shellfish and sediment samples (7.07%). Multiplex PCR analysis revealed the presence of shiga toxin genes (stx1, stx2), intimin (eae) gene and enterohemolysin gene (hlyA). The overall prevalence of toxigenic genes among the strains was 78.1%; in shellfish (84%), sediment (78.1%) and water (69%). Shiga toxin gene stx2, showed the highest prevalence (54.3%), followed by eae (36.2%), stx1(20%), and hlyA (6.7%) genes. To the best of our knowledge, the present study is the first report on the isolation of E. coli O157 from shellfish harvesting areas along the Indian coast. Higher levels of faecal contamination along with the presence of pathogenic E. coli may pose potential health hazards to the consumers.

Advanced molecular characterization of enteropathogenic Escherichia coli isolated from diarrheic camel neonates in Egypt.

Background and Aim:Camels are important livestock in Egypt on cultural and economic bases, but studies of etiological agents of camelid diseases are limited. The enteropathogen Escherichia coli is a cause of broad spectrum gastrointestinal infections among humans and animals, especially in developing countries. Severe infections can lead to death. The current study aimed to identify pathogenic E. coli strains that cause diarrhea in camel calves and characterize their virulence and drug resistance at a molecular level.Materials and Methods:Seventy fecal samples were collected from diarrheic neonatal camel calves in Giza Governorate during 2018-2019. Samples were cultured on a selective medium for E. coli, and positive colonies were confirmed biochemically, serotyped, and tested for antibiotic susceptibility. E. coli isolates were further confirmed through detection of the housekeeping gene, yaiO, and examined for the presence of virulence genes; traT and fimH and for genes responsible for antibiotic resistance, ampC, aadB, and mphA. The isolates in the important isolated serotype, E. coli O26, were examined for toxigenic genes and sequenced.Results:The bacteriological and biochemical examination identified 12 E. coli isolates from 70 fecal samples (17.1%). Serotyping of these isolates showed four types: O26, four isolates, 33.3%; O103, O111, three isolates each, 25%; and O45, two isolates, 16.7%. The isolates showed resistance to vancomycin (75%) and ampicillin (66.6%), but were highly susceptible to ciprofloxacin, norfloxacin, and tetracycline (100%). The structural gene, yaiO (115 bp), was amplified from all 12 E. coli isolates and traT and fimH genes were amplified from 10 and 8 isolates, respectively. Antibiotic resistance genes, ampC, mphA, and aadB, were harbored in 9 (75%), 8 (66.6%), and 5 (41.7%), respectively. Seven isolates (58.3%) were MDR. Real-time-polymerase chain reaction of the O26 isolates identified one isolate harboring vt1, two with vt2, and one isolate with neither gene. Sequencing of the isolates revealed similarities to E. coli O157 strains.Conclusion:Camels and other livestock suffer various diseases, including diarrhea often caused by microbial pathogens. Enteropathogenic E. coli serotypes were isolated from diarrheic neonatal camel calves. These isolates exhibited virulence and multiple drug resistance genes.

The Evolution of Phenotypic Properties and Molecular Genetic Organization of Genomes of Vibrio Cholerae O1 El Tor Variant Strains Isolated from Patients and Environmental Objects in the Caucasus in 1970–1998

Summary. Introduction: The formation of highly pathogenic strains of Vibrio cholerae resistant to antibiotics determines the pathogenesis, clinical picture, epidemiology, and laboratory diagnosis of cholera. It is necessary to study the structure of the genome of V. cholerae of any serological groups isolated during routine monitoring of environmental objects or patients with acute intestinal infections in order to detect toxigenic genes inherent to epidemic strains of V. cholerae O1. Our objective was to study changes in phenotypic properties and molecular genetic bases of persistence, epidemic and pathogenetic potential of El Tor variant of Vibrio cholerae O1 isolated from patients and environmental objects in the Caucasus in 1970–1998. Materials and methods: We studied phenotypic and molecular genetic characteristics of 32 V. cholerae O1 strains isolated from patients and environmental objects in the Caucasus (1970–1998) using a microbiological assay and PCR test, respectively. Results: We established that the following biotypes of V. cholerae O1 were found in the Caucasus: a) El Tor, ctxA–, and Hly+ nontoxigenic strains of V. cholerae O1, typical in their phenotypic and molecular genetic properties. Their toxigenic potential is represented by exo- and endotoxins different from the enterotoxin of toxigenic V. cholerae; b) toxigenic El Tor, ctxABEL+, rtхC+, and Hly– variant biotypes of V. cholerae O1, typical in their phenotypic, molecular and genetic properties during outbreaks of invasive cholera, producing a CT-2 enterotoxin and containing genes responsible for persistent, pathogenic and pandemic potential, and c) typical in terms of their main phenotypic characteristics but molecularly genetically altered variants (hybrids) of El Tor variant of Vibrio cholerae O1 including El Tor ctxABCL+, rtxC+, Hly–. Conclusions: The main manifestation of evolutionary changes in a typical toxigenic vibrio of the El Tor variant biotype was the replacement of the ctxBEl gene in its genome with the ctxBCL gene encoding the classical type of enterotoxin (CT-1), which significantly increased the persistent, toxic, epidemic, and pandemic potential of genetically modified variants of El Tor biotype Vibrio cholerae. The genome of these strains contains an integrative and conjugative element SXT with genes for antibiotic multiresistance.

Whole-genome sequencing of clinical Clostridioides difficile isolates reveals molecular epidemiology and discrepancies with conventional laboratory diagnostic testing

The high clinical burden of Clostridioides difficile infections merits rapid and sensitive identification of affected individuals. However, effective diagnosis remains challenging. Current best practice guidelines recommend molecular and/or direct toxin detection-based screening for symptomatic individuals, but previous work has called into question the concordance and performance of extant clinical assays. To better correlate the genomic and phenotypic properties of clinical C.difficile isolates with laboratory testing outcomes in both C.difficile-infected patients and asymptomatic carriers. Whole-genome sequencing of clinical C.difficile isolates collected from an inpatient population at a single healthcare institution was performed, enabling examination of their molecular epidemiology and toxigenic gene content. Genomic findings were compared with clinical testing outcomes, identifying multiple diagnostic discrepancies. Toxigenic culture, considered a 'reference standard', provided perfect sensitivity and specificity in predicting toxigenic gene content, whereas reduced performance was observed for Simplexa C.difficile Direct Assay (100% specificity, 88% sensitivity), Gene Xpert CD/Epi Assay (86% specificity, 83% sensitivity), and Quick Check Complete Tox A/B (100% specificity, 30% sensitivity). Genomic analysis additionally revealed variability in toxin gene sequences among C.difficile strains, phylogenomic equivalency between isolates from affected patients and carriers, and patient carriage with uncommon environmentally derived C.difficile lineages, as well as presenting opportunities for tracing pathogen transmission events. These results highlight the variable performance of clinical stool-based testing approaches as well as the potential diagnostic utility of whole-genome sequencing as an alternative to conventional testing algorithms.

Characterization of aflatoxigenic Aspergillus flavus associated with aflatoxin B1 (AFB1) production in maize kernel in India

Mycotoxin-producing fungi directly impact on the yield and quality of agricultural produce. Aspergillus flavus is predominant fungi to produce aflatoxin B1 (AFB1) mycotoxin in maize. Early detection and quantification of aflatoxin B1 producing A. flavus is indispensable to mitigate maize loss. With this aim, three different detection methods (PCR, qPCR and HPLC) deployed to screen highly toxigenic and atoxigenic isolates of A. flavus. PCR analysis of four A. flavus isolates revealed possession of six toxigenic genes (aflO, aflP, aflQ, aflM, aflS and aflD), among aflO, aflP and aflQ genes are most common. In qPCR, seven isolates tested representing 5 different maize growing agroclimatic zones of India, expressed nor-1 gene. In two of the isolates, higher mean expression levels were noticed. In HPLC, aflatoxin B1 (AFB1) production capacity was tested in all isolates. About 65% of the A. flavus isolates produced aflatoxin B1 (between 43.87 and 0.88 µg/g). This study exhibited that the highly toxic isolate observed from the sample collected from Hyderabad, where maize crop is growing year round, followed by nagaon’s sample from Assam, therefore these locations will need extra care during post harvest condition to minimize the risk of aflatoxin in storage.

Safety assessment of Streptococcus thermophilus IDCC 2201 used for product manufacturing in Korea.

Safety evaluation of probiotics has become increasingly important for human consumption in food industry. The aims of this study were to assess safety of Streptococcus thermophilus IDCC 2201 through in vitro and in vivo tests. In results, this strain was found to be negative for hemolytic and β‐glucuronidase activity. In addition, thermophilus IDCC 2201 was susceptible to nine antibiotics suggested by EFSA. In accordance with MIC tests, whole‐genome analysis indicated that S. thermophilus IDCC 2201 neither harbors antibiotic resistance nor toxigenic genes. Furthermore, none of the biogenic amines including tyramine and histamine was produced and negligible amounts of D‐lactate were produced by S. thermophilus IDCC 2201. Finally, it was confirmed that there was no mortality and toxicity throughout single‐dose oral toxicity tests in rats. Therefore, we report that S. thermophilus IDCC 2201 is considered to be safe for human consumption as probiotics.

Incidence and Profiles of Antibiotic Resistance and Putative Genes of the Clostridium difficile Recovered From Fish

Clostridium difficile (C. difficile) is a toxigenic bacterium with emergence of antibiotic resistance accountable for incidence of food poisoning. The purpose of this survey was to examine the antibiotic resistance profile and incidence of toxigenic genes amid the C. difficile bacteria recovered from dissimilar varieties of fish samples. One-hundred and eighty-four fish samples were obtained and examined by culture technique. C. difficile isolates were confirmed another time using the polymerase chain reaction. PCR and disk diffusion techniques were applied for detection of putative genes and phenotypic profile of resistance. Eleven out of 184 (5.97%) fish samples harbored C. difficile. Common carp (17.50%) had the uppermost incidence of C. difficile, though Scomberomorus guttatus (2.50%) had the lowermost. There were no positive results for Scomberomorus commerson and barracuda fish samples. TcdA (45.45%) was the most generally perceived toxigenic genes, though tcdC (18.18%) was the less frequently. There were no perceived cdtA and ctdB toxigenic genes. C. difficile bacteria displayed the uppermost incidence of resistance toward amoxicillin (63.60%), ampicillin (54.54%), moxifloxacin (54.54%) and piperacillin (54.54%). C. difficile bacteria displayed the uppermost incidence of susceptibility toward meropenem (90.90%%), vancomycin (90.90%) and metronidazole (72.72%). Common carp, rainbow trout and Scomberomorus guttatus may be reservoirs of C. difficile bacteria. Boost incidence of toxigenic and resistant C. difficile pose an imperative health threating issue rendering the consumption of raw fish samples.

Conventional and molecular diagnosis of clostridium difficile infections in a tertiary care hospital

Introduction: Indiscriminate use of broad-spectrum antibiotics has dramatically increased the incidence of Clostridium difficile-associated diarrhea (CDAD) in recent years. It is the most common cause of antibiotic-associated diarrhea (AAD) responsible for one-third of AAD cases. Aim: The aim was to study C. difficile in AAD. Objectives: The objectives were to study the prevalence of CDAD, to isolate C. difficile from AAD, and to study the molecular detection of toxin-producing strains of C. difficile. Materials and Methods: A total of 222 patients of AAD were assessed for C. difficile over a period of 2 years. Anaerobic culture for C. difficile was done on cycloserine-cefoxitin-fructose agar and brain–heart infusion agar. Enzyme-linked immunosorbent assay (ELISA) for toxin A and toxin B was used for detecting toxigenic strains of C. difficile. Identification of C. difficile and toxin-producing strains was done with the help of polymerase chain reaction (PCR). Observation and Results: Out of the total 222 cases of AAD, 20 (9%) were positive by culture and 70 (31.53%) were found to be toxin-producing C. difficile by ELISA. C. difficile was positive by PCR in 32 (14.41%); of these, 18 (56.25%) isolates were toxigenic, i.e., they possessed either the tcdA or the tcdB gene or both. Among the toxigenic isolates, 10 (31.25%) possessed both of the toxigenic genes (tcdA and tcdB) and the remaining 8 (25%) had one of the toxin genes. Only the toxin A (tcdA+ tcdB-) gene was found in 4 (12.5%) and only the toxin B (tcdA- tcdB+) gene in 4 (12.5%) of the toxigenic isolates. Conclusion: CDAD is an emerging nosocomial infection. Frequent and indiscriminate use of antibiotics has increased the prevalence of C. difficile infection. Conventional and molecular diagnosis can help to accurate diagnosis of these infections.

Occurrence and Abundance of Pathogenic Vibrio Species in Raw Oysters at Retail Seafood Markets in Northwestern Mexico

Seafood has frequently been associated with foodborne illness because pathogens are easily introduced during seafood cultivation, handling, and processing. Vibrio parahaemolyticus and Vibrio cholerae are human pathogens that cause gastroenteritis and cholera, respectively, and Vibrio vulnificus can cause fatal wound infections and septicemia. However, information about the occurrence of these pathogens in oysters from the Pacific coast of Mexico is limited to V. parahaemolyticus. In the present study, we evaluated the presence and abundance of these three Vibrio species in 68 raw oysters (Crassostrea corteziensis) obtained from retail seafood markets in Sinaloa, Mexico. The most probable number (MPN)-PCR assay was used for amplification of the tlh (thermolabile hemolysin), ompW (outer membrane protein), and vvhA (hemolytic cytolysin) genes that are specific to V. parahaemolyticus, V. cholerae, and V. vulnificus, respectively. All oyster samples were positive for at least one Vibrio species. V. parahaemolyticus, V. cholerae, and V. vulnificus prevalences were 77.9, 8.8, and 32.3% overall, respectively, and most species were present in all sample periods with increased prevalence in period 3. The tdh (thermostable direct hemolysin) gene was detected in 30.1%, trh (TDH-related hemolysin) was detected in 3.7%, and tdh/trh was detected in 7.5% of the total tlh-positive samples (53 of 68), whereas the pandemic serotype O3:K6 (orf8 positive) was detected in only 1 sample (1.8%). The total prevalence of tdh and/or trh was 41.5%. In none of the samples positive for V. cholerae were the cholera toxin (ctxA) and cholix (chxA) toxigenic genes or the rfb gene encoding the O1 and O139 antigens amplified, suggesting the presence of non-O1 non-O139 V. cholerae strains. Our results clearly indicated a high prevalence of pathogenic Vibrio species in raw oysters from retail seafood markets in Mexico. Consumption of these raw oysters carries the potential risk of foodborne illness, which can be limited by cooking.

Genomic and Toxigenic Heterogeneity of Bacillus cereus sensu lato Isolated from Ready-to-Eat Foods and Powdered Milk in Day Care Centers in Colombia.

Bacillus cereus sensu lato (s.l.) is a group of bacteria commonly found in diverse environments, including foods, with potential to cause emesis and diarrhea. In Colombia, it is one of the main foodborne pathogens. The aim of this study was to determine the genomic and toxigenic heterogeneity of B. cereus s.l. isolated from ready-to-eat foods and powdered milk collected in day care centers of Medellin, Colombia. Of 112 B. cereus s.l. isolates obtained, 94% were β-hemolytic. Toxigenic heterogeneity was established by the presence of nheABC, hblCDAB, cytK2, entFM, and cesB toxigenic genes. The nheABC operon and entFM gene were most frequently detected in the isolates, whereas the cesB gene was not found. According to the toxin genes content, nine toxigenic profiles were identified. A 44% of isolates had profiles with all genes for nonhemolytic enterotoxin, hemolysin BL, and enterotoxin FM production (profiles II and IV). Pulsed-field gel electrophoresis analysis indicated a high genomic heterogeneity among the B. cereus s.l., with 68 isolates grouping into 16 clusters and 33 placed separately in the dendrogram. This study provides useful information on the safety of ready-to-eat foods and powdered milk in day care centers where children, a susceptible population, are exposed and it should incentive for more studies to understand the distribution of different toxin-encoding genes among B. cereus s.l. isolates, enabling detailed risk assessment.

2372. PCR Ribotype and Antimicrobial Susceptibility of Clostridioides (Formerly Clostridium) difficile in Korea

Background Clostridioides difficile infection is a leading cause of healthcare-associated diarrhea. The epidemiology and characteristics of C. difficile vary geographically. We performed toxin enzyme immunoassay (EIA), toxigenic gene analysis, antimicrobial susceptibility tests (AST), and PCR ribotyping to elucidate the characteristics of C. difficile in Korea.MethodsBetween July 2017 and June 2018, C. difficile was prospectively isolated in 128 specimens from the culture of 1,182 unduplicated specimens. Seventy-five stool specimens with a positive toxin EIA between July 2016 and June 2017 were also included. We performed PCR for the tcdA and tcdB genes on these isolates, and AST and PCR ribotyping on the isolates with a positive toxin EIA.ResultsOlder patients tended to have a higher rate of positive toxin EIA and positive cultures than did younger patients. Ribotype 018 was predominantly identified (48.6%), followed by ribotype 014/020 (9.9%), and ribotype 002 (8.3%). All of A-B+ isolates were either ribotype 017 or B-2. Ribotypes 017, 018, and B-2 showed high resistance to various antibiotics. In contrast, ribotypes 002, 014/020 and C-4 demonstrated low resistance rates, except that to moxifloxacin in ribotype 002. Clindamycin and erythromycin showed a positive correlation. Most of the isolates resistant to rifampicin or tetracycline showed a high MIC to both erythromycin and clindamycin.ConclusionRibotype 018, which is highly transmissible and resistant to various antimicrobial agents, is predominant in Korea. Ribotype 002 has also been increasing in prevalence in Korea.Disclosures All authors: No reported disclosures.