Molecular characterization and toxigenic profiles of Bacillus cereus isolates from foodstuff and food poisoning outbreaks in Brazil.

Bacillus cereus sensu stricto (s.s.) is a well-known foodborne pathogen that produces a range of enterotoxins and is able to cause two different types of foodborne illnesses-the emetic and the diarrheal syndromes. In this study, 54 B. cereus s.s. strains isolated from foodstuff and foods involved in food poisoning outbreaks were characterized according to the presence of toxin-encoding genes, virulence-encoding genes, and panC typing. Most isolates were assigned to panC groups IV (61.1%) and III (25.9%), but members of groups II and V could also be found. Investigation of specific alleles revealed high numbers of isolates carrying toxin and other virulence genes including nheA (100%), nheB (100%), hblA (79.6%), hblC (79.6%), hblD (74.1%), cytK-2 (61.1%), clo (100%), pc-plc (75.9%), sph (68.5%), pi-plc (66.6%), hlyIII (62.9%), and hlyII (24.1%). All isolates were negative for ces and cytK-1. In summary, we detected various enterotoxin and other virulence factor genes associated with diarrheal syndrome in strains analyzed, implicated or not with food poisoning. Furthermore, the most isolates analyzed belong to high-risk phylogenetic groups' panC types III and IV. Our study provides a convenient molecular scheme for characterization of B. cereus s.s. strains responsible for food poisoning outbreaks in order to improve the monitoring and investigation and assess emerging clusters and diversity of strains.

Clostridioides difficile from Fecally Contaminated Environmental Sources: Resistance and Genetic Relatedness from a Molecular Epidemiological Perspective.

Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3')-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven "hypervirulent" RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential.

Prevalence, Antimicrobial Resistance and Toxin-Encoding Genes of Clostridioides difficile from Environmental Sources Contaminated by Feces.

Clostridioides difficile (C. difficile) is the most common pathogen causing antibiotic-associated intestinal diseases in humans and some animal species, but it can also be present in various environments outside hospitals. Thus, the objective of this study was to investigate the presence and the characteristics of toxin-encoding genes and antimicrobial resistance of C. difficile isolates from different environmental sources. C. difficile was found in 32 out of 81 samples (39.50%) after selective enrichment of spore-forming bacteria and in 45 samples (55.56%) using a TaqMan-based qPCR assay. A total of 169 C. difficile isolates were recovered from those 32 C. difficile-positive environmental samples. The majority of environmental C. difficile isolates were toxigenic, with many (88.75%) positive for tcdA and tcdB. Seventy-four isolates (43.78%) were positive for binary toxins, cdtA and cdtB, and 19 isolates were non-toxigenic. All the environmental C. difficile isolates were susceptible to vancomycin and metronidazole, and most isolates were resistant to ciprofloxacin (66.86%) and clindamycin (46.15%), followed by moxifloxacin (13.02%) and tetracycline (4.73%). Seventy-five isolates (44.38%) showed resistance to at least two of the tested antimicrobials. C. difficile strains are commonly present in various environmental sources contaminated by feces and could be a potential source of community-associated C. difficile infections.

A real-time multiplex PCR for the identification and typing of Vibrio cholerae.

We report the development and validation of a duo-triplex real-time polymerase chain reaction (PCR) for the rapid identification and typing of Vibrio cholerae. The PCR assay targets a species-specific toxR gene present in all strains of V. cholerae and used as a marker for the species wbeO1 and wbfO139, encoding the O1 and O139 somatic antigens, and ctxA, encoding cholera toxin (CT). The two tcpA variants associated with the classical and El-Tor biotypes are used to infer biotype. The assay was evaluated using 178 isolates comprising eight different Vibrio species, including 122 isolates of V. cholerae. The PCR results of 171/178 (96.1%) isolates were concordant with the serotyping, biotyping, and expected CT results. Variants of toxR (n=3), nonfunctional wbeO1 (n=1), and CT-negative isolates of V. cholerae O1 (n=3) were likely explanations for the mismatched results. This duo-triplex real-time PCR is a reproducible and robust assay for the rapid identification and typing of V. cholerae belonging to the highly pathogenic, pandemic lineages.