Enterotoxin Genes Research Articles

Pathogenic potential of ornithogenic <i>Escherichia coli</i> strains detected in the Earth's polar regions

Introduction. Pathogenic strains of Escherichia coli are an important object of surveillance within the One Health concept in the wild, agriculture and human society. Migratory bird colonies and high latitude avian colonies may be points of active intraspecies and interspecies contact between different animal species, accompanied by the spread of pathogens. At the same time, the phylogeography of E. coli in relation to the presence of natural foci of colibacillosis in polar regions remains virtually unstudied. The aim of this study was to assess the pathogenic potential of E. coli strains from the polar regions of the Earth, based on the analysis of the genomes of these bacteria from typical ornithogenic ecosystems of the Arctic and Antarctic. Materials and methods. The study used collections of E. coli isolated from ornithogenic biological material during expeditions to high latitude areas of the Arctic (archipelagos of Novaya Zemlya, Franz Josef Land, Svalbard) and Antarctic (Haswell Archipelago). 16 cultures associated with avian E. coli (12 polar and 4 temperate strains) were selected for genome-wide sequencing using BGI technology. The annotation of the genomes focused on the identification of genes for pathogenicity factors and antimicrobial resistance, as well as the identification of strains belonging to individual genetic lineages using the cgMLST method. Results. The annotation of the genomes allowed their assignment to different sequence types in the multilocus sequencing typing and genome-wide sequencing typing schemes. The analysis of the geographical distribution of the sequence types of polar E. coli strains determined by the cgMLST method showed their global representation in geographically distant regions of the planet. For example, cgST 133718 was observed in Antarctica (strain 17_1myr) and in the UK, and sequence 11903, to which strain 32-1 from the northernmost point of Novaya Zemlya belonged, was previously identified in the USA. All strains studied were characterized by the presence of extensive virulence. Among the pathogenicity factors identified were haemolysins A, E, F, siderophores, including the yersiniabactin gene cluster, a number of adhesion, colonization and invasion factors, as well as the thermostable enterotoxin EAST-1 and genes that characterize enteroaggregative strains of E. coli (the virulence regulator gene eilA and enteroaggregative protein (air)). One of the Arctic strains (33-1) had determinants of antibiotic resistance, in particular the extended-spectrum beta-lactamase gene TEM-1b and the Tn1721 transposon, including tetracycline resistance genes (tetA-TetR), were detected in its genome. Conclusion. The results of the study indicate the circulation of E. coli strains with strong pathogenic potential in high-latitude Arctic and Antarctic ornithogenic ecosystems. The analysis of genomic data indicates the presence of geographically widespread genetic lineages in these regions, which justifies the importance of monitoring epidemic clones of E. coli, along with monitoring for other pathogens, in bird colonies in high-latitude areas.

Molecular and genetic analysis of methicillin-resistant Staphylococcus aureus (MRSA) in a tertiary care hospital in Saudi Arabia.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to pose significant challenges in healthcare settings due to its multi-drug resistance (MDR) and virulence. This retrospective study examines the molecular and resistance profiles of MRSA isolates from a tertiary care hospital in Saudi Arabia, providing valuable insights into regional epidemiology. A total of 190 MRSA strains were analysed to assess antimicrobial susceptibility, genetic diversity, and virulence factors. Antimicrobial susceptibility testing was conducted according to CLSI guidelines, while molecular characterization involved spa typing, SCCmec typing, and DNA microarray analysis to determine clonal complexes (CCs), resistance genes, and virulence determinants. The isolates showed extensive resistance to beta-lactam antibiotics, with 78% classified as MDR. Notably, resistance to fusidic acid and ciprofloxacin was detected in 70% and 55% of isolates, respectively. The most prevalent clonal complexes-CC5, CC6, and CC22-comprised over 60% of the isolates and exhibited diverse spa types. The Panton-Valentine leukocidin (PVL) gene, linked to heightened virulence, was identified in approximately 20% of isolates, particularly within CC5, CC30, and CC80. Enterotoxin genes (sea and seb) and immune evasion genes (sak, chp, and scn) were also commonly detected, reflecting the isolates' capacity to adapt and persist within the hospital environment. These findings underscore the high burden of MDR MRSA with considerable genetic diversity and virulence potential. The study highlights the urgent need for strengthened molecular surveillance and targeted infection control measures to limit MRSA transmission and effectively manage infection risks in healthcare facilities.

Sample-to-answer microfluidic device towards the point-of-need detection of Staphylococcus aureus enterotoxin genes in ruminant milk.

Milk is commonly screened both for indicators of animal disease and health, but also for foodborne hazards. Included in these analyses is the detection of Staphylococcus aureus, that can produce an enterotoxin, causing staphylococcal food poisoning (SFP), which often leads to sudden onset of significant gastrointestinal symptoms in humans. Epidemiological data on SFP are limited, particularly in low- and middle-income countries. Many conventional assays for the detection of staphylococcal enterotoxins rely on the detection of the genes coding for them, either directly in food samples or after bacterial culture. Currently, many of the nucleic acid-based methods used require specific expertise and equipment, whilst bacterial culture takes 24-48 hours; both are contributory factors that limit efforts either during food safety emergencies or routine screening. Here we present the development of a "sample-to-answer" isothermal nucleic acid loop-mediated amplification (LAMP) assay in a microfluidic device for the detection of Staphylococcus aureus enterotoxin genes in ruminant milk. A multiplex LAMP assay targeting two of the most prevalent S. aureus enterotoxin-encoding genes (A and B) was integrated into a microfluidic device combining simple 1 : 10 dilution for sample preparation and a lateral flow assay for easy readout. We achieved a limit of detection of 104 colony forming units per ml in spiked cow and goat milk samples, an order of magnitude more sensitive than the European recommendation for the maximum allowable presence of coagulase-positive staphylococci in raw milk. The assay showed no cross-reactivity in detecting other tested non-enterotoxigenic S. aureus strains or associated foodborne pathogens. The test integrated the simplicity of use of microfluidic devices with the sensitivity, specificity and rapidity of a nucleic acid-based assay, and a simple lateral flow readout to provide an appropriate device to ensure the safety of milk for human consumption. To illustrate its potential for point-of-need practical applications, the test was performed in agricultural settings in rural Turkey in a limited feasibility exercise.

Vancomycin-resistant Staphylococcus aureus endangers Egyptian dairy herds.

The emergence of pandrug-resistant (PDR) and extensive drug-resistant (XDR) methicillin-resistant and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) isolates from bovine milk samples along with biofilm formation ability and harboring various virulence genes complicates the treatment of bovine mastitis and highlights the serious threat to public health. This study investigated for the first time the frequency, antimicrobial resistance profiles, biofilm-forming ability, virulence factors, spa and staphylococcal cassette chromosome mec (SCCmec) types of MRSA and VRSA isolated from clinical and subclinical bovine mastitis in Egypt. A total of 808 milk samples were collected from each quarter of 202 dairy animals, including 31 buffaloes and 171 cattle. The frequency of mastitis in the collected milk samples was 48.4% (60/124) in buffaloes and 29.2% (200/684) in cattle. A total of 65 Staphylococcus species isolates were recovered, including 27 coagulase-positive S. aureus (CoPS) isolates and 38 coagulase-negative staphylococci (CoNS). The CoNS included 27 mammaliicocci (20 Mammaliicoccus lentus and 7 M. sciuri) and 11 Non-aureus staphylococci (S.lugdunensis) isolates. All the CoPS isolates were mecA positive and resistant to 20-33 tested antimicrobials with multiple antibiotic resistance index ranging from 0.61 to 1. Three isolates were PDR, four were XDR, and 20 were multidrug resistant isolates. VRSA was detected in 85.2% of CoPS isolates with minimal inhibitory concentration (MIC) ranging from 64 to 1024µg/mL. The vanA gene was found in 60.8%, vanB in 73.9%, and both genes in 43.5% of VRSA isolates. All the CoPS isolates exhibited biofilm formation ability, with 55.6% being strong, and 44.4% moderate biofilm producers, and harbored icaA (74.1%) and icaD (74.1%) biofilm-forming genes. All S. aureus isolates harbored both beta-haemolysin (hlb) and leucotoxin (lukMF) genes, while 44.4% were positive for toxic shock syndrome toxin (tsst) gene. Enterotoxin genes sea, seb, sec, sed, and see were found in 59.3%, 40.7%, 18.5%, 33.3%, and 14.8% of isolates, respectively. Additionally, 70.4% of the isolates hadspa X-region gene, and exhibited eight different MRSA spa types (t127, t267, t037, t011, t843, t1081, t2663, and t1575), with spa t127 being the most common. Three SCCmec types (I, II and III) were identified, with SCCmec I being predominant, and were further classified into subtypes 1.1.1, 1.1.2, 1.n.1, and 4.1.1. The ability of MRSA and VRSA isolates to produce biofilms and resist antimicrobials highlights the serious threat these pathogens pose to bovine milk safety, animal welfare, and public health. Therefore, strict hygiene practices and antimicrobial surveillance are crucial to reduce the risk of MRSA and VRSA colonization and dissemination.

Enterotoxigenic profile, biofilm production, and antimicrobial resistance of Bacillus cereus isolated from rice-based food marketed in southern Mexico

Bacillus cereus is responsible for food poisoning worldwide; thus, the characterization of strains isolated from food, in this case rice, is essential. The objective of this study was to identify the toxigenic profile, lytic enzymes, antimicrobial resistance, and biofilm production of B. cereus strains isolated from rice. The genetic profile of toxins and biofilm-related genes in the strains was determined by endpoint PCR. Biofilm formation was visualized using safranin staining, and the evaluation of lytic enzymes was conducted in culture media. Psychrophilic characteristics were monitored by assessing the growth of the strains at refrigeration temperature. The GTG5 technique was employed to determine the genetic diversity of the strains, and their antimicrobial resistance was validated through minimum inhibitory concentration testing. The strains of B. cereus s.l. isolated from rice contained genes for enterotoxins and genes associated with biofilm production. However, the strains did not possess the cereulide gene. The strain isolated from fried rice was the only one that contained the hbl toxin gene and the eps2 operon. Interestingly, this strain was the only one that did not produce biofilm. It exhibited intermediate sensitivity to erythromycin, was positive for amylase activity, showed high lecithinase activity, and was capable of growing at refrigeration temperature.

High prevalence of CTX-M-15 producing Shigella spp. isolated from patients with gastroenteritis in Northeast Iran.

Shigellosis, a diarrheal disease caused by Shigella species, is a significant public health concern, particularly in developing countries with inadequate sanitation systems. This study aimed to investigate the patterns of antibiotic resistance, ESBL and AmpC genes, integrons, and enterotoxin genes in Shigella species isolated from patients with gastroenteritis in Northeast Iran. This cross-sectional study was conducted between January 2017 and December 2019 at a tertiary care hospital in Northeast Iran. Atotal of 110 Shigella isolates were collected from stool samples of patients with gastroenteritis. The isolates were identified using conventional biochemical tests and confirmed by PCR. The highest resistance rates were detected for ampicillin (88.2%) and cotrimoxazole (84.5%). Altogether 64.5% of isolates exhibited multidrug resistance however, ESBL and AmpC phenotypes were detected in 34.54% and 1.81% of isolates, respectively. Interestingly, blaCTX-M-15 and blaTEM were detected in all ESBL-positive isolates but integron class 1, 2, and 3 were identified in 97.3%, 76.4%, and 59.1% of isolates, respectively. The sen gene was present in 72.7% of the isolates. In this study CTX-M-15 production was detected in 31 strains of Shigella sonnei and in 7 strains of Shigella flexneri. The high prevalence of multidrug-resistant Shigella isolates is concerning and shows the need for continuous monitoring and rational use of antibiotics.

Prevalence and characterization of Staphylococcus aureus isolated from meat and milk in Northeastern Italy.

Staphylococcus aureus is a pathogenic microorganism often found in animal-derived foods and is known for its ability to readily develop resistance to antibiotic treatments. This study was designed to determine prevalence of S. aureus strains in raw milk and meat in Italy and to evaluate their antibiotic resistance profiles and biofilm production. Among the meat isolates, 41.67% were resistant to ampicillin, and 25% were methicillin-resistant S. aureus (MRSA). In milk, 20% of the isolates were resistant to gentamycin, while 5.71% were MRSA. The prevalence of multidrug-resistant strains was higher in meat (16.67%) compared to milk (5.71%). The biofilm formation capability was assessed in most of the isolates (80% in milk and 100% in meat). Representative strains exhibiting different antibiotic resistance profiles were all negative for the enterotoxin genes sea, seb, sec, sed, and see, but harboured potential virulence factors such as haemolytic activity, high pigmentation, low cell envelop permeability, charged and hydrophobicity. Finally, the interaction of representative strains with human Caco-2 intestinal cell line showed that most strains had an adhesion capacity. Our findings reveal that foodborne isolates of S. aureus present a considerable threat to consumers due to their production of virulence factors, which enhance their pathogenicity and increase the likelihood of antibiotic treatment failures.

A Recombinant Shigella flexneri Strain Expressing ETEC Heat-Labile Enterotoxin B Subunit Shows Promise for Vaccine Development via OMVs.

Diarrheal diseases caused by Shigella and enterotoxigenic Escherichia coli (ETEC) are significant health burdens, especially in resource-limited regions with high child mortality. In response to the lack of licensed vaccines and rising antibiotic resistance for these pathogens, this study developed a recombinant Shigella flexneri strain with the novel incorporation of the eltb gene for the heat-labile enterotoxin B (LTB) subunit of ETEC directly into Shigella's genome, enhancing stability and consistent production. This approach combines the immunogenic potential of LTB with the antigen delivery properties of S. flexneri outer membrane vesicles (OMVs), aiming to provide cross-protection against both bacterial pathogens in a stable, non-replicating vaccine platform. We confirmed successful expression through GM1-capture ELISA, achieving levels comparable to ETEC. Additionally, proteomic analysis verified that the isolated vesicles from the recombinant strains contain the LTB protein and the main outer membrane proteins and virulence factors from Shigella, including OmpA, OmpC, IcsA, SepA, and Ipa proteins, and increased expression of Slp and OmpX. Thus, our newly designed S. flexneri OMVs, engineered to carry ETEC's LTB toxin, represent a promising strategy to be considered as a subunit vaccine candidate against S. flexneri and ETEC.

Whole genome sequencing revealed high proportions of ST152 MRSA among clinical Staphylococcus aureus isolates from ten hospitals in Ghana.

Previous studies in Ghana indicated low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and predominance of ST152 methicillin-susceptible S. aureus (MSSA) among clinical isolates. ST152 MRSA clones are associated with severe infections and epidemics. Using whole genome sequencing (WGS), 159 S. aureus isolated from clinical sources (wound, blood, urine, ear, abscess, umbilical cord, eye, vaginal samples, and others) from 10 hospitals across Ghana were investigated. mecA (gene for methicillin resistance) was detected in 38% of the isolates. Panton-Valentine leucocidin toxin (PVL) gene occurred in 65% isolates, with 84% of the MRSA's harboring the PVL gene. ST152 was the major clone, with 74% harboring the mecA gene. Other MRSA clones detected were ST5, ST5204, ST852, and ST1. MSSA clones included ST3249, ST152, ST5, ST1, and ST8. Twenty-three genes encoding resistance to 12 antimicrobial classes were observed with blaZ (97%) being the most prevalent. Other predominant resistance genes included tetK (46%), cat (42%), and dfrG (36%) encoding resistance for tetracyclines, phenicols, and diaminopyrimidine, respectively. Virulence genes for enterotoxins, biofilms, toxic-shock-syndrome toxins, hemolysins, and leukotoxins were also detected. Phylogenetic analysis revealed a shift in the dominant clone from MSSA ST152 to MRSA ST152 over the past decade. The study provides valuable insights into the genomic content of S. aureus from clinical sources in Ghana. The finding of ST152 MRSA in high numbers suggests a shifting epidemiological landscape of these pathogens and continuous surveillance using robust tools like WGS is needed to monitor the rise and spread of these epidemic clones in the country.IMPORTANCESince its emergence in 1959, MRSA has been a significant public health concern, causing infections in both clinical and community settings. Patients with MRSA-related infections experience higher mortality rates due to its ability to evade antimicrobials and immune defenses. In Ghana, understanding the molecular epidemiology of MRSA has been hindered by the lack of appropriate laboratory infrastructure and the limited capacity for molecular data analysis. This study, the largest genomic study of S. aureus in Ghana, addresses this gap by utilizing whole genome sequencing to examine the diversity of circulating S. aureus strains from 10 hospitals. Our findings highlight the predominance of pandemic clones, particularly ST152, and the notable transition of ST152 MSSA to ST152 MRSA over the past decade. The findings from this study supports AMR surveillance efforts in Ghana and emphasize the importance of implementing genomic surveillance using WGS to comprehensively monitor the rise and spread of multi-drug-resitant organisms such as MRSA in the country.

Diversity of Staphylococcus aureus enterotoxin genes and its potential impact on severity of mastitis in dairy cows

Staphylococcus aureus enterotoxins (SEs) can lead to food poisoning and appear to play a pivotal role in the development and severity of mastitis. This study aimed to evaluate whether the presence and diversity of S. aureus Enterotoxin genes mastitis-causing are associated with the development and severity of mastitis in dairy cows. Comparative analysis of S. aureus isolates from cows with clinical and subclinical mastitis (103 isolates each, CM and SM respectively) was conducted, examining the occurrence of enterotoxins (sea-see, seg-sez, sel26, sel27, sel01 and se02). Clinical isolates exhibited greater diversity, with 14 genes compared to four in subclinical isolates, and higher gene frequency (100 % vs. 86.4 %). Among the classical SEs, only sec (1 %) and sed (4.9 %) were detected in clinical isolates, suggesting a potential insignificance in the development of mastitis. Conversely, the selw and selx genes were among the most prevalent in both clinical and subclinical mastitis isolates. While their role in human food poisoning has not been definitively established, they appear to influence mastitis development. Statistical analysis revealed significant differences in clinical isolates carrying seg, seh, sei, seo, seul1, seul2, selw, and selx genes compared to subclinical isolates, suggesting their potential involvement in the severity of mastitis. Determining the frequency of SE gene and its role in mastitis establishment and in clinical mastitis severity, as well as understanding how enterotoxins contribute to pathogenicity in clinical and subclinical mastitis, is crucial for guiding optimal medical therapy for animals and establishing effective veterinary treatments to mitigate economic losses in the dairy industry.

Methicillin- and vancomycin-resistant Staphylococcus aureus in chicken carcasses, ready-to-eat chicken meat sandwiches, and buffalo milk

Methicillin-resistant Staphylococcus aureus (MRSA) is a growing public health concern; however, there is limited information about MRSA and VRSA (Vancomycin-resistant S. aureus) among animal-origin food. Therefore, this study intended to elucidate the prevalence, enterotoxin existence, antimicrobial resistance profiles, and antimicrobial resistance genes of S. aureus strains isolated from chicken carcasses, ready-to-eat (RTE) chicken meat sandwiches, and buffalo milk samples marketed in Mansoura City, Egypt. Of the 240 samples examined, 52.1 % were contaminated with S. aureus, with a mean count of 4.11 log10 CFU/g. A total of 250 isolates were verified as S. aureus by PCR targeting nuc gene, of which 39.2 % (98/250) harbored at least one S. aureus enterotoxin (SE) gene. The predominant SE genes were sea (61.2 %, 60/98), followed by sed (58.2 %, 57/98), sec (38.8 %, 38/98), and seb (27.6 %, 27/98). All isolates were resistant to at least one antimicrobial, with an average MAR (multiple antibiotic resistance) index of 0.530. Four isolates exhibited resistance to all antimicrobial agents tested. Interestingly, 100 %, 76.4 %, 35.6 %, 30.8, 10.4 %, 6 %, and 1.6 % of isolates were resistant to penicillin, azithromycin, oxacillin, ceftaroline, vancomycin, linezolid, and daptomycin, respectively. Of the 250 S. aureus strains tested, 38 % were confirmed as MRSA by mecA gene, while 10.4 % were identified as VRSA by vanA gene. The high prevalence of MRSA and VRSA isolates among samples tested is worrisome. Hence, monitoring antimicrobial usage in veterinary medicine and applying strict hygienic measures during food handling and processing is imperative to prevent the spread of such resistant bacteria and protect public health.

An Investigation of Virulence Genes of Staphylococcus aureus in Autologous Vaccines Against Sheep Mastitis.

Staphylococcus aureus is well known to be the primary causal agent of clinical or subclinical mastitis in dairy sheep. The production of virulence factors allows S. aureus strains to cause mastitis. In the present study, 96 strains isolated from dairy sheep farms used for the production of autologous vaccines were tested for enterotoxin and hemolysin genes by PCR. In addition, 14 strains isolated from half udders of ewes with subclinical mastitis belonging to a single farm were also tested for the same genes. The phylogenetic trees were constructed, and spatial analysis was performed. Overall, 20 gene patterns were identified, but 43.64% of the tested strains showed the same profile (sec+, sel+, hla+, hld+, hlgAC+). Considering only the enterotoxin genes, four profiles were identified while the evaluation of the hemolysin genes revealed the presence of 12 gene patterns. In the farm with subclinical mastitis, six gene profiles were found. Spatial analysis of the isolated strains and their virulence genes did not show a specific pattern. The present study highlights the importance of identifying and analyzing virulence genes of S. aureus strains involved in dairy sheep mastitis, and the presence of different strains in the same farm.

Production of bacteriocin-like inhibitory substance (BLIS) by Staphylococcus spp. isolates from dogs.

In the present study, 39 canine isolates of Staphylococcus spp. were tested for antimicrobial substance (AMS) production. Seven AMS producers were identified, whose products exhibited a non-acidic character and a proteinaceous nature, therefore being considered bacteriocin-like inhibitory substances (BLIS). The producer strains of BLIS P1, P16 and I3 showed a broad spectrum of antimicrobial activity. Human, veterinary and plant pathogens, such as Listeria monocytogenes, Klebsiella pneumoniae, Staphylococcus spp. and Clavibacter michiganensis, were among the inhibited micro-organisms, suggesting the potential biotechnological application of these peptides. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified the producer strains of BLIS P1, P16 and I3 as Staphylococcus pseudintermedius P1, Staphylococcus schleiferi P16 and Staphylococcus pseudintermedius I3. The plasmid profile of these strains suggests that the BLIS production is linked to biosynthetic genes located on plasmids. PCR analyses revealed that BLIS P1, P16 and I3 are different from 11 staphylococcins already described in the literature and that their genomic DNAs do not carry the most prevalent staphylococcal enterotoxin genes. The highest levels of BLIS production were achieved after 18-24h of growth of the producer strains in TSB medium. Moreover, BLIS P1 and I3 exhibited high resistance to temperature and pH variations, and BLIS P16 maintained 100% of its activity in almost all conditions tested. The characteristics associated with BLIS P1, P16 and I3 described in this work encourage further investigation of these substances, in addition to this study being the first report of BLIS production by a strain of S. schleiferi.

Investigation of Various Toxigenic Genes and Antibiotic and Disinfectant Resistance Profiles of Staphylococcus aureus Originating from Raw Milk.

This study investigated the toxigenic genes and antimicrobial resistance profiles of Staphylococcus aureus strains isolated from 260 raw milk samples collected from dairy farms in Türkiye. The results indicated that 60.7% of staphylococcal enterotoxin genes (sea, seb, sed, seg, sei, sej, sek, seq, sem, seo, and seu) and 21.4% of the tst and eta genes were positive, with most enterotoxin-positive samples carrying more than one gene. The sec, see, seh, sel, sen, sep, and etb genes were not identified in any samples. The prevalence of antibiotic resistance genes (mecA, blaR, blaI, blaZ, vanA, ermT, tetK, aac/aph, ant, dfrA, tcaR, IS256, and IS257) was high at 89.2%, with bla being the most frequently detected gene (75%). The mecA gene was present in 14.2% of samples, while tcaR was detected in 78.5%. Nevertheless, the mecC was not identified. Disinfectant resistance genes (qacA/B, qacC, qacJ, smr) were detected in 21.4% of the samples. The results of the disk diffusion test showed that 64.2% of strains were resistant to penicillin G and ampicillin, with additional resistance found for cefoxitin, teicoplanin, levofloxacin, norfloxacin, and other antibiotics. These findings highlight a significant public health and food safety risk associated with raw milk due to the presence of S. aureus strains with toxigenic genes and high antimicrobial resistance.

Virulence characterization of Staphylococcus spp. isolated from mozzarella cheeses and cheese-slicers

Different species of the Staphylococcus genus can be found as contaminants in foods and may carry virulence factors that could potentially make them The enterotoxigenic potential ofpathogenic or transfer genes to other bacteria. Therefore, the aim of the present study was to isolate and identify species of Staphylococcus genus isolated from sliced mozzarella cheeses and cold-cut slicers and characterize biofilm formation in vitro, presence of enterotoxins and antibiotic resistance genes, and β-lactamase production. Sampling was carried out in 44 retail markets and isolates identified on species level. Resistance genes blaZ and mecA, as well as enterotoxin genes sea, seb, sec, and sed, were identified using PCR. Phenotypic evaluation of biofilm formation and detection of β-lactamase enzyme were also analyzed. Nine species of coagulase-negative Staphylococcus (CNS) were identified from 85 bacterial isolates. Of this total, 67.0% were positive for the presence of one or both antimicrobial resistance genes blaZ and mecA, while the β-lactamase enzyme was detected in 52.9% isolates. Regarding biofilm production, 48.2% of isolates were positive and 51.8% were negative. However, none of the isolates were identified with the enterotoxin genes. The results demonstrate the potential risk of cross-contamination, as biofilm-producing CNS could persist in the cheese retail environment, highlighting the danger of resistance genes transferring to other commensal or pathogenic bacteria.

Characterization of Staphylococcus aureus isolated from milk samples for their virulence, biofilm, and antimicrobial resistance

The Staphylococcus aureus (S. aureus) one of the important food borne pathogen from milk, which was investigated in this study. The isolates were screened for antimicrobial resistance, enterotoxin genes, biofilm formation, spa typing, coagulase gene polymorphism and accessory gene regulator types. The prevalence of S. aureus in milk samples was 34.4% (89/259). Methicillin resistant S. aureus (MRSA) was found at 27% (24/89) of the isolates, were classified as community acquired based on SCCmec typing. The 24.71% (22/89) isolates demonstrated multiple antimicrobial resistance (MAR) pattern. However, none of the isolates carried vancomycin and mupirocin resistance genes. The isolates were positive for sea and sed enterotoxin genes and exhibited high frequency of biofilm formation. The High-Resolution Melting and conventional spa typing revealed that the isolates had both animal and community-associated S. aureus clustered origins. Coagulase gene polymorphism and agr typing demonstrated variable genotypic patterns. The finding of this study establishes the prevalence of community associated, enterotoxigenic, biofilm forming and antimicrobial resistance among S. aureus from milk in Chennai city. This emphasizing a potential threat to public health which needs a continuous monitoring system and strategies to mitigate their spread across the food chain and achieve food safety.

Identification of a new Clostridium perfringens variant with a chromosomally encoded enterotoxin gene in a suspected persistent food poisoning outbreak in Eritrea.

Clostridium perfringens is a causative agent of various human and animal enteric diseases including food poisoning. In this study, we describe an interesting case of a persistent food poisoning outbreak among Finnish peacekeepers in Eritrea, possibly caused by Clostridium perfringens carrying a new variant of the chromosomally encoded enterotoxin gene. C. perfringens strains causing food poisoning carry the enterotoxin gene, cpe, in its chromosome (c-cpe) or on a plasmid (p-cpe). PCR assays are widely used for toxinotype C. perfringens strains. The integration sites for the cpe gene are highly conserved, and PCR assays targeting the cpe gene and the adjacent IS elements (the IS1470 in c-cpe and the IS1470-like or IS1151 in p-cpe strains) are used to further determine the genetic location of the cpe gene. We sequenced nine enteropathogenic C. perfringens strains related to a persistent food poisoning outbreak among Finnish peacekeepers in Eritrea. Six of these strains produced non-typeable cpe results in the standard PCR assay due to divergence in the enterotoxin integration site. The gene order of the new variant of the chromosomal cpe insertion site with an additional IS1470 element impairing genotyping PCR assay for the location of cpe is described. In addition, variant c-cpe strains carried 58-81 copies of IS1470 in their genomes, compared to 9-23 copies found in previously described c-cpe strains. Thus, the present study represents an untraditional type of C. perfringens food poisoning caused by variant c-cpe strains, and the sequenced strains bring geographic variation to the existing strain collection of sequenced C. perfringens.

Predominance of enterotoxigenic Escherichia coli among ESBL/plasmid-mediated AmpC-producing strains isolated from diarrheic foals: a public health concern

BackgroundThe upsurge of diarrheagenic E. coli pathotypes carrying extended-spectrum beta-lactamases (ESBLs)/plasmid-mediated AmpC β-lactamase (pAmpC) among animals constitutes an emerging threat for humans and animals. This study investigated the burden of ESBL-/pAmpC-producing diarrheagenic E. coli among diarrheic foals and its potential public health implications. Rectal swabs were collected from 80 diarrheic foals. These swabs were processed to isolate and identify ESBL/pAmpC-producing E. coli using a selective culture medium, biochemical tests, phenotypic identification, and molecular identification of ESBL- and pAmpC-encoding genes. Moreover, all ESBL-/pAmpC-producing E. coli isolates were examined for different virulence genes related to diarrheagenic E. coli pathotypes.ResultsOut of 80 examined foals, 26 (32.5%) were confirmed as ESBL-/pAmpC-producing E. coli, of which 14 (17.5%) animals carried only ESBL-producing E. coli, whereas 12 (15%) animals possessed ESBL-pAmpC-producing E. coli. The only detected diarrheagenic pathotype was enterotoxigenic, encoded by the heat-stable enterotoxin gene (ST) with a prevalence rate of 80.8% (21/26). The ST gene was further characterized where STa, STb, and STa + STb were found in one, four, and 16 strains, respectively. Moreover, all enterotoxigenic E. coli (ETEC) isolates exhibited a multidrug-resistance pattern. The phylogenetic analysis of 3 obtained partial STb sequences revealed high genetic relatedness to ETEC isolates retrieved from humans, conferring such sequences' public health significance.ConclusionsThese findings highlight that diarrheic foals could serve as a potential reservoir for multidrug-resistant ESBL-/pAmpC-producing enterotoxigenic E. coli.

Heat resistance differences are common between both vegetative cells and spores of Clostridium perfringens type F isolates carrying a chromosomal vs plasmid-borne enterotoxin gene.

Clostridium perfringens type F isolates utilize C. perfringens enterotoxin (CPE) to cause food poisoning (FP) and nonfoodborne gastrointestinal diseases. The enterotoxin gene (cpe) can be located on either the chromosome or plasmids, but most FP isolates carry a chromosomal cpe (c-cpe) gene. Our 2000 article in Applied and Environmental Microbiology (66:3234-3240, 2000, https://doi.org/10.1128/aem.66.8.3234-3240.2000https://doi.org/10.1128/AEM.66.8.3234-3240.2000) determined that vegetative cells and spores of c-cpe isolates are more heat resistant than those of plasmid cpe (p-cpe) isolates, which is favorable for their survival in improperly cooked or held food. However, that 2000 article was recently retracted (90:e00249-24, 2024, https://doi.org/10.1128/aem.00249-24). To our knowledge, the 2000 article remains the only study reporting that heat resistance differences are common between both vegetative cells and spores of type F c-cpe isolates vs type F p-cpe isolates. To confirm and preserve this information in the literature, the heat resistance portion of the 2000 study has been repeated. The 2024 results reproduced the 2000 results by indicating that, relative to the surveyed type F p-cpe isolates, the vegetative cells of surveyed type F c-cpe isolates are ~2-fold more heat resistant and the spores of most surveyed c-cpe isolates are ~30-fold more heat resistant. However, consistent with several reports since our 2000 paper, one surveyed type F c-cpe isolate (which did not appreciably sporulate in 2000 but sporulated in 2024) produced spores with intermediate heat sensitivity, confirming that spores of some type F c-cpe isolates lack exceptional heat resistance.IMPORTANCEClostridium perfringens type F food poisoning (FP), which is the second most common bacterial cause of FP, involves the production of C. perfringens enterotoxin. While the enterotoxin gene (cpe) can be located on either the chromosome or plasmids in type F isolates, most FP cases are caused by chromosomal cpe isolates. The current results support the conclusion that the vegetative cells and spores of type F chromosomal cpe isolates are often more heat resistant than vegetative cells and spores of type F plasmid cpe isolates. Greater heat resistance should favor the survival of the spores and vegetative cells of those chromosomal cpe isolates in temperature-abused food, which may help explain the strong association of type F chromosomal cpe strains with FP.

Analysis of genetic signatures of virulence and resistance in foodborne Staphylococcus aureus isolates from Algeria

Staphylococcus aureus, a frequent commensal of the human microbiota, also frequently associated with various infections. This study aimed to genetically characterize foodborne methicillin resistant S. aureus (MRSA) in Oran, Algeria. From a total of 474 food matrices, 30 MRSA strains were isolated and characterized using whole genome sequencing (WGS) and bioinformatics. The genomes were assessed for antimicrobial resistance and virulence genes, sequence and spa types and single nucleotide polymorphism (SNP) to establish their relationship. The prevalence of S. aureus was 34.38% with MRSA accounting for 18.40%. MRSA strains showed high resistance to penicillinG, ampicillin and tetracycline. However, inconsistencies were noted between phenotypic and genotypic resistance for methicillin, aminoglycosides, and phenicolates, with mecA gene identified in three isolates. The isolates revealed eight different sequence types (predominantly ST97 and ST45) and eleven spa types (mainly t230 and t8503), correlating with SNP clusters except for isolates belonging to ST7200.86.66% of isolates harboured at least one enterotoxin gene underscores their potential public health threat. This investigation highlights the genetic heterogeneity among S. aureus regarding virulence and antimicrobial resistance, shedding light into food safety in Algeria and the ease of using WGS approach to rapidly characterize bacterial pathogens in community environments.