Enterotoxigenic methicillin resistant Staphylococcus aureus contamination in salted fish from Gwadar Balochistan

Staphylococcus aureus is an important foodborne pathogen associated to food intoxication and other multiple infections in human being. Its presence in salted food is a serious issue due to its salt tolerance potential. A study was conducted to analyze the presence of enterotoxins producing drug resistance S. aureus in salted sea fish from Gwadar. Freshly persevered samples (n=50) of salted fish were subjected to analyze the presence of S. aureus using 16S rRNA and Nuc genes primers. The isolates were then evaluated for drug resistance and enterotoxins producing potential using specific primers for MecA (methicillin resistance gene), (SEA) staphylococcal enterotoxin A and (SEB) staphylococcal enterotoxin B genes. Total 13/50 (26%) of the samples were found positive for the presence of S. aureus, preliminary confirmed with biochemical profiling and finally with the help of target genes presence. The isolates were found showing 100% resistant to methicillin, which were molecularly confirmed by the presence of MecA gene present in genome. The isolates 5/13 (38%) were positive for SEA and 3/13 (23%) for SEB genes, whereas 2/13 (15%) were confirmed having both SEA and SEB genes in its genome. It was also confirmed that all the isolates were capable to form biofilm over the glass surfaces. It was concluded that the study confirmed the presence of enterotoxigenic methicillin resistance Staphylococcus aurous (MRSA) in salted fish product, that poses gross food safety concern. Preventive and control measures are necessary to handle this serious food safety concern.

Staphylococcal Enterotoxin A Induces Intestinal Barrier Dysfunction and Activates NLRP3 Inflammasome via NF-κB/MAPK Signaling Pathways in Mice

Staphylococcal enterotoxin A (SEA), the toxin protein secreted by Staphylococcus aureus, can cause staphylococcal food poisoning outbreaks and seriously threaten global public health. However, little is known about the pathogenesis of SEA in staphylococcal foodborne diseases. In this study, the effect of SEA on intestinal barrier injury and NLRP3 inflammasome activation was investigated by exposing BALB/c mice to SEA with increasing doses and a potential toxic mechanism was elucidated. Our findings suggested that SEA exposure provoked villi injury and suppressed the expression of ZO-1 and occludin proteins, thereby inducing intestinal barrier dysfunction and small intestinal injury in mice. Concurrently, SEA significantly up-regulated the expression of NLRP3 inflammasome-associated proteins and triggered the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways in jejunum tissues. Notably, selective inhibitors of MAPKs and NF-κB p65 ameliorated the activation of NLRP3 inflammasome stimulated by SEA, which further indicated that SEA could activate NLRP3 inflammasome through NF-κB/MAPK pathways. In summary, SEA was first confirmed to induce intestinal barrier dysfunction and activate NLRP3 inflammasome via NF-κB/MAPK signaling pathways. These findings will contribute to a more comprehensive understanding of the pathogenesis of SEA and related drug-screening for the treatment and prevention of bacteriotoxin-caused foodborne diseases via targeting specific pathways.

A comparative characterization of nasal and clinical isolates of Staphylococcus aureus from west of Iran

Background and Objectives: Recently, the rise of methicillin-resistant Staphylococcus aureus (MRSA) isolated from hos- pital healthcare workers (HCWs) and various infectious samples has become one of the main concerns in hospital settings. Therefore, epidemiological studies are necessary to monitor antibiotic resistance patterns in each region and to study the pathogenesis of this strain to control infections. Materials and Methods: In this cross-sectional study, a total of 100 S. aureus isolates, including 50 isolates obtained from the anterior nares of healthcare workers, as well as 50 other isolates cultured from the various clinical specimens from the referral hospitals in Khorramabad (West of Iran) were tested. All isolates were examined to determine antibiotic resistance pattern, and the presence of staphylococcal enterotoxin A (sea), staphylococcal enterotoxin B (seb) and mecA genes. Results: The mecA gene was found among 36% (18/50) of the clinical S. aureus isolates (CSIs) and 14% (7/50) of nasal S. aureus isolates (NSIs), with statistically significant difference (X2 = 6.53; p = 0.011). The difference between the frequency rate of sea gene among MRSA strains isolated from clinical specimens (46.6%, 7/15) was significant compared to strains isolated from nostrils (14.3%, 1/7) (X2 = 3.85; p = 0.049). Conclusion: The frequency of mecA, sea, and seb genes among the clinical samples was more than strains isolated from the nostrils of healthcare personnel.

Computational Construction of a Single-Chain Bi-Paratopic Antibody Allosterically Inhibiting TCR-Staphylococcal Enterotoxin B Binding

Staphylococcal enterotoxin B (SEB) simultaneously crosslinks MHC class II antigen and TCR, promoting proliferation of T cells and releasing a large number of toxic cytokines. In this report, we computationally examined the possibility of using a single-chain biparatopic bispecific antibody to target SEB and prevent TCR binding. The design was inspired by the observation that mixing two anti-SEB antibodies 14G8 and 6D3 can block SEB-TCR activation, and we used 14G8-6D3-SEB tertiary crystal structure as a template. Twelve simulation systems were constructed to systematically examine the effects of the designed bispecific scFV MB102a, including isolated SEB, MB102a with different linkers, MB102a-SEB complex, MB102a-SEB-TCRβ complex, MB102a-SEB-TCR-MHC II complex, and MB102a-SEB-MHC II. Our all atom molecular dynamics simulations (total 18,900 ns) confirmed that the designed single-chain bispecific antibody may allosterically prevent SEB-TCRβ chain binding and inhibit SEB-TCR-MHC II formation. Subsequent analysis indicated that the binding of scFV to SEB correlates with SEB-TCR binding site motion and weakens SEB-TCR interactions.

Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products

Staphylococcal enterotoxins (SEs) represent the leading reason for staphylococcal food poisoning (SFP) and various other diseases. Reports often indicate Staphylococcal enterotoxin C (SEC) as the most frequently found enterotoxin in dairy products. To minimize consumer exposure to SEC, this paper aimed to create a sandwich enzyme-linked immunosorbent assay (ELISA) based on nanobodies (sandwich Nbs-ELISA) to accurately detect SEC in dairy products without the influence of staphylococcal protein A (SpA). Therefore, after inoculating a Bactrian camel with SEC, a phage display Nb library was created. Eleven Nbs against SEC were identified in three biopanning steps. Based on their affinity and pairing level, a sandwich Nbs-ELISA was developed using the C6 anti-SEC Nb as the capture antibody, while the detection antibody was represented by the C11 phage display anti-SEC Nb. In optimal conditions, the quantitative range of the present sandwich ELISA was 4-250 ng/mL with a detection limit (LOD) of 2.47 ng/mL, obtained according to the blank value plus three standard deviations. The developed technique was subjected to specific measurements, revealing minimal cross-reactivity with Staphylococcus aureus (S. aureus), Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), and SpA. The proposed method exhibited high specificity and an excellent recovery rate of 84.52~108.06% in dairy products. Therefore, the sandwich Nbs-ELISA showed significant potential for developing a specific, sensitive technique for SEC detection in dairy products.

Occurrence, enterotoxin production and antimicrobic susceptibility of Staphylococcus aureus isolates from domestic cheeses in the Dubrovnik area

Staphylococcus aureus is a well-known op¬portunistic pathogen that causes a wide range of acute and chronic diseases in both humans and animals. Staphylococcal food poisoning is just one of many diseases caused by this bacterium. The causative agents are entero¬toxins produced by enterotoxigenic strains of S. aureus during its growth in favourable conditions in food. Epidemiological data show that S. aureus is often found in raw milk cheeses and accordingly, cheeses are often implicated in food poisoning outbreaks. Since there are no data on the nature of S. aureus isolated from cheese produced in Croatia, the aim of this study was to determine the occur¬rence, enterotoxin production capability and antimicrobic susceptibility of S. aureus iso¬lates from domestic cheeses produced in the Dubrovnik area. A total of 30 cheese samples were analysed, and 18 samples (60%) were highly contaminated with S. aureus strains. The contamination level ranged from 3.94 to 6.26 log10 cfu/g. Such a high level of contami¬nation was supported by an inappropriate temperature regime above 8°C during market sale. Although highly contaminated, staphy¬lococcal enterotoxins were not detected in any of the cheese samples. A total of 180 coagu¬lase-positive isolates were collected from 18 cheese samples, 175 of which were confirmed as S. aureus by the latex agglutination test. En¬terotoxin production was detected in 35 iso¬lates (20%), and of these 32 isolates produced staphylococcal enterotoxin C. The other three isolates presumably produced enterotoxin E. Antibiotic resistance was detected in 1.1% of isolates and only to mupirocin. However, a full comprehensive conclusion on the nature of S. aureus isolates cannot be achieved with¬out determining its genotype characteristics for the presence of staphylococcal enterotoxin genes and molecular typing, to determine the origin of the isolates.