Detection of Enterotoxin Genes in Bacillus species Isolated from Cassava Processing Environment in Nigeria.

Introduction: The health challenges associated with cassava products as a common staple food for approximately 70% of Africans and part of Asia pose a looming danger due to Bacillus enterotoxins’ presence in the processing environment. Objective: This study investigated the presence of enterotoxigenic genes namely, Bacillus cereus enterotoxin T (bceT), hemolysin bl (hblC, hblD) and non-haemolytic enterotoxin (nheA, nheB and nheC) from Bacillus species isolated from soil of cassava processing environment. Methods: Soil samples from 20 cassava processing sites in Ile-Ife and Modakeke, Nigeria were collected and cultured on Nutrient agar at 37 ºC for 24 hours. Colonies phenotypically identified as Bacillus were identified using Bacillus-specific 16S rRNA-targeted PCR technique. Screened Bacillus spp were assessed for the presence of enterotoxigenic genes using PCR with previously reported primers. Results: A total of 100 Bacillus isolates were selected from this study with Bacillus macerans (33 %) showing the highest frequency of occurrence among the identified species, however, 74 isolates were molecularly confirmed as Bacillus. Amongst the 74 molecularly confirmed Bacillus isolates, 28 (37.84%), 35 (47.30 %) and 37 (50 %) has nhe, hbl and bceT genes respectively. Investigation showed that 42 (56.76 %) of the Bacillus species encoded at least one of the screened enterotoxin genes. Conclusion: The presence of these 3 sets of enterotoxin genes in Bacillus isolated from cassava processing sites calls for immediate attention as they could be pivotal in the release of toxins in cassava products, cause lethal effects via consumption. This study demonstrates the possibility of foodborne disease outbreaks in Bacillus toxin-laden cassava products processed under unhygienic conditions.

Characterization of Virulence Factors in Enterotoxin-Producing Staphylococcus Aureus From Bulk Tank Milk

Background: Staphylococcus aureus, a persistent and chronic mastitis-causing pathogen, produces various virulence factors, including enterotoxins. This study analyzed the genetic characteristics of bovine mastitis-related virulence factors to evaluate potential pathogenesis in S. aureus isolated from bulk tank milk.Results: Among 93 S. aureus isolates from 396 dairy farms in six factories operated by three dairy companies in Korea, 40 (43.0%) isolates carried at least one or more enterotoxin genes and there were significant differences between factories within the same company (p < 0.05). Moreover, S. aureus carrying enterotoxin genes showed a higher prevalence in all virulence genes tested in this study except for pvl and lukM, which were not detected in any isolate, than the isolates without enterotoxin genes. In particular, the prevalence of six genes (hla, hlb, lukED, fnbA, clfA, and clfB) was significantly higher in S. aureus carrying enterotoxin genes than isolates without enterotoxin genes (p < 0.05). The most common multilocus sequence type (ST) of 40 enterotoxin-producing isolates was ST188, and all isolates of ST188 harbored the see gene. However, none of the isolates of ST1 and ST72 carried the see gene, and all isolates of ST1 carried the seh gene.Conclusions: Although S. aureus isolated from bulk tank milk, not from mastitis, had a high prevalence of enterotoxins and virulence factors simultaneously, posing a public health threat. Moreover, high enterotoxins in bulk tank milk may be reflected by poor hygiene; therefore, it is important to develop strong monitoring and sanitation programs to ensure that dairy factories produce hygienic milk.

Prevalence of enterotoxin genes (SEA to SEE) and antibacterial resistant pattern of Staphylococcus aureus isolated from clinical specimens in Assiut city of Egypt

Background Infections in communities and hospitals are mostly caused by Staphylococcus aureus strains. This study aimed to determine the prevalence of five genes (SEA, SEB, SEC, SED and SEE) encoding staphylococcal enterotoxins in S. aureus isolates from various clinical specimens, as well as to assess the relationship of these isolates with antibiotic susceptibility. Traditional PCR was used to detect enterotoxin genes, and the ability of isolates expressing these genes was determined using Q.RT-PCR. Results Overall; 61.3% (n = 46) of the samples were positive for S. aureus out of 75 clinical specimens, including urine, abscess, wounds, and nasal swabs. The prevalence of antibiotic resistance showed S. aureus isolates were resistant to Nalidixic acid, Ampicillin and Amoxicillin (100%), Cefuroxime (94%), Ceftriaxone (89%), Ciprofloxacin (87%), Erythromycin and Ceftaxime (85%), Cephalexin and Clarithromycin (83%), Cefaclor (81%), Gentamicin (74%), Ofloxacin (72%), Chloramphenicol(59%), Amoxicillin/Clavulanic acid (54%), while all isolates sensitive to Imipinem (100%). By employing specific PCR, about 39.1% of isolates were harbored enterotoxin genes, enterotoxin A was the most predominant toxin in 32.6% of isolates, enterotoxin B with 4.3% of isolates and enterotoxin A and B were detected jointly in 2.1% of isolates, while enterotoxin C, D and E weren’t detected in any isolate. Conclusion This study revealed a high prevalence of S. aureus among clinical specimens. The isolates were also multidrug resistant to several tested antibiotics. Enterotoxin A was the most prevalent gene among isolates. The presence of antibiotic resistance and enterotoxin genes may facilitate the spread of S. aureus strains and pose a potential threat to public health.

Ready-to-eat foods: A potential vehicle for spread of coagulase-positive staphylococci and antibiotic-resistant Staphylococcus aureus in Buea municipality, South West Cameroon

Background The consumption of ready-to-eat (RTE) foods contaminated with coagulase-positive staphylococci (CoPS) and especially Staphylococcus aureus puts consumers at potential risk of foodborne disease or colonization and subsequent infection. This cross-sectional study determined the levels of CoPS and the presence of S. aureus in RTE foods sold in Buea municipality. Method A total of 420 RTE food samples comprising 70 each of cake, bread, fruit salad, meat-hot-pot, suya and boiled rice were randomly purchased from February to August 2020. The CoPS counts were determined by culturing on Baird-Parker agar and S. aureus identified by amplification of the nuc gene using polymerase chain reaction. All S. aureus isolates were screened for the presence of classical staphylococcal enterotoxin genes and each isolate challenged with 11 antibiotics to determine their antibiotic susceptibility profiles. Oxacillin-resistant S. aureus were analyzed for the presence of mecA gene. Result Overall, 161 (38.3%) samples had detectable levels of coagulase-positive staphylococci ranging from 2.0-5.81 log10CFU/g. Based on CoPS levels, 37 (8.81%) of the 420 RTE food samples, only fruit salad and meat-hot-pot, had unsatisfactory microbiological quality. A total of 72 S. aureus isolates, comprising 52.78% from fruit salad, 16.67% from meat-hot-pot, 12.5% from boiled rice, 9.72% from suya, 5.56% from bread and 4.17% from cake were recovered. None of the S. aureus isolates possessed any of the classical enterotoxin genes. All the isolates were sensitive to vancomycin and ofloxacin while 68 (94.44%) and 66 (91.67%) were sensitive to oxacillin and ciprofloxacin, respectively. Resistance to penicillin (93.06%) was highest followed by amoxicillin (91.67%) and erythromycin (79.17%). Four isolates were identified as methicillin-resistant S. aureus all of which carried the mecA gene. A total of 24 antibiotypes were identified. Conclusion Our findings showed that RTE foods sold in the Buea municipality are likely vehicles for transmission of CoPS and antibiotic-resistant S. aureus.

Effect of Temperature on the Expression of Classical Enterotoxin Genes among Staphylococci Associated with Bovine Mastitis

Staphylococcal food poisoning (SFP), caused by the contamination of staphylococcal enterotoxins, is a common foodborne disease worldwide. The aims of this study were: (1) to investigate classical staphylococcal enterotoxin genes, sea, seb, sec, sed, and see, among Staphylococcus aureus and coagulase-negative staphylococci (CNS) associated with bovine mastitis; (2) to determine the effect of temperature on the expression of classical staphylococcal enterotoxin genes in staphylococci in milk. The detection of classical staphylococcal enterotoxin genes was performed using S. aureus (n = 51) and CNS (n = 47). The expression of classical enterotoxin genes, including sea, seb, sec, and see, was determined during the growth of staphylococci in milk subjected to ultra-high-temperature processing at two different temperatures: 8 °C and room temperature. Classical staphylococcal enterotoxin genes were expressed more frequently in S. aureus (35.30%) than in CNS (12.77%). The sec gene was most frequently detected in S. aureus (29.41%) and CNS (6.38%). Moreover, the expression of sea and sec was significantly higher at room temperature than at 8 °C after 16 h of incubation (p < 0.05). These results emphasize the importance of maintaining the storage temperature of milk below 8 °C to reduce the risk of SFP.

Characterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC

While many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.

Prevalence and distribution of multilocus sequence types of Staphylococcus aureus isolated from bulk tank milk and cows with mastitis in Pennsylvania

A total of 163 S. aureus isolates; 113 from mastitic milk (MM) and 50 from bulk tank milk (BTM) (2008, 2013–2015) submitted for bacteriologic analysis at the Penn State Animal Diagnostic Laboratory were examined for their phenotypic and genotypic characteristics. Multi-locus sequence typing (MLST) analysis identified 16 unique sequence types (STs) which belonged to eight clonal complexes (CCs). Majority of the isolates were variants of CC97 (68.7%) and CC151 (25.1%). CC97 comprised of seven STs, of which two were new STs (ST3273, ST3274), while CC151 comprised of three STs of which ST3272 was identified for the first time. Several farms had more than one ST type that were either members of the same clonal complex or unrelated STs. On one farm, six different STs of both categories were seen over the years within the farm. It was observed that ST352 and ST151 were the two main clonal populations in cattle not only in Pennsylvania but also globally. Most isolates were susceptible to all the antibiotics evaluated. 6.7% of isolates showed resistance to vancomycin and penicillin. Two isolates of ST398 showed multidrug resistance (>3 antibiotics) against clindamycin, erythromycin, tetracycline, and penicillin. It was noted that 59 of 163 (36.2%) isolates encoded for enterotoxigenic genes. Enterotoxin genes seg/sei accounted for ~85% of enterotoxin positive isolates. Toxic shock syndrome gene tsst-1 alone was positive in two isolates (ST352, ST 2187). 97.5% of CC151 isolates were enterotoxin seg/sei positive. Most isolates were positive for lukED (95%) and lukAB (96.3%) leukotoxin genes. Bovine specific bi-component leucocidin lukMF’ was present in 54% of isolates. A prominent observation of this study was the explicit association of lukMF’ with lineages ST151 and ST352. In conclusion, the findings of the study, suggest that small number of S. aureus STs types (ST352, ST2187, ST3028, and ST151) are associated with majority of cases of bovine mastitis in Pennsylvania dairy farms. It was observed that one ST of S. aureus predominated in the herd and this ST can coexist with several other ST types of S. aureus strains. When STs were interpreted along with virulence, leucocidin genes and antimicrobial resistance, ST-variants allowed better interpretation of the S. aureus molecular epidemiologic findings specifically for tracing recurrence or persistence of infections in cow over time, among cows in the herd, and between herds in Pennsylvania.