Characterisation of Staphylococcus aureus strains from milk and goat cheese and evaluation of their inhibition by gallic acid,nisin and velame of the Brazilian caatinga

Twenty isolates from milk and goat cheese were confirmed as Staphylococcus aureus. These isolates were characterised for phenotypic properties related to cell adhesion and for the presence of enterotoxin production, intercellular adhesion and β‐lactam resistance genes. Staphylococcus aureus L47 showed cell adhesion ability and positivity for the sec, sed, icaD, mecA and blaZ genes. Three antimicrobial compounds were tested singly or in pairs for growth control of strain L47: gallic acid (GA), nisin and essential oil (EO) of Croton heliotropiifolius (velame). At 24 h, EO and EO + nisin showed higher inhibitory activity against S. aureus L47 in goat milk.     

建立金黄色葡萄球菌肠毒素B悬浮芯片定量检测方法

目的建立金黄色葡萄球菌肠毒素B悬浮芯片定量检测方法。方法利用双抗体夹心法免疫学原理,以金黄色葡萄球菌肠毒素B的特异性抗体包覆微球为载体,利用悬浮芯片(Bio-Plex)系统建立检测模型;检测不同浓度金黄色葡萄球菌肠毒素B,测定方法的灵敏性;通过该方法对大肠杆菌、普通变型杆菌、蓖麻毒素和禽流感病毒HA、NH蛋白和金黄色葡萄球菌热休克毒素的检测,判断方法的特异性;将不同浓度金黄色葡萄球菌肠毒素B添加到奶粉中验证方法的实用性和稳定性。结果悬浮芯片方法对金黄色葡萄球菌肠毒素B的检测线性范围为0.2～1653.4ng/ml,最低检测值为203pg/ml,均优于酶联免疫吸附实验;除与2.3μg/ml金黄色葡萄球菌热休克毒素有交叉反应外,和其他几种细菌、毒素、蛋白均无交叉反应;对添加相同浓度金黄色葡萄球菌肠毒素B奶粉的检测的相对标准偏差在1.30%～16.93%。结论悬浮芯片定量检测方法对于模拟添加的金黄色葡萄球菌肠毒素B具有良好的检测效果。     

Prevalence of staphylococcal enterotoxins, toxin genes and genetic-relatedness of foodborne Staphylococcus aureus strains isolated in the Marmara Region of Turkey

Staphylococcus aureus is a major foodborne pathogen and it has the ability to produce a number of extracellular toxins. We analyzed 1070 food samples obtained from retail markets and dairy farms in the Marmara Region of Turkey for the presence of S. aureus. Out of 147 isolates, 92 (62.6%) were enterotoxigenic. PCR was used to investigate the presence of staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu), exfoliative toxin genes (eta and etb) and the toxic − shock syndrome toxin gene (tst). The PCR results showed that 53.3% of the isolates contained staphylococcal enterotoxin-like (SEl) toxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu) which were more frequent than classical enterotoxin genes (sea to see). Furthermore, seo, sei, sem, seg, seu and sec were found in 37.0, 32.7, 30.4, 29.3, 29.3 and 27.2% of the isolates, respectively. The tst gene was detected and confirmed by DNA sequencing in 9 isolates. The presence of eta and etb were not found in the isolates. Enterotoxigenic capabilities of isolates with SEA-SEE were investigated by ELISA. Enterotoxigenic S. aureus isolates produced one to three enterotoxins, with the most frequently produced types being enterotoxin A and C. There was a correlation of 72.1% between production of a specific toxin and the presence of the respective genes. PFGE analysis was used to identify genetic-relatedness of enterotoxigenic S. aureus isolates and the results revealed that 13 groups of isolates from different or the same origin that contained the same genes showed 100% homology with indistinguishable band patterns. The other enterotoxigenic isolates showed related band patterns with 72-86% homology in sea-, 61-90% homology in sec-, 80-96% homology in seh-, and 69-96% homology in sep-positive isolates. To our knowledge, this is the first study to examine enterotoxins and related gene contents of S. aureus food isolates in the Marmara Region of Turkey.     

Formation of cereulide and enterotoxins by Bacillus cereus in fermented African locust beans

Afitin, iru and sonru are three spontaneously fermented African locust bean Benin condiments. The fermentation processes are exothermic, with temperatures mostly being above 40 °C. A total of 19 predominant Bacillus cereus isolates from afitin, iru and sonru, were investigated. The enterotoxin genes nhe (A, B, C) were present in all 19 isolates, the hbl (A, C, D) in one (afitin), and the cytK gene in three isolates (afitin). Levels of cytotoxicity to Vero cells and NheA production in BHI-broth was within the range of known diarrheal outbreak strains. Autoclaved cooked African locust beans inoculated with emetic (cereulide producing) B. cereus Ba18H2/RIF supported growth at 25, 30 and 40 °C with highly different maximum cereulide productions of 6 ± 5, 97 ± 3 and 0.04 ± 0.02 μg/g beans, respectively (48 h). For non-autoclaved cooked beans inoculated with 2, 4 and 6 log₁₀B. cereus Ba18H2/RIF spores/g beans, cereulide production was 5 ± 4, 64 ± 8 and 69 ± 34 μg/g beans, respectively at 24 h, while it was 70 ± 43, 92 ± 53 and 99 ± 31 μg/g at 48 h of fermentation at 30 °C. Even though high toxin levels were observed, to date there are no known reports on diarrhea or vomiting due to the consumption or afitin, iru and sonru in Benin, which also according to the present study is likely to be expected from the low levels of cereulide produced at 40 °C.     

Characterization for enterotoxin production,virulence factors,and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal

Staphylococcus aureus represents a public health challenge worldwide. The aim of this study was the characterization of different food isolates of S. aureus on the basis of their production of enterotoxins, hemolysins and resistance to antibiotics. A total of 148 coagulase-positive staphylococcal strains isolated from different food origins were identified to the species level. By multiplex PCR, 69% of the isolates were shown to be enterotoxigenic (SEs); the most common were sea seg, sea seg sei and seg sei. According to CLSI [CLSI, Clinical and Laboratory Standards Institute, 2007. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. CLSI document M100-S15. Clinical and Laboratory Standards Institute, Wayne, PA], 38% of the isolates were resistant to oxacillin (≥6 μg/mL; MRSA positives) but only 0.68% showed the presence of mecA gene. 70 and 73% of the S. aureus strains were resistant to β-lactams, ampicillin and penicillin, respectively. The virulence pattern was demonstrated to be origin and strain dependent. These findings emphasise the need to prevent the presence of S. aureus strains and SEs production in foods.     

食品中金黄色葡萄球菌及肠毒素的检测方法

食品中金黄色葡萄球菌及肠毒素的常规检测方法包括传统的微生物分离培养及生化鉴别,肠毒素的免疫学检测等。本文简要介绍了各种方法及其原理和优缺点。     

The influence of manufacturing and drying conditions on the survival and toxinogenesis of Staphylococcus aureus in two Spanish dry sausages (chorizo and salchichón)

The effects of formulation, starter culture and fermentation temperature on growth and synthesis of toxin A (SEA) and TNase by Staphylococcus aureus during fermentation and drying of Spanish chorizo were investigated. Inhibitory factors able to inhibit SEA synthesis in culture media were unable to prevent SEA production in chorizo fermented at 20 and 30°C, though a lower temperature and starter culture SP318 (Lactobacillus sake, Pediococcus pentosaceous and Staphylococcus xylosus) decreased staphylococcal growth and SEA formation. Reduction and even disappearance of the SEA during ripening was observed. In most batches, TNase was a reliable indicator of staphylococcal growth and SEA production. Dextrose added to the salchichón formulation repressed S. aureus growth during drying. Lactobacillus curvatus in combination with dextrose was an effective anti-staphylococcal agent during fermentation.     

Microbiological quality assessment of garri with reference to thermonuclease activity

The microbiological quality of garri obtained from open markets and traditional processing industry in Benin City with reference to staphylococcal contamination was investigated. Enumeration of the total heterotrophic bacteria, total fungal propagules and Staphylococcus aureus in food samples was carried out using appropriate media. The staphylococcal thermonuclease activity determination was used as an indicator of previous (substantial) growth of S. aureus and possible presence of staphylococcal enterotoxins. The total heterotrophic bacterial count of the open markets’ samples was 7.75±2.87×10³ cfu/g with corresponding staphylococcal count of 1.65±0.82×10³ and fungal propagule count of 2.50±1.14×10² cfu/g. Samples obtained immediately after processing from the traditional garri industry revealed total heterotrophic bacterial count of 5.00×10¹ cfu/g; staphylococcal count of 5.00×10⁰ cfu/g and no fungal count. The zone diameter of thermonuclease activity was 8.13±0.52 mm for open markets’ samples while traditional processing industry samples have 7.83±0.54 mm. From the study, it was found that there was contamination and growth of S. aureus and possibly the presence of staphylococcal enterotoxin in the product. The public health significance of the microorganisms with the production of thermonuclease and enterotoxins by S. aureus are discussed.