Occurrence of toxigenic Staphylococcus aureus in ready-to-eat food in Korea

Toxigenic Staphylococcus aureus contamination in ready-to-eat (RTE) food is a leading cause of foodborne illness in Korea. To monitor food contamination by S. aureus, a total of 3332 RTE food samples were selected from nationwide wholesale marts between 2003 and 2004 and examined. A total of 285 (8.6%) of the overall samples were contaminated by S. aureus. According to the analysis, 31.6% of the tested cream-cakes, 19.8% of the raw fish, and 19.3% of the rice cakes with filling were contaminated with S. aureus. Forty-seven percent of the strains isolated from the contaminated food were enterotoxigenic S. aureus. The phenotypic result of the strain isolated from food showed that 48% of the strains produced one or more toxins, such as staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC). At least one SEA was produced by over 90% of the toxigenic strains. Other toxins, such as SEB, SEC, SED, SEA+SEC, and SEC+SED, were each detected. Toxic shock syndrome toxin 1 (TSST-1), a causative agent of toxic shock syndrome, was detected in 13 strains of the toxigenic isolates from the food. As the result of genotyping, 22 strains with a toxin gene that was not detected in the phenotypic analysis were also detected. Sixty-nine percent of the toxigenic strains had at least one sea gene, and the most prevalent genotype was sea+seh (34.4%), followed by sea (18.8%) and sea+seg+sei (15.6%). The tst gene encoding TSST-1 was found in 13 strains (13.5%). The genes (eta and etb) encoding exfoliative toxins A and B were not detected in any of the samples.     

噬菌体编码的金黄色葡萄球菌肠毒素A的 研究进展

金黄色葡萄球菌(Staphylococcus aureus)是一种人畜共患的食源性致病菌,并且能够产生不同种类的毒素,其中金黄色葡萄球菌肠毒素(staphylococcal enterotoxin,SE)是引起食物中毒的一类主要毒素。金黄色葡萄球菌肠毒素A(staphylococcal enterotoxin A,SEA)在食物中毒事件中的检出率最高,毒性最强,成为目前研究的热点。编码金黄色葡萄球菌肠毒素A的基因sea是由噬菌体携带并在菌体之间传播的。本研究主要综述了编码SEA的噬菌体与SEA之间的关系、SEA的分类、在不同的食品基质中环境因子对SEA产生量、sea基因相对表达量的影响及SEA新型的检测方法等,并介绍了目前金黄色葡萄球菌肠毒素研究的难点及研究方向的分析。     

THE NATURE OF TOXINS PRODUCED BY BACILLUS CEREUS BIS-59

Eight strains of Bacillus spp. were examined for production of toxin(s). Three strains of Bacillus cereus produced toxins lethal to mice. The culture filtrate of Bacillus cereus BIS-59 contained two lethal toxins. One was a hemolysin and the other a nonhemolytic glycoprotein. The growth of cells in fresh beef infusion broth resulted in high toxin production, compared to growth in tryptone-glucose-yeast extract or nutrient broth. Separate identities for the two toxins from B. vcereus BIS-59 were established on the basis of hemolytic activity, separation profiles on Sephadex gel filtration, responses to heat and radiation and kinetics of their production during the growth of the organism.     

Characterization of Staphylococcus aureus isolates from white-brined Urfa cheese

The aim of this study was to investigate the presence of Staphylococcus aureus and staphylococcal enterotoxin (SE) genes in Urfa cheese samples and to characterize the enterotoxigenic potential of these isolates. From a total of 127 Urfa cheese samples, 53 isolates (from 41.7% of the samples) were identified by a species-specific PCR assay as S. aureus. Of these isolates, 40 (75.5%) gave positive PCR results for the 3' end of the coa gene. The coa-positive S. aureus strains were characterized for their population levels and enterotoxigenic properties, including slime factor, β-lactamase, antibiotic susceptibilities, production of the classical SEs (SEA through SEE), in both cheese and liquid cultures by enzyme-linked immunosorbent assay (ELISA) and for the presence of specific genes, including classical SE genes (sea through see), mecA, femA, and spa, by PCR. The genetic relatedness among the coa-positive S. aureus isolates was investigated by PCR-based restriction fragment length polymorphism (RFLP) analysis and the 23S rRNA gene spacer. The 23S rRNA gene spacer and coa RFLP analysis using AluI and Hin6I revealed 14 different patterns. SEB, SEC, and SEA and SEE were detected by ELISA in three cheese samples. Fourteen S. aureus strains harbored enterotoxin genes sea through see, and three strains carried multiple toxin genes. The most commonly detected toxin gene was sec (25% of tested strains). Of the 40 analyzed S. aureus strains, 3 (7.5%) were mecA positive. Based on tandem repeats, four coa and spa types were identified. The results of this study indicate that S. aureus and SEs are present at significant levels in Urfa cheese. These toxins can cause staphylococcal food poisoning, creating a serious hazard for public health.     

DNA microarray based detection of genes involved in safety and technologically relevant properties of food associated coagulase-negative staphylococci

Aim of the work was to design a polynucleotide based DNA microarray as screening tool to detect genes in food associated coagulase-negative staphylococci (CNS). A focus was laid on genes with potential health concern and technological relevance. The microarray contained 220 probes for genes encoding antibiotic resistances, hemolysins, toxins, amino acid decarboxylases (e.g. biogenic amine formation), binding proteins to extracellular matrix (ECM), lipases, proteases, stress response factors, or nitrate dissimilation. Hybridization of genomic DNA isolated from 32 phenotypically characterized CNS permitted to detect numerous genes, corresponding with the phenotype. However, numerous hybridization signals were obtained for genes without any detectable phenotype. The antibiotic resistance genes blaZ, lnuA, and tetK involved in ?-lactam, lincomycin and tetracycline resistance, respectively, were rarely identified in CNS, however, all species contained some strains with such resistance genes. Decarboxylase genes involved in biogenic amine formation were detected regularly in Staphylococcus carnosus, S. condimenti, S. piscifermentans and S. equorum, but was rarely correlated with the phenotype. The same applied for the fibrinogen (clf) and fibronectin (fbp) binding protein genes, whose phenotype (binding assay) was only correlated in S. equorum and Staphylococcus succinus. Although some CNS showed hemolytic activity and enterotoxin production (Immunoblot) the corresponding genes could not be verified. Technological relevant genes such as proteases or lipases revealed good hybridization signals. In addition, genes involved in nitrate dissimilation (nre, nar, nir), catalase (kat), or superoxide dismutase (sod) were well detected. Interestingly, genes involved in dissimilatory nitrate reduction were more prevalent in strains of S. carnosus, S. condimenti and S. piscifermentans than of S. equorum, S. succinus and S. xylosus.     

Anti-Pathogenic and Technological Traits of Coagulase-Negative Staphylococci Isolated from Koozh,a Fermented Food Product of South India

The aim of this study was to assess the biopotency of fermented food product Koozh associated coagulase-negative staphylococci (CNS) against Mycobacterium tuberculosis H37Rv and non-mycobacterial pathogens, and to document some technologically relevant attributes. The anti-tubercular property of six CNS strains was exhibited using luciferase reporter phage assay. All strains revealed pronounced mycobactericidal property in terms of higher percentage of relative light unit reduction (> 90%). The antagonistic role of CNS strains against non-mycobacterial pathogens was evaluated using agar well diffusion assay. Staphylococcus hominis strain MANF2 was found to be the most effective against Staphylococcus aureus MTCC 96 and Yersinia enterocolitica MTCC 840 with arbitrary units of 1600.31 ± 22.3 and 1500.23 ± 22.3 AU/mL, respectively. Further, various technological attributes of staphylococci were determined using standard methodology. All strains exhibited promising carbohydrate fermentation ability at standard in vitro conditions. CNS strains were positive for exopolysaccharide production and expressed strain specific lipase activity. All the isolates lacked amylase activity and biofilm formation ability. All six CNS strains, particularly strain MANF2 exhibited significant rate of autolytic and catalase activity, and showed the potential to reduce nitrate to nitrite. Most importantly, these strains were categorized for safe utilization based on in vitro safety hazards test. Overall, the present investigation indicated the persuasive biopotency of CNS against M. tuberculosis H37Rv and non-mycobacterial pathogens, and revealed adaptable technological attributes that would make them relevant for formulation of staphylococcal starter cultures for the development of quality fermented food products.     

Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food

Extensive analysis of the Staphylococcus aureus genome has allowed the identification of new genes encoding enterotoxin-like superantigens (SEls). Some of these are thought to be involved in staphylococcal food poisoning, while others do not elicit any emetic effect. The potential impact of these members of the enterotoxin-like family on the human organism seems to rely mainly on their superantigenic activity. In this paper the distribution of the genes coding for enterotoxin-like superantigens in S. aureus isolated from food was studied. Fifty isolates of S. aureus were examined and 27 were shown to be enterotoxigenic. Only 9 of the 27 strains carried genes encoding enterotoxins SEA-SEE. In 18 SEA-SEE-negative strains the presence of newly described enterotoxin genes was detected. All SEA-SEE-positive strains simultaneously carried genes of new SEls. We show that the gene encoding SElH (staphylococcal enterotoxin-like enterotoxin H) was the most frequently detected (n=14), while genes encoding SElI together with SElG accompanied by the other genes of the egc locus were detected in three strains. We also detected the presence of three less investigated genes: sep, sel, and sek. These genes were present in eight, two, and one isolate, respectively. In one strain, sep was accompanied by genes of other SEls, while in the remaining seven it was the only enterotoxin-like gene detected. The high prevalence of newly discovered enterotoxin genes, including the genes encoding emetic toxins, was demonstrated in food-derived strains. This supports the need for additional work on its role in food poisoning and, alternatively, to monitor its presence in S. aureus isolated from food. Our results suggest that yet unknown genetic elements encoding enterotoxin genes may exist.     

Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production

Staphylococcus aureus is an important agent of bacterial mastitis in milking animals and of foodborne intoxication in humans. The purpose of this study was to examine the genetic and phenotypic diversity, enterotoxigenicity, and antimicrobial resistance of S. aureus strains isolated from raw milk used for the production of artisan cheese in Vermont. Cross-tabulations revealed that the 16 ribotypes identified among the 90 milk isolates examined were typically associated with a specific animal species and that more than half of these ribotypes were unique to individual farms. In general, specific EcoRI ribotypes were commonly associated with specific phenotypical characteristics, including staphylococcal enterotoxin production or the lack thereof. Limited antimicrobial resistance was observed among the isolates, with resistance to ampicillin (12.51%) or penicillin (17.04%) most common. Two isolates of the same ribotype obtained from the same farm were resistant to oxacillin with 2% NaCl. More than half (52.22%) of isolates produced toxin, and 31 of the 32 isolates solely produced staphylococcal enterotoxin type C. Although these data demonstrate that S. aureus strains found in raw milk intended for artisan cheese manufacture are capable of enterotoxin production, staphylococcal enterotoxin C is not typically linked to foodborne illness. Because S. aureus is a common contaminant of cheese, an understanding of the ecology of this pathogen and of the antimicrobial susceptibility and toxigenicity of various strains will ultimately contribute to the development of control practices needed to enhance the safety of artisan and farmstead cheese production.     

Genotypes and enterotoxicity of Staphylococcus aureus isolated from the hands and nasal cavities of flight-catering employees

Hand and nasal samples of flight-catering staff were collected from 1995 to 1997 to find employees carrying Staphylococcus aureus. Altogether 153 hand samples and 136 nose samples were taken. Nasal sampling showed a higher prevalence of S. aureus among food handlers (29%) than hand sampling (9%). A high proportion of the strains (46%) were enterotoxigenic, and a considerable amount of food handlers carried enterotoxigenic S. aureus, 6% and 12% according to hand and nasal sampling, respectively. Pulsed-field gel electrophoresis macrorestriction profiles revealed a total of 32 different types associated with the 35 employees carrying S. aureus. In most cases, the same type colonized both the hand and nose of a person. Despite the wide variety of types found, one strain colonized five persons and the second most common strain was associated with four food handlers. The predominant toxin produced was B, which was produced by the most common strain. The results showed that nasal sampling is a good way to detect S. aureus carriers, whereas hand sampling may fail to reveal carriers. The high proportion of enterotoxigenic strains show that a food handler harboring S. aureus must be considered a potential source of enterotoxigenic strains for airline meals.     

The detection of enterotoxins and toxic shock syndrome toxin genes in Staphylococcus aureus by polymerase chain reaction

A simple polymerase chain reaction (PCR)-based procedure was developed for the detection of fragments of staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, and SEI together with the toxic shock syndrome toxin (TSST-1) genes of Staphylococcus aureus. One hundred and twenty-nine cultures of S. aureus were selected, 39 of which were recovered from 38 suspected staphylococcal food-poisoning incidents. The method was reproducible, and 32 different toxin genotypes were recognized. The presence of SE genes was associated with S. aureus strains reacting with phages in group III, and the TSST-1 gene with phages in group I. There was a 96% agreement between the PCR results for detection of SEA-D and TSST-1 as compared with a commercial reverse passive latex agglutination assay for the detection of SEs from cultures grown in vitro. Enterotoxin gene fragments were detected in S. aureus cultures recovered from 32 of the 38 suspected staphylococcal food poisoning incidents, and of these, 17 were associated with SEE, SEG, SEH, and SEI in the absence of SEA-D. Simple PCR procedures were also developed for the detection of SE directly in spiked food samples, and this was most successfully achieved in mushroom soup and ham. Detection was less successful in three types of cheese and in cream. SEA or SEB were detected by enzyme-linked immunosorbent assay in three food samples (two of which were associated with food poisoning incidents) naturally heavily contaminated with S. aureus: the appropriate SEA or SEB gene fragments were detected directly in these three foods by PCR.     

Detection of toxins involved in foodborne diseases caused by Gram‐positive bacteria

Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram‐positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus, neurotoxins of Clostridium botulinum, enterotoxin of Clostridium perfringens, and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose‐response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens, and S. aureus. Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.     

Genetic diversity, antimicrobial resistance, and toxigenic profiles of Bacillus cereus strains isolated from Sunsik

Bacillus cereus can cause emetic and diarrheal types of food poisoning, but little study has been done on the toxins and toxin-encoding genes of B. cereus strains isolated from Sunsik, a Korean ready-to-eat food prepared from grains, fruits, and vegetables. In this study, 39 unique B. cereus strains were isolated and identified from Sunsik samples, with an average contamination level of 10 to 200 CFU/g. The detection rates of the hblACD, cytK, and bceT genes among all the strains were 48.7, 66.7, and 87.1%, respectively. All 39 B. cereus strains carried nheABC and entFM genes, and 36 strains also had the ces gene, which encodes an emetic toxin. Nonhemolytic enterotoxin and hemolysin BL enterotoxin were produced by 39 and 26 strains, respectively. The strains were separated into 13 profiles based on the presence or absence of toxins and their genes, as determined by antibody tests and PCR analysis. Profile 1 was the largest group, comprising 30.7% (12 of 39) of the B. cereus strains tested; these strains harbored all toxins and their genes. The B. cereus strains were susceptible to most of the antibiotics tested but were highly resistant to b -lactam antibiotics. The repetitive element sequence polymorphism PCR fingerprints of the B. cereus strains were not influenced by the presence of toxin genes or antibiotic resistance profiles. Our results suggest that B. cereus strains from Sunsik could cause either the diarrheal or emetic types of food poisoning because all strains isolated contained at least one toxin and its gene, although the level of B. cereus contamination in Sunsik was low.     

Novel multiplex PCR for the detection of the Staphylococcus aureus superantigen and its application to raw meat isolates in Korea

A multiplex PCR assay that allows for the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs) (sea to see, and seg to sei), SE-like (SEl) toxins (sej to ser, and seu), and toxic shock syndrome toxin-1 (TSST-1) (tst) was developed in this study. These toxins are included in the pyrogenic toxin superantigen (PTSAg) family and are responsible for many diseases such as staphylococcal food poisoning (SFP) and TSS. The primers were designed based on dual priming oligonucleotide (DPO) technology to detect all of the 19 SAg genes in three sets of PCR. The developed multiplex PCR was applied to 143 Staphylococcus aureus strains isolated from pork and chicken meat in Korea. Almost 50% of the strains possessed at least one of the 19 SAg genes. The most frequently found genes were seg, sei, sem, and sen (53 isolates, 37%), which were often found simultaneously in the same isolate. In those isolates, the seo (39 isolates, 27%) or seu (6 isolates, 4%) genes were frequently found together and this combination (seg, sei, sem, sen, and seo or seu) was considered to be a part of the enterotoxin gene cluster (egc). The sea gene (10 isolates, 7%) was the gene most frequently detected out of all the classical SE genes (sea to see). Although these classical SEs are considered to be major etiological factors in SFP, newly described SE or SEl genes (seg to ser, and seu) were more frequently detected than the classical SE genes in this study. There was no isolate detected containing the seb, sec, sek, sel, or seq genes. S. aureus possessing mobile genetic elements known to encode these SAg genes, such as egc, were presumed to be widely distributed among pork and chicken meats in Korea. The multiplex PCR developed in this study could be applied to the investigation of SAg genes in S. aureus strains isolated from various sources.     

A Study on the Growth Potential of Staphylococcus aureus In Boletus edulis, A Wild Edible Mushroom, Prompted by a Food Poisoning Outbreak

A dish of Boletus edulis, a wild edible mushroom, in vinegar caused staphylococcal food poisoning in 13 of 35 diners in a restaurant. Enterotoxicosis was confirmed by detection of toxins A and D in the dish. Staphylococcus aureus growth potential in B. edulis was studied by inoculating fresh and frozen and thawed bolete with S. aureus strains VTTE 530, 757, 793 and 805 and storing for 3 days at 15 and/or 21°C. Essentially no staphylococcal growth was observed in frozen and thawed mushrooms contaminated with strains 530, 793 or 805 and stored at 15 or 21°C. In fresh B. edulis the same strains showed slight growth at 21°C. Frozen and thawed bolete inoculated with strains 530 and 757 (isolated from mushroom soup, nonenterotoxigenic) supported staphylococcal growth in 2 days at 21°C from a level of 4.8±10⁴ and 5.4±10³ to 2.0±10⁶ and 7.0±10₇ cfu/g, respectively. Enterotoxin was not detected in these samples.     

DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN A FROM RICOTTA CHEESE BY CELL CULTURE

Staphylococcal food poisoning (SFP) is a very common foodborne disease. Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which are often involved in SFP. These foods may become contaminated owing to subclinical staphylococcal mastitis or during handling, storage and trade. For sanitary purposes, an effective approach is to detect the staphylococcal enterotoxins directly in foods, rather than detect, count and type the isolated S. aureus strains, because the toxin is the causative agent of foodborne illness. This paper illustrates a cell‐based bioassay that uses the PEB (bovine embryo lung cells) cell line to detect staphylococcal enterotoxin type A (SEA) directly from artificially contaminated ricotta cheese. The test was able to detect SEA in the contaminated samples after 24 and 48 h of incubation at 37C, while results were uncertain when the samples were incubated for 72 h. The test proved to be easy to use and rapidly provided results.     

Adherence, invasion, toxigenic, and chemotactic properties of Mexican campylobacter strains

To determine the virulence factors of Mexican wild-type strains of Campylobacter jejuni and Campylobacter coli, 31 wild-type strains were isolated from food and from humans. The production of cytolethal distending toxin and the adherence and invasion capabilities of these strains were assayed in Vero cells. Hard agar plugs with repellents and attractants were used to examine chemotaxis. Mueller-Hinton agar with supplements was used for motility analysis and to measure hemolytic activity. Nine strains of C. jejuni and eight strains of C. coli exhibited motility, most within a diameter of 2 to 13 mm. Most of the strains reacted to the repellent compounds analyzed, and α- and β-like hemolysis and cytotoxicity in Vero cells were observed for all strains. Isolates adhered to and invaded Vero cells to various degrees. Although strains of C. jejuni exhibited stronger adherence but less invasion compared with strains of C. coli, the difference was not significant (P > 0.05). The strains of C. jejuni and C. coli isolated from food and from patients in Mexico could have major impacts on public health.     

Characteristics of enterotoxin H-producing Staphylococcus aureus isolated from clinical cases and properties of the enterotoxin productivity

Staphylococcal enterotoxin H (SEH) is predicted to be involved in staphylococcal food poisoning. To characterize SEH-producing Staphylococcus aureus isolates from staphylococcal food poisoning cases in Japan, we investigated the relationship between SEH production and coagulase serotype, which is an epidemiological marker, and compared the properties of SEH production with those of staphylococcal enterotoxins A (SEA) and B (SEB). SEH production was determined by a newly developed sandwich enzyme-linked immunosorbent assay. Eighty-six (59.7%) of 144 isolates from staphylococcal food poisoning cases produced SEH. Seventy-one of the SEH-producing isolates simultaneously produced SEA, SEB, or both. All SEH-producing isolates belonged to coagulase type VII, which was the predominant type, representing 99 (68.8%) of 144 isolates. The amount of SEH produced in brain heart infusion was almost the same as the amount of SEA and approximately 10-fold lower than that of SEB. SEH and SEA were produced mainly during the late exponential phase of growth, whereas SEB was produced mostly during the stationary phase. The production levels of SEH and SEA were gradually affected by decreases in water activity, but the production of SEB was greatly reduced under conditions of low water activity. These findings indicate that SEH-producing S. aureus isolates are of high prevalence in staphylococcal food poisoning cases. Given the unique epidemiological characteristic of these isolates, SEH and SEA probably are responsible for food poisoning.     

Staphylococcus aureus isolates from Irish domestic refrigerators possess novel enterotoxin and enterotoxin-like genes and are clonal in nature

A previous study carried out by the National Food Centre in Dublin on bacterial contamination of Irish domestic refrigeration systems revealed that 41% were contaminated with Staphylococcus aureus. One hundred fifty-seven S. aureus isolates were screened by multiplex PCR analysis for the presence of 15 staphylococcal enterotoxin and enterotoxin-like genes (sea-see, seg-sei, selj-selo, and selq) and the toxic shock toxin superantigen tst gene. Of the refrigerator isolates, 64.3% possessed more than one staphylococcal enterotoxin or staphylococcal enterotoxin-like gene. All bar one of the 101 staphylococcal enterotoxin or staphylococcal enterotoxin-like gene-positive strains possessed the egc locus bearing the seg, sei, selm, seln, and selo genes. Twelve random amplified polymorphic DNA (RAPD) types accounted for 119 (75.8%) of the strains, two of these types accounting for 25 (RAPD type 1, 15.9%) and 52 (RAPD type 5, 33.1%), respectively. All of the RAPD type 5 isolates possessed the egc gene cluster only. The RAPD type 5 amplicon profile was identical to that of S. aureus isolates associated with osteomyelitis in broiler chickens in Northern Ireland that also possessed the egc locus only. However, the RAPD type 5 domestic refrigerator and chicken isolates differed in penicillin G sensitivity, production of Protein A and staphylokinase, and crystal violet agar growth type. These findings highlight that the average Irish household refrigerator harbors potential enterotoxin-producing S. aureus that may or may not be of animal origin and, accordingly, is a potential reservoir for staphylococcal food poisoning.     

Characterization of coagulase-negative staphylococci from brining baths in Germany

Brining is an important step in cheese making, and using brine baths for this purpose is common practice in German dairies. Time of brining, brine concentration, and composition of the complex and heterogeneous microbiota, including coagulase-negative staphylococci (CNS), contribute to the ripening and taste of cheese. As well as producing staphylococcal enterotoxins, some CNS show antibiotic resistance; therefore, we isolated 52 strains of presumptive CNS from cheese brines from 13 factories in Germany. Species identification by sodA gene sequencing revealed that 50 isolates were CNS: 31 Staphylococcus saprophyticus, 4 Staphylococcus carnosus, 4 Staphylococcus equorum, 3 Staphylococcus sciuri, 2 Staphylococcus hominis, and 2 Staphylococcus warneri. One isolate each was identified as Staphylococcus epidermidis, Staphylococcus pasteurii, Staphylococcus succinus, and Staphylococcus xylosus. Further subtyping of the Staph. saprophyticus isolates to the subspecies level revealed the presence of 6 Staph. saprophyticus ssp. saprophyticus. Using pulsed-field gel electrophoresis with the identified Staph. saprophyticus strains, 12 independent clones were identified, resulting in the exclusion of 18 strains from further testing. In 19 of the remaining 32 CNS isolates, resistance to antibiotics was observed. Resistance was found against oxacillin (17), penicillin (5), and cefoxitin (1). Four isolates expressed resistance to both oxacillin and penicillin. No resistance was found to enrofloxacin, tetracycline, gentamicin, or erythromycin. Then, PCR analysis for antibiotic resistance genes was performed for 22 different genes. Only genes blaZ and bla_TEM were found in 7 isolates. These isolates were selected for challenge tests with different concentrations of lactic acid and NaCl to examine whether expression of antibiotic resistance was influenced by these stressors. An increase in the minimal inhibitory concentration from 0 to 2.0 µg/mL was seen for trimethoprim/sulfamethoxazole only in one isolate of Staph. saprophyticus at an increased lactic acid concentration. Finally, all isolates were tested for genetic determinants (entA, entB, entC, entD, and entE) of the most common staphylococcal enterotoxins; none of these genes were detected. We found no indication for unacceptable risks originating from the isolated CNS.     

Putative virulence factors of the Aeromonas spp. isolated from food and environment in Abu Dhabi, United Arab Emirates

Thirty randomly selected Aeromonas isolates from food and the environment in Abu Dhabi, United Arab Emirates, were characterized for putative virulence determinants, such as production of cytotoxin, cytotonic toxin, and hemolysin and their capacity to adhere to and invade Henle 407 cells in vitro. Seventy percent of the tested isolates were cytotoxin producers, and 80% were hemolytic. Cytotoxin was produced by 6 of 7 A. hydrophila strains, 6 of 13 A. caviae strains, and 6 of 7 A. veronii bv. sobria strains, mostly from food sources. A. schubertii, A. jandaei, and A. trota also produced both cytotoxin and hemolysin. All of the 30 isolates tested adhered to Henle 407 cells, but none were able to invade the cells, as determined with the in vitro assay. However, no significant correlation of the presence of these putative virulence factors was found among these aeromonad food isolates.