Tst Genes Research Articles

Virulence properties of methicillin-susceptible Staphylococcus aureus food isolates encoding Panton–Valentine Leukocidin gene

In this study, three Panton–Valentine Leukocidin gene carrying methicillin-susceptible Staphylococcus aureus (MSSA) strains (M1-AAG42B, PY30C-b and YF1B-b) were isolated from different food samples in Kesan—Edirne, Turkey. These strains were characterized on the basis of MLST type, spa type, virulence factor gene contents, antibiotic susceptibilities against 21 antibiotics and biofilm formation. The genetic relatedness of the strains was determined by PFGE. In addition, the complete gene sequences of lukS-PV and lukF-PV were also investigated. All strains were found to be susceptible to tested antibiotics and they were mecA negative. Three strains showed the same PFGE band pattern, ST152 clonal type and t355 spa type. In the detection of virulence factor genes, sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, seu, eta, etb, set1, geh and tst genes were not detected. All strains showed the positive results for α- and β-haemolysin genes ( hla and hlb), protease encoding genes ( sspA, sspB and aur), lukE and lukD leukocidin genes ( lukED). The strains were found to be non-biofilm formers. By this study, the virulence properties of the strains were described and this is one of the first reports regarding PVL-positive MSSA strains from food.

Genetic Diagnosis of Community-Acquired MRSA: A Multiplex Real-Time PCR Method for Staphylococcal Cassette Chromosome mec Typing and Detecting Toxin Genes

Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of infections in health care settings and community environments. In particular, community-acquired MRSA (CA-MRSA) is important for clinicians because many fatal cases in healthy populations have been reported. Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element and carries the central determinant for broad-spectrum beta-lactam resistance encoded by the mecA gene. The emergence of MRSA is due to the acquisition and insertion of the SCCmec element into the chromosome. CA-MRSA is characterized as SCCmec type IV. Thus, we aimed to establish a novel multiplex real-time PCR method to distinguish SCCmec type, which enables us to evaluate the pathogenicity of MRSA. A total of 778 MRSA were isolated at Nagasaki University Hospital from 2000 to 2007. All isolates were subjected to minimal inhibitory concentration testing and PCR for SCCmec typing and detecting genes of toxins: tst (toxic shock syndrome toxin 1), sec (encoded enterotoxin type c), etb (exfoliative toxin type b), and lukS/F-PV (Panton-Valentine leukocidin). PCR was performed to amplify a total of 10 genes in the same run. The 667 MRSA clones detected from pus in 778 clones were classified as SCCmec type II (77.7%), type IV (19.2%), and type I (3.0%). 87.5% of SCCmec type II clone had tst and sec genes. No isolate was lukS/F-PV positive. The present study indicates the high rate of lukS/F-PV-negative SCCmec type IV in Nagasaki. Our PCR method is convenient for typing MRSA and detecting toxins in Japan.

Forte prévalence des infections communautaires et nosocomiales à Staphylococus aureus résistant à la méticilline et portant le gène de la leucocidine de Panton-Valentine dans l’Algérois

Objectives To determine the prevalence of community acquired and hospital methicillin-resistant Staphylococcus aureus ( S. aureus) infections and the Panton-Valentine leukocidin. Patients and methods Seven hundred S. aureus strains were collected during 21 months period in Mustapha Bacha hospital. Bacterial identification was based on standard methods and susceptibilities were tested by disk diffusion method. Molecular study (toxins, mecA gene and agr alleles) were determined for 221 S. aureus isolates by multiplex PCR. Results The global MRSA prevalence was 42 %, 35 % in the community and 49 % in hospital setting. The frequency of strains containing PVL genes (PVL+) was 36 %, their molecular profile was: agr3, mecA+, etd, edin, which correspond to the C-MRSA major ST80 clone in Europe and the Maghreb. The H-MRSA-PVL+ were multidrug resistant. Among the MSSA, 13 strains contained the tst gene and five contained the exfoliatine genes ETA and ETB. Conclusion Our results show a high rate of MRSA-PVL+ in the community and the hospital setting. The H-MRSA-PVL+ were multidrug resistant complicating their antibiotic treatment options.

COMPARISON OF THE PREVALENCE OF GENES CODING FOR ENTEROTOXINS, EXFOLIATINS, PANTON-VALENTINE LEUKOCIDIN AND TSST-1 BETWEEN METHICILLIN-RESISTANT AND METHICILLIN-SUSCEPTIBLE ISOLATES OF STAPHYLOCOCCUS AUREUS AT THE UNIVERSITY HOSPITAL IN OLOMOUC

Staphylococcus aureus is an important pathogen characterised by its potential to express many virulence factors. Currently, special attention is being paid to methicillin-resistant strains of S. aureus (MRSA). The aim of this study was to compare the prevalence of 13 selected virulence factor genes in methicillin-resistant versus methicillin-susceptible S. aureus (MSSA) isolates and to investigate their accumulation in the same isolate. Real-time PCR was used to detect the presence of genes in 200 isolates of S. aureus (100 MRSA and 100 MSSA) from the University Hospital Olomouc collected in 2005-2006. Six out of the 13 monitored genes were detected more frequently in MRSA isolates: sea, seb, seg, sei, sej and eta, coding for the production of the enterotoxins A, B, G, I, J and the exfoliative toxin A. On the other hand, the pvl and tst genes coding for Panton-Valentine leukocidin and TSST-1 were more frequent in MSSA. Statistical analysis (chi-squared test) of the prevalence of virulence factors in the two groups showed a significant difference (P<0.05) in two cases (seg, sei). A higher prevalence of selected virulence genes was not confirmed in the methicillin-resistant S. aureus group. This indicates no further increase in their threat.

Nationwide epidemiological study revealed the dissemination of meticillin-resistant Staphylococcus aureus carrying a specific set of virulence-associated genes in Japanese hospitals

To study comprehensive toxin profiles and the chromosomal diversity of current Japanese hospital-associated meticillin-resistant Staphylococcus aureus (HA-MRSA) strains, we conducted PCR-based identification of 28 toxin genes, and staphylococcal cassette chromosome mec (SCCmec) typing and PFGE analysis of 208 MRSA strains isolated from 100 hospitals throughout Japan. Of the tested HA-MRSA strains, 80.3 % were tst-positive. The most frequent toxin gene profile was characterized by the carriage of 13 genes, tst, sec, seg, sei, sel, sem, sen, seo, lukED, hla, hlb, hld and hlg-2. Ninety of the 208 strains had this profile, which was named pattern A. Among the 118 non-pattern A strains, 100 had similar toxin gene profiles, the concordance rates to pattern A of which were more than 80 %. Consequently, 91.3 % of the examined HA-MRSA strains carried similar toxin profiles, although PFGE patterns showed a wide variation. These strains belonged to SCCmec type II, agr II and coagulase type II. We concluded that, unlike MRSA from many other countries, most of the Japanese HA-MRSA strains belonged to, or were related to, a specific group carrying the set of 13 toxin genes, irrespective of chromosomal diversity. In addition, among the 13 toxin genes, the coexistence rates of tst, sec and sel, and those of seg, sei, sem, sen and seo, were higher than for the other toxin genes. High coexistence rates of tst, sec and sel genes suggested the presence of the pathogenicity island SaPIn1 in these strains.

Molecular epidemiology of Staphylococcus aureus in asymptomatic carriers

Microarrays were used to extensively characterise 155 Staphylococcus aureus isolates obtained from asymptomatic carriers from Saxony, Germany, in order to determine clonal complex affiliation, as well as the carriage of clinically relevant genes. Isolates belonged to 20 different clonal complexes (CCs). The most common CC was CC8 (18.71%), followed by CCs 15, 30 and 45. Three isolates (1.94%) were methicillin-resistant S. aureus (MRSA). Beta-lactamase was common (70.97%), but other resistance genes were found only sporadically. Genes encoding superantigens were abundant. The enterotoxin cluster egc was found in 45.81% of isolates. The toxic shock syndrome toxin gene tst was detected in 14.84% of isolates and 17.42% harboured enterotoxin A alleles (sea, sea-N315). Contrarily, Panton-Valentine leukocidin (lukS/F-PV) was rare, being found in only one methicillin-susceptible CC30 isolate. Its low prevalence in asymptomatic carriers might emphasise a pathogenetic significance in patients with skin and soft tissue infections. Most microbial surface components recognising adhesive matrix molecules of the host (MSCRAMMs) genes were nearly ubiquitously present. However, two MSCRAMM genes, cna (collagen adhesion) and sasG (surface protein G), were detected in only some CCs. These data provide an insight into its pathogenesis, especially when compared to isolates from patients with defined clinical conditions. They might also be helpful for the design of a future vaccine.

Methicillin-resistant Staphylococcus aureus among a network of French private-sector community-based-medical laboratories

ObjectiveThe aim of this study was to estimate the frequency of methicillin-resistant Staphylococcus aureus (MRSA) strains in the French community and the proportion of Panton-Valentine (PVL)-MRSA. DesignA cross-sectional study was made during a 3-month period in 2003 through a network of private-sector, community-based medical laboratories selected throughout France: the Labville network. Each MRSA isolate was included and characterized by French National Reference Center for Staphylococci. The total number of S. aureus isolates was also collected. ResultsAmong the 283 patients infected or colonized by MRSA, 166 (59%) were considered as healthcare-associated, 14 (5%) as nursing-associated and 39 (14%) as community-acquired. The proportion of methicillin resistance among S. aureus was 14%. Taking into account the sampling design, the incidence of MRSA cases in French outpatients was estimated to be 0.50 [CI95%: 0.41–0.60] per 10,000 inhabitants. The molecular analysis confirmed that 80.6% belong to the Lyon clone, the most prevalent hospital MRSA clone spreading in France and 10.6% to a closely related clone. An emerging MRSA clone containing the tst1 gene was detected in six patients and the PVL-positive ST80 clone only in one, 22-year-old, patient. ConclusionMost of MRSA cases diagnosed in the community in France, in 2003, were elderly with specific risk factors and harbored hospital strains. The prevalence of PVL-MRSA remained low.

Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations.

SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage-SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction.

Comparison of Genotypes and Enterotoxin Genes Between Staphylococcus aureus Isolates from Blood and Nasal Colonizers in a Korean Hospital

In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness.

Variations in amount of TSST-1 produced by clinical methicillin resistant Staphylococcus aureus (MRSA) isolates and allelic variation in accessory gene regulator (agr) locus

BackgroundStaphylococcus aureus (S. aureus) is an important pathogen associated with both nosocomial and community-acquired infections and its pathogenicity is attributed to its potential to produce virulence factors. Since the amount of toxin produced is related to virulence, evaluating toxin production should be useful for controlling S. aureus infection. We previously found that some strains produce relatively large amounts of TSST-1; however, no reports have described the amount of TSST-1 produced by clinical isolates.MethodsAmounts of TSST-1 produced by clinical methicillin resistant S. aureus (MRSA) isolates were measured by Western blotting. We determined their accessory gene regulator (agr) class by PCR and investigated whether TSST-1 production correlates with variations in the class and structure of the agr.ResultsWe found that 75% of surveyed MRSA isolates (n = 152) possessed the tst gene and that 96.7% belonged to agr class 2. The concentrations of TSST-1 secreted into culture supernatants by 34 strains measured by Western blotting differed 170-fold. Sequencing the entire agr locus (n = 9) revealed that some had allelic variations regardless of the amount of TSST-1 produced whereas sequencing the sar, sigma factor B and the tst promoter region revealed no significant changes.ConclusionThe amounts of TSST-1 produced by clinical MRSA isolates varied. The present results suggest that TSST-1 production is not directly associated with the agr structure, but is instead controlled by unknown transcriptional/translational regulatory systems, or synthesized by multiple regulatory mechanisms that are interlinked in a complex manner.

Prevalence of the toxic shock gene (TST) in an Australian Staphylococcus aureus cohort and changes in strain prevalence and virulence genes, 1989–2003

Aim To determine the prevalence of toxic shock gene tst in an Australian Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) cohort collected over the last two decades. Method The identity of 300 S. aureus isolates was confirmed by real-time polymerase chain reaction (PCR) assays for mecA, nuc and 16S rRNA. Isolates were assayed for eight single nucleotide polymorphisms (SNPs) and for five binary genes (pvl, cna, sdrE, pUB110, pT181). SNP profiles are concordant with multilocus sequence typing (MLST) clonal complexes (CCs) and/or sequence types (STs). Two real-time PCR assays were developed for tst, based on primer sequences previously described. Results SNP profiles correlated with 21 CCs/STs. The 90 MRSA isolates correlated with three CCs: CC239, CC1 and CC22. We found 18 tst positive isolates, 3/91 (1989), 6/104 (1996) and 9/105 (2003). Of these, CC30 was predominant. Of 210 methicillin-sensitive S. aureus isolates, 26 were CC1, 24 CC5 and 23 CC78. Conclusions The proportion of tst positive isolates and binary genes including pvl was low, and has not increased significantly. Most tst positive isolates belong to CC30 which accords with overseas publications. In 1989 and 1996 CC239 MRSA (AUS-2/3) was the sole MRSA strain. MRSA decreased significantly in 2003 in spite of the appearance of CC1 MRSA (WA-1) and CC22 MRSA (EMRSA-15).

Comparative prevalence of superantigenic toxin genes in meticillin-resistant and meticillin-susceptible Staphylococcus aureus isolates

A total of 118 meticillin-resistant Staphylococcus aureus (MRSA) and 140 meticillin-susceptible S. aureus (MSSA) isolates from different patients in the same time period were comprehensively searched using a multiplex PCR for the classical and recently described superantigenic toxin gene family comprising the staphylococcal enterotoxin genes sea to ser and the toxic shock syndrome toxin 1 gene, tst-1. Both MRSA and MSSA isolates carried a number of superantigenic toxin genes, but the MRSA isolates harboured more superantigenic toxin genes than the MSSA isolates. The most frequent genotype of the MRSA isolates was sec, sell and tst-1 together with the gene combination seg, sei, selm, seln and selo, which was found strictly in combination in 69.5% of the isolates tested. In contrast, possession of the sec, sell and tst-1 genes in MSSA isolates was significantly less than in MRSA (2.1 vs 77.1%, respectively), although they also often contained the combination genes (25.0%). This notable higher prevalence in MRSA isolates indicated that possession of the sec, sell and tst-1 genes in particular appeared to be a habitual feature of MRSA. Moreover, these were mainly due to the fixed combinations of the mobile genetic elements type I nuSa4 encoding sec, sell and tst-1, and type I nuSabeta encoding seg, sei, selm, seln and selo. Analysis of the relationship between toxin genotypes and the toxin gene-encoding profiles of mobile genetic elements has a possible role in determining superantigenic toxin genotypes in S. aureus.

Molecular Typing ofStaphylococcus aureusIsolated from Blood on the Basis of Coagulase Gene Polymorphism and Toxin Genes

Coagulase is produced by all strains of Staphylococcus aureus. The 3' coding region of the coagulase (coa) gene contains varying numbers of 81 bp tandem repeats. S. aureus produces a variety of extracellular protein toxins. Here, we typed S. aureus strains isolated from blood by coa gene restriction fragment length polymorphism (RFLP) patterns and toxin gene profiles. A total of 120 strains of S. aureus were isolated from blood cultures during 2003-2006 at Kangdong Sacred Heart Hospital. The isolates were typed by PCR RFLP analysis of the coa gene and by multiplex PCR for detection of genes encoding enterotoxins (sea, seb, sec, sed, and see), toxic shock syndrome toxin-1 (tst), exfoliative toxins (eta and etb), mecA and femA. All the S. aureus strains were classified into 16 types on the basis of coa gene RFLP and could be further differentiated into 34 types according to the combined patterns of coa gene RFLP and toxin gene profiles. Of 85 methicillin-resistant S. aureus (MRSA) strains, 43 (50.6%) and 36 (42.4%) belonged to the RFLP pattern L5 and pattern L1, respectively. MRSA strains belonging to pattern L5 frequently carried tst (93.0%) or sec gene (81.4%), and strains belonging to pattern L1 frequently carried sea (88.9%) or see gene (44.4%). The rate of the pattern L5 in MRSA strains increased over the past few years and was higher in intensive care unit than in other wards. We typed S. aureus strains isolated from blood on the basis of coa gene RFLP and toxin genes. The strains belonging to coa gene RFLP pattern L5 and L1 appeared to be the major types of MRSA isolasted from bacteremia and revealed specific toxin gene profiles according to the coa gene RFLP patterns.

Molecular typing of nasal carriage isolates of Staphylococcus aureus from an Irish university student population based on toxin gene PCR, agr locus types and multiple locus, variable number tandem repeat analysis

Forty-eight Staphylococcus aureus isolates collected from a young, healthy, Irish university student population from 1995 to 2004 were screened for 16 enterotoxin (SE) and enterotoxin-like (SEl) genes (sea-see, seg-sei, selj-selo, selq, selu), and for the toxic shock toxin syndrome toxin-1 gene, tst. All of the isolates harboured at least one SE or SEl gene and 66.7 % possessed a classical SE gene (sea, seb, sec), the commonest being the seb gene. Most of the isolates (85.4 %) had a complete egc locus (selo, selm, sei, seln, seg). The intergenic sei-seln region of the egc locus was typed by PCR-RFLP in 34 isolates, 15 possessing pseudogenes psient1 and psient2 and 19 having the selu gene. The seh and sell genes, the selk-selq gene combination, and the tst gene were each found in <15 % of isolates. The agr genotype distribution was agr type III, 37.5 %; agr type I, 35.4 %; agr type II, 25 %; and agr type IV, 2.1 %. There was no association between SE-SEl genotype and agr type. All tst gene-positive isolates were of agr type III and harboured a classical SE gene. Multiple locus, variable number tandem repeat analysis (MLVA) produced 47 different patterns. While the sdr locus was present in all isolates, half of them lacked one or two of the sdr gene amplimers. Twenty isolates harboured the bbp gene, its presence being associated with agr type III, but not with the SE-SEl gene profile. The agr types of isolates were associated with MLVA subclusters. Selective MLST analysis revealed seven novel sequence types and a new aroE allele. Five clonal clusters (CCs), including CCs comprising major pandemic clones CC30, CC5 and CC22 and minor lineages CC6 and CC9, and three singletons were identified.

Staphylococcus aureus of phage type 187 isolated from people occurred to be a genes carrier of eneterotoxin C and toxic shock syndrome toxin-1 (TSST-1)

The aim of this study was to examine the genotype properties of Staphylococcus aureus of phage type 187 strains that constitute a separate group among the strains of S. aureus. Sixteen strains were collected from the hospital patients (n=12) and the healthy carriers (n=4) in 13 medical centres in Poland during 1991 and 2005. Biotyping, antibiotic susceptibility, phage typing, detection the genes of enterotoxins and toxic shock syndrome toxin, genotyping of chromosomal DNA by pulsed-field gel electrophoresis (PFGE), also amplification and restriction analysis of the coagulase (coa) and the protein A genes (spa) (PCR/restriction fragment length polymorphism (RFLP)) was tested. The results of this study showed that all staphylococcus of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Finding out the toxin genes showed that almost all of them (93.8%) had the enterotoxin C gene (sec) and TSST-1 gene (tst). The PFGE typing proved that the phage type 187 strains (except for one) constitute one PFGE type. These results and the identical restriction patterns in the PCR/RFLP method, also the same biotype, sensitivity to antibiotics and the presence genes of the same type of toxins confirmed that the phage type 187 strains constitute one clone within our country. Additionally, the fact that almost all of them have the enterotoxin genes and tst gene allows to consider them the strains of potentially high virulence.

One in five mortality in non-menstrual toxic shock syndrome versus no mortality in menstrual cases in a balanced French series of 55 cases

Staphylococcus aureus superantigenic toxins are responsible for menstrual and non-menstrual toxic shock syndrome (TSS). We compared the clinical and biological characteristics of 21 cases of menstrual TSS (MTSS) with 34 cases of non-menstrual TSS (NMTSS) diagnosed in France from December 2003 to June 2006. All 55 S. aureus isolates had been spontaneously referred to the French National Staphylococcal Reference Center, where they were screened for superantigenic toxin gene sequences. Most of the patients had previously been in good health. The most striking differences between MTSS and NMTSS were the higher frequency in NMTSS of neurological disorders (p=0.028), of S. aureus isolation by blood culture (50% versus 0% in MTSS), and the higher mortality rate in NMTSS (22% versus 0% in MTSS). The tst and sea genes were less frequent in isolates causing NMTSS than in those causing MTSS (p<0.001 and 0.051, respectively). Higher mortality was significantly associated with the presence of the sed gene (p=0.041), but when considering NMTSS survivors and non-survivors, no clinical or bacteriological factors predictive of vital outcome were identified. Specific antitoxinic therapy was rarely prescribed, and never in fatal cases. Higher mortality was observed in NMTSS than in MTSS, and no definite factors could explain the higher severity of NMTSS. NMTSS would require more aggressive therapy, comprising systematic rapid wound debridement, antistaphylococcal agents, including an antitoxin antibiotics, and intravenous immunoglobulin.

Absolute and relative real-time PCR in the quantification of tst gene expression among methicillin-resistant Staphylococcus aureus: evaluation by two mathematical models

Absolute and relative quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) by the use of two mathematical models were applied in order to study the expression of tst gene encoding the toxic shock syndrome toxin-1 (TSST-1), among methicillin-resistant Staphylococcus aureus (MRSA). Thirteen epidemic MRSA belonging to different clones and carrying a variety of toxin genes were selected. tst gene expression was achieved by using absolute and relative quantitative real-time RT-PCR and the SYBR Green I. Absolute RT-PCR showed a statistically significant higher level of tst expression among strains isolated from soft tissue infections. Relative quantification was performed in relation to 23S rRNA expression by the application of two mathematical models, the 2(-DeltaDeltaCt) and the Pfaffl analysis methods. tst gene expression was best calculated by the relative real-time RT-PCR analysis applying the Pfaffl analysis method, taking into account the reactions' efficiencies. Level of tst expression was related to patients' infection and did not depend on the MRSA genetic profile. The results indicate that the application of the Pfaffl analysis method in the evaluation of relative real-time RT-PCR is more adequate.

Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary

Staphylococcus aureus is a major foodborne pathogen due to its capability to produce a wide range of heat-stable enterotoxins. The primary purpose of this research was to characterize S. aureus isolates recovered from mammary quarter milk of mastitic cows and from bulk tank milk produced on Hungarian dairy farms of different sizes. Macrorestriction analysis of chromosomal DNA from S. aureus isolates was performed using the restriction enzyme SmaI followed by pulsed-field gel electrophoresis (PFGE). The prevalence rates of nine S. aureus enterotoxin genes ( sea, seb, sec, sed, see, seg, seh, sei, and sej) and of the toxic shock syndrome toxin 1 gene ( tst) were determined by multiplex polymerase chain reaction (PCR). The bulk tank milks of 14 out of 20 farms were contaminated with S. aureus at levels of up to 6.0 × 10 3 CFU/ml. Farm size had no significant effect ( P > 0.05) on the S. aureus counts in bulk milk. The prevalence rates of penicillin resistance were 88.9% and 20.0% among the S. aureus recovered from mastitic quarter milk and bulk tank milk, respectively. After phenotypic characterization, a total of 59 S. aureus isolates were selected for genotyping. PFGE analysis revealed 22 distinct pulsotypes, including 14 main types and 8 subtypes, at a similarity level of 86%. Only one or two main types were observed on each of the farms tested, indicating a lack of genetic diversity among S. aureus isolates within farms, and there were only two pulsotypes which occurred on more than one farm. The PFGE patterns showed genetic relatedness between the S. aureus strains recovered from quarter milk and bulk milk on two large farms, implying that on farms having a high number of mastitic cows, S. aureus from infected udders may contaminate bulk milk and, subsequently, raw milk products. Sixteen (27.1%) of the S. aureus isolates tested by multiplex PCR were found to be positive for enterotoxin genes, with 15 of them carrying just one gene and one strain carrying two genes ( seg and sei). The most commonly detected toxin genes were seb, sea, and sec, whereas none of our isolates possessed the see, seh, sej, or tst genes. On 75% of the dairy farms surveyed, no enterotoxigenic staphylococci were recovered from either mastitic quarter milk or bulk tank milk.

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 3,332 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.

Molecular Characterization of Methicillin‐Resistant Staphylococcus aureus in Hospitals in Niigata, Japan: Divergence and Transmission

The major methicillin-resistant Staphylococcus aureus(MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCC mec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCC mec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst(+) sec(+) strains represented a majority of 86.5% and 9.4% were tst(-) sec(-). SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCC mec IV and seemed to originate in the community, while ST8 strains exhibited SCC mec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type.