Identification and functional activity of a staphylocoagulase type XI variant originating from staphylococcal food poisoning isolates.

UNLABELLED: Staphylocoagulase, an extracellular protein secreted by Staphylococcus aureus, has been used as an epidemiological marker. At least 12 serotypes and 24 genotypes subdivided on the basis of nucleotide sequence have been reported to date. In this study, we identified a novel staphylocoagulase nucleotide sequence, coa310, from staphylococcal food poisoning isolates that had the ability to coagulate plasma, but could not be typed using the conventional method. The protein encoded by coa310 contained the six fundamental conserved domains of staphylocoagulase. The full-length nucleotide sequence of coa310 shared the highest similarity (77·5%) with that of staphylocoagulase-type (SCT) XIa. The sequence of the D1 region, which would be responsible for the determination of SCT, shared the highest similarity (91·8%) with that of SCT XIa. These results suggest that coa310 is a novel variant of SCT XI. Moreover, we demonstrated that coa310 encodes a functioning coagulase, by confirming the coagulating activity of the recombinant protein expressed from coa310. This is the first study to directly demonstrate that Coa310, a putative SCT XI, has coagulating activity. These findings may be useful for the improvement of the staphylocoagulase-typing method, including serotyping and genotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to identify a novel variant of staphylocoagulase type XI based on its nucleotide sequence and to demonstrate coagulating activity in the variant using a recombinant protein. Elucidation of the variety of staphylocoagulases will provide suggestions for further improvement of the staphylocoagulase-typing method and contribute to our understanding of the epidemiologic characterization of Staphylococcus aureus.

Clonal distribution of enterotoxigenic Staphylococcus aureus on handles of handheld shopping baskets in supermarkets.

AIMS: Shopping carts and handheld shopping baskets in supermarkets are subject to accidental bacterial contamination through contacts with a variety of food. We investigated the prevalence of Staphylococcus aureus on the handles of handheld shopping baskets in four supermarkets distantly located in Osaka district, Japan. METHODS AND RESULTS: Fifty two strains of Staph. aureus were isolated from 760 basket handles. Among these, six strains were positive for staphylococcal enterotoxin B (SEB) production, representing 12% of total. This SEB producer ratio is considerably higher than among Staph. aureus isolated from nasal swabs of the supermarket workers (2%) and from independently collected clinical specimens (4%). These SEB-producing Staph. aureus strains from the basket handles are clonal and belong to ST12. Coagulase typing showed that they are in group VII, which is the most common cause of food poisoning in Japan. Biofilm assays indicated that SEB gene (seb)-positive strains including this clone produced a significantly higher amount of biofilm than seb-negative strains. CONCLUSIONS: The frequent isolation of seb-positive Staph. aureus on shopping basket handles raises the possibility that they could be a hidden reservoir for Staph. aureus with a potential to cause food poisoning and draws attention to the importance of shopping basket sanitation.

Genetic variation among Staphylococcus aureus strains from bovine milk and their relevance to methicillin-resistant isolates from humans.

In genetic analysis of bovine Staphylococcus aureus isolates that are recognized as an important pathogenic bacterium in bovine mastitis, multilocus sequence typing (MLST) showed strong correlation to the results of pulsed-field gel electrophoresis, coa PCR-restriction fragment length polymorphism (RFLP), spa typing, and the coagulase serotyping method. According to MLST results, strains derived from sequence type 97 (ST97) and ST705 were suggested as not only dominant bovine S. aureus lineages in Japan but also pandemic bovine S. aureus lineages. Although both lineages seem to be distantly related to each other by phylogenetic analysis, both had common characteristics, i.e., lukM/lukF'-PV and coagulase serotype VI. These characteristics were very rare among minor bovine strains and human strains and may contribute to the host specificity of these lineages. Four methicillin-resistant S. aureus (MRSA) isolates were first confirmed from bovine milk in Japan; these isolates showed geno- and serotypes that were identical or similar to those of human MRSA isolates in Japan (ST5, staphylococcal cassette chromosome mec type II [SCCmec II], Spa type t002 or t375, and coagulase serotype II, and ST89, SCCmec IIIa, Spa type t5266, and coagulase serotype I). ST5 and ST89 are uncommon among bovine isolates in the world, whereas these STs are common among human MRSA isolates in Japan.

Simultaneous detection and differentiation of Staphylococcus species in blood cultures using fluorescence in situ hybridization.

OBJECTIVE: To develop a new protocol for the simultaneous detection and differentiation of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in blood cultures using fluorescence in situ hybridization (FISH), without cultivation and biotyping. MATERIALS AND METHODS: Oligonucleotide probes were used to target the variable regions of the 16S rRNA of S. aureus and CoNS, the probes were labeled with fluorochrome Cy3 (red signal) and fluorescein isothiocyanate (green signal), respectively. It is not possible to design one probe that will hybridize to all CoNS species. Therefore, in this study two differentially labeled probes (STA and SAU) were mixed and used to detect and differentiate S. aureus from CoNS rapidly in a single smear. Samples of 189 positive blood cultures with Gram-positive cocci in clusters and 11 Gram-positive cocci in chains or pairs were included. RESULTS: The FISH assay showed 91.8% sensitivity and 100% specificity for S. aureus and 100% sensitivity and 93.3% specificity for CoNS when hybridized with STA and SAU probes. Meanwhile, the assay showed 91.8% sensitivity and 100% specificity for S. aureus and 100% sensitivity and 90% specificity for CoNS, when hybridized with the SAU probe only. CONCLUSION: Our data showed that the FISH technique was suitable for the simultaneous detection and differentiation of Staphylococcus sp. in blood cultures.

Diversity of staphylocoagulase and identification of novel variants of staphylocoagulase gene in Staphylococcus aureus.

Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Serotype of SC (coagulase type) is used as an epidemiological marker and 10 types (I-X) have been discriminated so far. To clarify genetic diversity of SC within a single and among different serotype(s), we determined approximately 1500 bp-nucleotide sequences of SC gene encoding D1, D2, and central regions (N-terminal half and central regions of SC; SC(NC)) for a total of 33 S. aureus strains comprising two to three strains from individual coagulase types (I-VIII, X) and 10 strains which were not determined as previously known SC serotypes (ND-strains). Amino acid sequence identities of SC(NC) among strains with a single coagulase type of II, III, IV, V, VI and X were extremely high (more than 99%), whereas lower identity (56-87%) was observed among different types. In contrast, within a single coagulase type of I, VII, or VIII, sequence divergence was found (lowest identity; 82%). SC(NC) sequences from the ND-strains were discriminated into two genetic groups with an identity of 71% to each other (tentatively assigned to genotypes [XI] and [XII]), and exhibited less than 86% sequence identities to those of most known coagulase types. All the types [XI] and [XII] strains were methicillin susceptible and belonged to different sequence types from those of coagulase types I-X strains reported so far by multilocus sequence typing. These findings indicated genetic heterogeneity of SC in coagulase types I, VII, and VIII strains, and the presence of two novel SC genotypes related to antigenicity of SC serotypes.

Multiplex PCRs for assignment of Staphylocoagulase types and subtypes of type VI Staphylocoagulase.

Staphylocoagulases (SCs) have been classified by the differences in antigenicity using a serological method. We have developed a system to classify them based on the nucleotide differences in SC genes (coa). The system was composed of three multiplex PCRs (M-PCRs): M-PCR:A, identifying types III, IV, VII, and VIII; M-PCR:B, identifying types I, II, V, and VI; M-PCR:C, identifying three subtypes of type VI. In this study, we found that coa genes of the serotype VI were not identical, but classified into three subtypes based on the nucleotide differences, especially in D2 and the central region: VIa, the coa gene carried by stp12 from human; and VIb and VIc, the coa genes carried by strains IFH556 and IFH514 isolated from bovine raw milk. The primer pair used in M-PCR:B was designed to identify all three subtypes of type VI coa. The results showed that coa types of 154 out of 155 Staphylococcus aureus strains from various origins assigned by M-PCR:A and B were identical to those obtained by serological methods, leaving a serotype IV strain unclassifiable. All 73 type VI strains were classified into one of three subtypes by M-PCR:C. Furthermore, we found that type VIa and VIb strains carried characteristic pyrogenic toxin superantigen genes, while no toxin genes were identified in type VIc strains, suggesting the correlation between the subtype of type VI coa gene and the carriage of genomic islands. Our results showed that these M-PCRs are convenient methods for SC typing that might be useful for epidemiological studies.

Compatibility of pulsed-field gel electrophoresis findings and clinical criteria commonly used to distinguish between true coagulase-negative staphylococcal bacteremia and contamination.

OBJECTIVES: To evaluate the specificity and sensitivity of the clinical criteria widely used to differentiate true coagulase-negative staphylococcal (CoNS) bacteremia from contamination, using pulsed-field gel electrophoresis (PFGE) as the reference test. DESIGN: The study sample consisted of 79 CoNS isolates recovered from cultures of blood from 38 patients. Medical charts of the patients were reviewed for demographic and clinical information. The relatedness of CoNS strains recovered from 2 or more successive blood cultures was analyzed by PFGE. Patients from whom similar strains were recovered were assumed to have true bacteremia, whereas patients from whom different strains were recovered were considered to have contaminated blood cultures. The clinical criteria comprised Centers for Disease Control and Prevention (CDC) surveillance definitions for bloodstream infection (BSI), as well as an alternative criterion based on the presence of fever, the presence of leukocytosis, the absence of another recognized infection, and the recovery of CoNS from 2 or more successive blood cultures. RESULTS: Nineteen (50%) of the 38 patients had bacteremia due to similar strains; the remaining patients had bacteremia due to different strains. Criterion 2a of the CDC definition for BSI had a sensitivity of 100% and a specificity of 31.6% for distinguishing between true bacteremia and contamination. CDC criterion 2b had a sensitivity of 78.9% and a specificity of 52.6%. CONCLUSIONS: Molecular typing correlated poorly with the clinical criteria for true bacteremia. In view of the limited applicability of clinical criteria, more studies are needed to improve them. Periodic cross-sectional studies based on PFGE findings might be useful to estimate local contamination rates in an institution, which in turn can be used to improve the accuracy of the clinical diagnosis of bacteremia.

The microbiology of infected hip arthroplasty.

Infection following joint replacement surgery although rare presents a challenging problem. Bacterial resistance to antibiotics is an emerging problem. We analysed the microbiology of 337 single-stage revisions for deep infection. Coagulase negative staphylococcus was found to be the predominant organism, although staphylococcus aureus is gaining importance. Gentamicin only provides cover for 64.1% of organisms. Resistance to this commonly used antibiotic prophylaxis is escalating. Fusidic acid and erythromycin provide improved cover. We would suggest on a microbiological basis that these antibiotics be considered for addition to acrylic bone cement. This will provide local antibiotic delivery when performing a revision for deep infection.

A clone of coagulase-negative staphylococci among patients with post-cardiac surgery infections.

Coagulase-negative staphylococci (CoNS) are important causes of hospital-acquired infections such as infections after cardiac surgery. Efforts to reduce these infections are hampered by the lack of knowledge concerning the epidemiology of CoNS in this setting. Forty strains of CoNS collected during the surgical revision of 27 patients operated on between 1997 and 2000 were analysed. Strains were also collected from the ambient air in the operating suite. Their pulsed-field gel electrophoresis (PFGE) characteristics and antibiotic resistance were analysed. Using PFGE 19 of 40 strains from 15 of 27 patients were shown to belong to one clone, and strains from this clone were also isolated from the ambient air. This clone had caused infections throughout the period. Antibiotic resistance did not correlate with PFGE patterns. Using PFGE one clone could be identified that caused 56% of the CoNS infections during this period. A strain from this clone was also found in the air of the operating suite suggesting the origin of the CoNS causing infections was the hospital environment.

Epidemiological study of methicillin-resistant Staphylococcus aureus at Fukuoka University Hospital.

To clarify what types of methicillin-resistant Staphylococcus aureus (MRSA) strains were easily transmitted and colonized in the inpatients of the emergency center and the neonatal intensive-care unit (NICU) at Fukuoka University Hospital, 70 MRSA isolates obtained from February to November 1995 (the first survey) and from November 1996 to March 1997 (the second survey) were investigated biologically and genetically. Pulsed-field gel electrophoresis (PFGE) showed 12 types (PFGE types A-L) of DNA patterns for the MRSA isolates. At the emergency center, the PFGE types A and B strains were isolated from 40.9% and 27.2% of the MRSA-excreting patients in the first survey, respectively, while type E was isolated from 66.7% in the second survey. At the NICU, type A and J strains were isolated from 33.3% and 55.6% of the MRSA-excreting patients in the first survey, while the types A and B were isolated from 25% and 50%, respectively, in the second survey. Type A-D strains were isolated in both wards, while other epidemic types strains were isolated in only one ward. These results suggest that the type A and B strains have been colonized in the two wards for a long time and these strains might spread and colonize easily in the patients. Type C and D strains have also been colonized, but only in a small population over the two wards.

Analysis of mec regulator genes in clinical methicillin-resistant Staphylococcus aureus isolates according to the production of coagulase, types of enterotoxin, and toxic shock syndrome toxin-1.

The intrinsic resistance of methicillin-resistant Staphylococcus aureus (MRSA) is frequently explained by the production of an additional penicillin-binding protein (PBP), which is encoded by the mecA gene. The mec regulator genes, mecR1 and mecI, was identified in mecA-carrying Staphylococcus aureus N315. Between February and March, 1993, 179 clinical MRSA isolates were collected from institutions in Hiroshima prefecture. According to serological types of coagulase, enterotoxins, and toxic shock syndrome toxin-1 (TSST-1) productions, these strains were classified into 6 groups. In 53 strains chosen from all groups, mec regulatory gene distributions were divided into two groups; one with whole regulatory genes and another with the lacking region, including 3'-partial region of the mecR1 gene and mecI gene. This same deletion was detected across the different groups, suggesting that the deletion occurred at the ancestral strain before branching according to coagulase or enterotoxin productions. The strains with this lacking region showed a high-level of resistance to methicillin, while the strains with whole regulatory genes consisted of low and high levels of resistant strains. The highly resistant strains with whole regulatory genes were found to harbor a point mutation in the mecI gene. The basal levels of mecA gene transcription were elevated in the strains with the lacking region or the mecI point mutations. These data suggest that deletion or mutation of the mecI gene, the repressor on the mecA gene, might play an important role in methicillin resistance in clinical isolates of MRSA.

Producibility of exfoliative toxin and staphylococcal coagulase types of Staphylococcus aureus strains isolated from skin infections and atopic dermatitis.

BACKGROUND: Strains of Staphylococcus aureus which cause staphylococcal scalded skin syndrome (SSSS) and bullous impetigo secrete exfoliative toxin (ET). Two antigenically distinct serotypes of ET, ETA and ETB, have been reported. MATERIALS AND METHODS: Two hundred eighty-three strains of S. aureus isolated from impetigo, SSSS, furuncles (including furunculosis) and atopic dermatitis were examined in terms of the producibility of ET, ET serotypes and coagulase types. We examined ET production and ET serotypes using the polymerase chain reaction with the oligonucleotide primers for eta and etb. RESULTS: The incidence of ET producers was 69.4% (100/144) in impetigo, 100% (6/6) in SSSS, 2.8% (3/112) in atopic dermatitis and 0% (0/21) in furuncles. ETA alone was produced by 57 strains from impetigo and by 3 strains from atopic dermatitis. ETB alone was produced by 36 strains from impetigo and by all 6 strains from SSSS. Seven strains from impetigo produced both ETA and ETB. Most ETA producers belonged to coagulase type V and most ETB producers to coagulase type I. Impetigo strains mostly belonged to type I or V. All strains from SSSS were classified as type I. Type IV was most frequent among S. aureus isolates from furuncles. CONCLUSION: These results add to the epidemiological information as to ET producibility and ET serotypes of S. aureus strains isolated from impetigo, SSSS, furunculosis and atopic dermatitis. We have found that there is a relationship between the ET serotypes and the coagulase types of ET-producing strains.

An improved method for the serotyping of free coagulase from Staphylococcus aureus.

The serotyping of free coagulase, one of the most reliable ways to identify strains of Staphylococcus aureus, and widely employed in Japan, has been improved by adding magnetite sand to the reaction mixture. Culture medium supernatant and a type-specific antibody are mixed in a well of a microtiter plate, and plasma-enriched bovine fibrinogen is treated with magnetite sand. The use of tranexamic acid and gum arabic in the reaction mixture also increases the sensitivity of the reaction. Finally, the plate is placed on a magnetic stirrer. If the type of the coagulase corresponds to that of the antibody, no clot formation will occur, and this is easily confirmed by the movement of the sand. Although the amount of reaction mixture required is much less than that for the conventional tube method, our new method is able to detect slight increases in viscosity of the reaction mixture due to fibrin formation even before complete clotting occurs, thus providing very high sensitivity. Clot formation can also be judged by observing a turbid mass of fibrin in the well (Hwang's method), but this approach is a little slower than our method involving immobilization of magnetite sand.

Esquema simplificado de identificacion de las especies de staphylococcus coagulasa-negativa aislados de infecciones humanas / Identificacion simplified scheme for coagulase-negative staphylococcus strains isolated from human infections

Neste trabalho se identificaram 100 cepas de estafilococos coagulasa negativa de diferentes mostras clínicas. Buscando a correlaçäo entre homologia de DNA e características fenotípicas foi dividido em 2 grandes grupos pela sensibilidade à novobiocina e agregando a detecçäo de beta-galactosidasa e oxidasa em que se determinou 4 grupos (quadro 1): grupo de espécies de S. epidermidis, grupo de espécies de S.simulans: grupo de espécies de S. sapiophyticus e grupo de espécie de S. sciuri que säo factíveis de resolver em todo laboratório de microbiologia clínica. Por agregado de outras provas simples: detecçäo de ureasa, fosfatasa alcalina e beta-glucosidasa e detecçäo de aeróbica de acidez de maltosa, trehalosa e manitol se determinam as espécies de grupo S. epidermidis (quadro 2). Geralmente dada a maior frequência do S. epidermidis suficiente somente a detecäo de ureasa e fosfatasa. As espécies do grupo S. simulans se identificam com a determinaçäo de ureasa: fosfatasa alcalina e acidez de manitol (quadro 3): com a realizaçäo de ureasa e acidificaçäo de sacarosa e arabinosa se estabelece as espécies do grupo S. saprophyticus e unicamente por meio da rafinosa as espécies do grupo S. sciuri (quadro 5). A correlaçäo destas mínimas provas obtidas em nossas determinaçöes com as numerosissimas realizadas segundo apresenta o Manual de Bergey 9a. ediçäo, nos permite considerar a aplicabilidade deste esquema simples e rápido para diferenciar as espécies coagulasa negativa do gênero Staphyloccocus na maioria dos laboratórios de bacteriologia clínica