Latex agglutination and hemagglutination tests for the rapid identification of methicillin sensitive and methicillin resistant Staphylococcus aureus

Ten latex agglutination (LA) and hemagglutination (HA) kits for the identification of Staphylococcus aureus were compared with reference methods for their reliability and performance. The ten commercial kits consisted of Accu-Staph, Bacto-Staph, Hemastaph, Staphaurex, Staph-Latex, Staphylochrome, Staphyloslide, Staph-Rapid, Sero-Stat and Veri-Staph. The conventional methods included slide coagulase test, tube coagulase test (4 hr, 24 hr), thermonuclease and growth on mannitol salt agar (MSA). A total of 583 clinical isolates of staphylococci were used and all the kits correlated well with the conventional methods (93.1-99.4% sensitivity) in their ability to identify both methicillin sensitive (MSSA) and methicillin resistant S. aureus (MRSA). Although all were rapid, easy to perform and simple to interpret, Staphaurex and Staphyloslide gave the best sensitivities and specificities.     

The differentiation of Staphylococcus aureus from other Micrococcaceae isolated from bovine mammary glands

Haemolysin production, the slide coagulase test and the tube coagulase test were assessed for their capability to differentiate Staphylococcus aureus among other Micrococcaceae in 199 isolates from udders of cows in herds with a low bulk milk somatic cell count. The API-Staph test was used as a reference.
Haemolysin production was less effective in identifying Staph. aureus among Micrococcaceae than a combination of other tests. Differences were found in the predictive values of results from diagnostic protocols in which the slide coagulase test was performed on all Micrococcaceae, or on β-haemolysin-negative Micrococcaceae only. Diagnostic protocols in which haemolysin production was combined with the results of the other tests resulted in excellent diagnostic performance and a reduction in diagnostic procedures. Recommendations for routine Staph. aureus identification in bovine mastitis bacteriology are given.     

Occurrence and clonal relatedness of sec/tst-gene positive Staphylococcus aureus isolates of quartermilk samples of cows suffering from mastitis

AIMS: To investigate the prevalence of sec/tst-gene positive Staphylococcus aureus in bovine mastitis and to get information about the clonal relatedness of these clinical isolates. METHODS AND RESULTS: A total of 533 Staph. aureus strains isolated from bovine mastitic quartermilk samples at 493 randomized dairy farms in Hessia, Germany, from January 1997 until June 1998 were examined for enterotoxin C (sec) gene and toxic shock syndrome toxin (tst) gene by multiplex polymerase chain reaction. Fifty-three (9.3%) of the strains were sec/tst-gene positive. Phenotypic TSST-1 production was found in all positive strains by reversed passive latex agglutination test. With DNA macrorestriction analysis, sec/tst-gene positive strains were divided into five different macrorestriction types. Type I (10 isolates) and III (40 isolates) were found to be the predominant types in terms of frequency of isolation in the investigated area. These DNA macrorestriction types differed in only two bands in the 500 and 270 bp region. CONCLUSIONS: Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.     

Evaluation of Three Slide Agglutination Tests for Rapid Identification of Staphylococcus aureus

Three slide agglutination tests for identification of Staphylococcus aureus were compared. The agglutination tests used for evaluation were Staphaurex (Wellcome Diagnostics), Staphyslide-Test (BioMerieux), and ANI S. aureus TEST (Ani Biotech Oy). A total of 347 isolates were analyzed, including 288 strains of S. aureus, 49 of S. epidermis, 11 of S. intermedius, 12 strains of other staphylococci and 14 non-staphylococcal strains. One hundred of the S. aureus strains were isolates from cases of food poisoning, 129 from mastitis and 59 from other clinical cases. The sensitivities of the tests were also compared using diluted suspensions of S. aureus strains and with purified Protein A dilutions. The results showed that the sensitivities of the tests were 98.6 %, 97.9 % and 99.0 % for Staphaurex, Staphyslide-test and ANI S. aureus TEST, respectively. The specificities were 100 % for the Staphyslide test and 98.8 % for both the ANI S. aureus TEST and the Staphaurex test. The sensitivities measured with diluted S. aureus strain suspensions and Protein A solutions were equal with the Staphaurex and ANI S. aureus TEST. All the agglutination tests studied proved to be practical, easy to use and accurate for the rapid identification of S. aureus strains from culture isolates.     

Plasmid profiles of Staphylococcus aureus causing bovine mastitis

A. B aumgartner , J. N icolet & M. E ggimann 1984. Plasmid profiles of Staphylococcus aureus causing bovine mastitis. Journal of Applied Bacteriology 56 , 159–163.
Plasmid profiles of Staphylococcus aureus , isolated from herds affected with chronic bovine mastitis, are heterogeneous, as shown by the study of 85 strains from 18 different farms. The strains isolated within a herd sometimes show related plasmid patterns, although even strains isolated from different quarters of the same udder may show variations in their plasmid content. Strains without antibiotic resistance are frequently free of plasmids. Therefore, the existence of mastitis Staph. aureus virulence plasmids is unlikely. Staphylococcus aureus , resistant to penicillin and streptomycin from 9 different farms show related plasmid patterns. This result confirms a geographic spread of certain mastitis Staph. aureus strains.     

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.     

Helichrysum italicum extract interferes with the production of enterotoxins by Staphylococcus aureus

AIMS: The purpose of this work was to evaluate the effect of Helichrysum italicum extract on enterotoxin (A-D) production by Staphylococcus aureus strains. METHODS AND RESULTS: The production of enterotoxins A-D in the presence or absence of H.italicum diethyl ether extract was estimated in microtiter plates using a reversed passive latex agglutination (SET-RPLA) kit (Oxoid, Basingstoke, UK). The results indicate that, in culture medium, inhibition of staphylococcal growth and enterotoxins appeared with 250-125 microg ml(-1) of the extract. Lower concentrations of the extract (62.5-31.25 microg ml(-1)) did not affect the final viable count of Staph. aureus but reduced the production of enterotoxins B and C. CONCLUSIONS: H. italicum interferes with growth and production of enterotoxins by Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: There is considerable interest in the use of natural compounds as alternative methods to control undesirable pathogenic micro-organisms.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37 degrees C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Aureocin A70 production is disseminated amongst genetically unrelated Staphylococcus aureus involved in bovine mastitis

Aims: The main aim of this study was to analyse the genetic relationship amongst 46 Staphylococcus aureus Bac⁺ strains isolated in Brazil from 12 geographically distant dairy herds, including 34 isolates that produce the antimicrobial peptide aureocin A70. Methods and Results: The comparison of 46 Staph. aureus Bac⁺ strains was performed by pulsed‐field gel electrophoresis (PFGE). Thirteen different pulsotypes were identified, and the subtype A₁ was the most prevalent one. Nine strains belong to pulsotype F, the second most prevalent and mostly confined to a single herd. The PFGE patterns of the 34 Staph. aureus aureocin A70‐producers, isolated in Brazil, were also compared with those of strains isolated from bovine mastitis cases in Argentina and revealed that these strains are not genetically related. Conclusions: Although a previous study has suggested that a prevalent pulsotype of aureocin A70‐producer Staph. aureus involved in bovine mastitis is disseminated in Argentina, this does not occur in Brazil. Additionally, it was possible to demonstrate that closely related staphylococcal strains can produce distinct staphylococcins. Significance and Impact of the Study: This study corroborates the hypothesis of horizontal gene transfer of aureocin A70 genes amongst distinct staphylococcal strains involved in bovine mastitis, giving them a selective advantage when colonizing the mammary glands.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37°C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Application of current methods for isolation and identification of staphylococci in raw bovine milk

Samples of raw milk were examined for counts of somatic cells, total viable bacteria, staphylococci (Schleifer & Kramer's medium) and Staphylococcus aureus (Baird-Parker medium, Baird-Parker medium with pig plasma and Baird-Parker medium with additional antibiotics). For the isolation of staphylococci from raw milk, Schleifer & Kramer's medium was found to be very selective and in general performed satisfactorily. From the results obtained with the three remaining media the continued use of Baird-Parker medium for isolation of Staph. aureus from raw milk is recommended with the proviso that colonies selected for identification should include those that clear and do not clear the egg yolk and are not limited to colonies with diameters greater than 1 mm. Staphylococci isolated from raw milk were identified by key tests using a multipoint inoculation procedure. A selected number were also examined by the API STAPH system in conjunction with the API LAB computer programme for identification of staphylococci. Of the staphylococci examined, 90.0% were identified using the multipoint procedure. For strains identified as Staph. aureus, Staph. hyicus subsp. hyicus, Staph. epidermidis, Staph. simulans, Staph. xylosus or members of the Staph. hominis/Staph. warneri/Staph haemolyticus group, the API system provided confirmatory evidence. With strains identified by the multipoint procedure as Staph. hyicus subsp. chromogenes, Staph. sciuri subsp. sciuri and Staph. sciuri subsp. lentus the API system did not always provide concurring results. Several strains which could not be identified by the multipoint procedure could be identified by the API system. Staph. aureus, Staph. hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains isolated from milk were examined for production of enterotoxin A-E. Only 3.9% of Staph. aureus strains examined produced detectable enterotoxin (type C). None of the Staph. hyicus subsp. hyicus or Staph. hyicus subsp. chromogenes strains produced any of the known enterotoxins.     

Peptostreptococcus micros coaggregates with Fusobacterium nucleatum and non-encapsulated Porphyromonas gingivalis

Numerous in vitro studies have demonstrated that Staphylococcus aureus may be internalized and survive in a bovine mammary epithelial cell line. We report here the presence of internalized and living S. aureus in alveolar cells and macrophages in milk samples of bovine mastitis. We used fluorochrome labeled monoclonal antibodies, specifically recognizing surface cell markers of bovine alveolar cells and macrophages, to isolate these two types of cells using fluorescence activated cell sorting. Extracellular bacteria and DNA were previously eliminated to exclude possible contamination. In order to detect intracellular bacterial DNA inside the isolated cells, we used PCR amplification of bacterial DNA and the PCR products were analyzed by Southern blot with a specific probe for Staphylococcus. The results showed the presence of Staphylococcus DNA inside the two isolated populations of cells, confirming that S. aureus could penetrate alveolar cells and macrophages. The demonstration of the presence of intracellular living S. aureus was determined by bacteriological culture of positive samples plated onto blood agar plates and by its further identification. Our results showed for the first time that living S. aureus and its DNA are present in both alveolar cells and macrophages in chronically infected cow milk.     

VITEK-2 compact全自动微生物鉴定仪对葡萄球菌鉴定能力的评价

目的评价VITEK-2 compact全自动微生物鉴定仪对葡萄球菌的鉴定能力。方法收集从我院病人标本中分离的葡萄球菌81株。常规细菌培养后,用VITEK-2 compact和API Staph系统进行检测,以API Staph系统为参照,评价VITEK-2 compact的优势和不足。结果 VITEK-2 compact和API Staph系统的总体鉴定符合率为95.1%,其中金黄色葡萄球菌的鉴定符合率为100%,凝固酶阴性葡萄球菌的鉴定符合率为90.5%。结论 VITEK-2 compact鉴定系统能够满足临床工作的需求,其中对金黄色葡萄球菌的鉴定率较高,在进行凝固酶阴性葡萄球菌鉴定时有一定的局限性,需要其他方法予以补充。     

Lack of Staphylococcal Enterotoxin Production among Strains of Staphylococcus aureus from Bovine Mastitis in Denmark

Staphylococcal enterotoxins (SE’s) are a group of small exoproteins produced by some strains of Staphylococcus aureus. The SE’s, designated A to E according to their antigenic specificities, are important causes of food poisoning worldwide. Milk and dairy products are frequently associated with S. aureus enter-otoxin food poisoning, and it is supposed that infected milk from mastitic animals constitute the main source of enterotoxigenic S. aureus of animal origin (Bryon 1983, Gilmour & Harvey 1990, Bergdoll 1989). Indeed, S. aureus is the most common cause of bovine mastitis worldwide, and if mastitis strains produce SE this makes up an enormous reservoir of potential enterotoxin producers. The production of SE by S. aureus isolated from bovine mastitis have been investigated in several countries (Matsunaga et al. 1993, Kenny et al. 1993, Olson et al 1970, Orden et al. 1992, Olsvik et al. 1981, Adekeye 1980, Garcia et al. 1980, Abbar 1986, Harvey & Gilmour 1985). Since no studies have been performed on the prevalence of enterotoxigenic strains of S. aureus isolated from bovine mastitis in Denmark, a well characterized collection of S. aureus (Aarestrup et al. 1995) was investigated with respect to this property.     

Evaluation of a rapid culture-based screening test for detection of methicillin resistant Staphylococcus aureus

The performance of a culture based assay, BacLite Rapid MRSA for the rapid detection (5 hours) of methicillin resistant Staphylococcus aureus (MRSA) from specimens (n = 377) obtained from nares, throat, wounds and perineum was investigated. Compared to culture based reference methods (chromogenic MRSA ID (bioMerieux)), selective enrichment broth, PBP2' latex agglutination (Oxoid) and VITEK 2 identification (bioMerieux), an overall sensitivity of 71% with a 82% specificity and a negative predictive value (NPV) of 95% was provided. The Baclite test is rapid and easy to use and has the advantage of a culture-based detection method for MRSA.     

Potential problems in the use of oligonucleotide probes for staphylococcal enterotoxin genes

Oligonucleotide probes unique to the five major enterotoxin genes of Staphylococcus aureus were synthesized and used to detect DNA sequences homologous to these genes in 27 non-clinical isolates of Staph. aureus isolated from nasal swabs of 74 healthy human volunteers. Genomic DNA from all 27 isolates reacted with at least one of the probes. In a phenotypic assay for toxin production by a reverse passive latex agglutination test however, only 15 of the 27 isolates produced enterotoxin in culture. The results raise the possibility that a number of Staph. aureus isolates harbour DNA sequences that are apparently silent or mutant copies of the enterotoxin genes. This complicates the identification of enterotoxin producers by tests which depend on oligonucleotide or DNA hybridization.     

The pathogenesis and control of Staphylococcus aureus-induced mastitis: study models in the mouse

The intramammary colonisation by Staphylococcus aureus provokes mastitis in the cow. Once established, the infection is difficult to eradicate with available therapies and may become chronic. The present article focuses on the use of the experimental mouse model of S. aureus-induced mastitis as a practical approach for the study of bovine mastitis. Results obtained regarding the pathogenesis of S. aureus and the development of new therapeutic approaches are discussed.     

Enterotoxigenic Staphylococcus aureus in bulk milk in Norway

AIMS: To investigate the presence of enterotoxigenic Staphylococcus aureus in bulk milk and in a selection of raw milk products. METHODS AND RESULTS: Samples of bovine (n = 220) and caprine (n = 213) bulk milk, and raw milk products (n = 82) were analysed for S. aureus. Isolates were tested for staphylococcal enterotoxin (SE) production (SEA-SED) by reversed passive latex agglutination and for SE genes (sea-see, seg-sej) by multiplex PCR. Staphylococcus aureus was detected in 165 (75%) bovine and 205 (96.2%) caprine bulk milk samples and in 31 (37.8%) raw milk product samples. Enterotoxin production was observed in 22.1% and 57.3% of S. aureus isolates from bovine and caprine bulk milk, respectively, while SE genes were detected in 52.5% of the bovine and 55.8% of the caprine bulk milk isolates. SEC and sec were most commonly detected. A greater diversity of SE genes were observed in bovine vs caprine isolates. CONCLUSIONS: Staphylococcus aureus seems highly prevalent in Norwegian bulk milk and isolates frequently produce SEs and contain SE genes. Enterotoxigenic S. aureus were also found in raw milk products. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus in Norwegian bovine and caprine bulk milk may constitute a risk with respect to staphylococcal food poisoning from raw milk products.     

Production of enterotoxins and toxic shock syndrome toxin by Staphylococcus aureus isolated from bovine mastitis in Brazil

The production of toxic shock syndrome toxin 1 (TSST-1) and enterotoxins (SE) A, B, C and D by bovine mastitis isolates of Staphylococcus aureus was evaluated by immunodiffusion using the Optimum-Sensitivity Plate method. S. aureus strains were isolated from bovine mastitis in 23 dairy herds in the state of Minas Gerais, Brazil, during 1994-9. Of 127 isolates, 83 (65.04%) produced one or several toxins, and among them production of SE was found in 54 (43.0%) isolates, of which 1138 (29.09%) secreted enterotoxin identified as type D. TSST-1 was found in 5829 (45.723.0%) isolates.