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.     

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis

AIMS: Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. METHODS AND RESULTS: Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. CONCLUSIONS: The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.     

Genotyping of Staphylococcus aureus isolated from humans, bovine subclinical mastitis and food samples in Argentina

The aim of the present study was to characterize genotypically 45 Staphylococcus aureus strains isolated from humans, bovine subclinical mastitis and food samples in Argentina by rep-PCR and PCR amplification of virulence genes. Resistances to various antibiotics could be observed for the human S. aureus, less pronounced for the bovine strains, but not for the eight S. aureus isolated from food samples. The strains could be classified genotypically by rep-PCR and by amplification of the genes encoding protein A, coagulase, clumping factor, the collagen adhesin domains A and B, capsular polysaccharide 5 and 8, the accessory gene regulator agr classes I to III, and the S. aureus gene regulator sae. rep-PCR analyses and the different gene patterns revealed that the strains could be divided into seven groups mostly matching with the origin of the isolates. The present study describes genotypic variations of S. aureus strains isolated from different origins in Argentina. The study provides a valuable insight into molecular specificities of this important pathogen.     

Characterization of Staphylococcus aureus strains isolated from raw milk of bovine subclinical mastitis in Tehran and Mashhad

Staphylococcus aureus is considered one of the most important food borne pathogens. A total of 111 isolates of S. aureus were cultured from raw milk samples during January 2009 to June 2009 from Tehran and Mashhad. The coagulase gene polymorphism and the prevalence of classical enterotoxin genes of S. aureus strains were determined by PCR-RFLP (restriction fragment length polymorphism) and Multiplex-PCR. Disk diffusion method was used to determine the susceptibility of isolates to antimicrobial agents as instructed by Clinical and Laboratory Standards Institute. Sixty-seven % of the isolates harboured one or more enterotoxin genes. The most prevalent gene was sec, found in 59 % of the isolates. Approximately 8% of the isolates were positive for sea, seb and sed genes. Only one isolate had see gene. The rate of coexistence of enterotoxin genes was 14%. All S. aureus isolates were susceptible to ciprofloxacin, gentamicin, imipenem, minocycline, oxacillin and vancomycin. They were resistant to ampicillin (64%), penicillin (56%), clindamycin (22%), tetracycline (22%), doxycycline (19%), teicoplanin (13%), rifampin (2%) and trimethoprim-sulfamethoxazole (2%). On the basis of coagulase gene analysis of 111 S. aureus isolates, the PCR products of 56 isolates were digested with Alu I that produced three distinct patterns. These data indicate the high prevalence of enterotoxigenic S. aureus in raw bovine milk in Tehran and Mashhad, and highlight the importance of proper quality control of dairy products for public health.     

The persistence of biofilm-associated antibiotic resistance of Staphylococcus aureus isolated from clinical bovine mastitis cases in Australia

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3–4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.     

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.     

Effect of milk on surface properties of Staphylococcus aureus from bovine mastitis

Abstract The surface hydrophobicity of cells of Staphylococcus aureus strains isolated from bovine mastitis grown on conventional agar and broth media was drastically reduced after incubation with bovine milk. Strains grown in high carbohydrate-high salt media yielded cells with reduced surface hydrophobicity compared to cells grown in conventional media, and adding bovine milk to minimal medium also yielded cells with reduced surface hydrophobicity, as determined by hydrophobic interaction chromatography and the salt aggregation test. Incubation of strains in milk and growth in a medium supplemented with bovine milk also significantly changed bacterial surface charge as determined by free-zone electrophoresis. Strains with high or with decreased adsorptive and aggregating properties did not produce surface capsule or slime. Heat treatment (60° C or 80° C) of the bacterial suspensions did not significantly change their adsorptive and aggregating properties.     

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller–Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline’s activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.     

Staphylococcus aureus strains isolated from bovine mastitis: virulence,antibody production and protection from challenge in a mouse model

Septic arthritis in mice was used as a model to evaluate the virulence of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from cases of bovine mastitis. In addition, the model was used to evaluate the cross protection elicited by heterologous antibodies. Mice were intramuscularly inoculated with serial bacterial doses of different strains of S. aureus or CNS, for virulence determination; they were monitored for arthritis, gangrene or death up to 20 days. Antibody response, cross reactivity and resistance to challenge were tested by subcutaneous inoculation with a low dose of one of the S. aureus or CNS strains followed by challenge with two S. aureus strains. S. aureus alpha-hemolysin isolate was the most virulent, followed by alpha+beta-hemolysin and beta-hemolysin isolates. The least virulent isolates were the non-hemolytic S. aureus strains but even they were more virulent than the CNS strains tested. Antibodies against three different S. aureus antigens were detected by the ELISA in all mice that were inoculated with the S. aureus strains but not in any of those with the CNS strains. Immunoblot test against various S. aureus strains as antigens showed high cross-reactivity among the S. aureus strains but only a slight similarity, restricted to the bands above 36 kDa, with the CNS sera. Low-dose inoculation of alpha or alpha+beta strains before challenge with homologous and heterologous strains protected the mice, whereas the two beta strains provided only partial protection. The inoculations of non-hemolytic S. aureus or the CNS strains did not elicit any protection. Our findings demonstrate that pre-exposure of mice to a low dose of certain S. aureus strains could provide protection and that the antibodies produced could have an important protective role.     

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.     

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.     

Characterization of Staphylococcus aureus strains involved in human and bovine mastitis

Staphylococcus aureus is one of the main etiological agents of mastitis in different mammalian species. At present, it is unknown whether strains isolated from human mastitis cases share phenotypic properties and genetic background with those obtained from animal mastitis cases. Therefore, the objective of this study was to characterize S. aureus strains isolated from women with lactational mastitis and to compare them with the strains responsible for bovine mastitis and noninfectious strains. All the strains were genotyped by both pulsed field gel electrophoresis and multilocus sequence typing and submitted to a characterization scheme that included diverse assays related to pathogenic potential and antibiotic resistance. Apart from siderophore production, no significant association was observed between the strains from bovine and human mastitis. Statistical differences between human- and bovine-mastitis-associated strains were detected for some traits and virulence determinants, such as the presence of prophages and cna and hlb genes, which were more frequently found within the bovine group. On the contrary, resistance to penicillin was significantly higher among strains isolated from human lactational mastitis, probably related to the common presence of the blaZ gene. A high genetic diversity was found among the strains involved in mastitis in breastfeeding women.     

Coagulase gene polymorphism of Staphylococcus aureus isolated from goat mastitis in Brazilian dairy herds

AIMS: To investigate the coagulase gene polymorphism of Staphylococcus aureus isolated from mastitic goat's milk. METHODS AND RESULTS: A typing procedure based on coagulase gene polymorphism was used to discriminate S. aureus isolated from goat mastitis. Thirty-six strains collected from goats belonging to herds from northern Ceara State and Serrana region of Rio de Janeiro State were analysed. Based on restriction fragment length polymorphism of 3' end coagulase gene, the goat strains were grouped into 11 types. In northern Ceara herds, the predominant type was found in 60% of the strains, while in the Serrana region herds the two most common accounting for 62.5% of the strains. CONCLUSIONS: The analysis of the coa gene proved to be useful for typing S. aureus from goat mastitis. Although the results showed that goats from the studied regions were infected by S. aureus strains harbouring more than one coagulase genotype, only one or two types predominated. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of the prevalent strains within a herd or region is a necessity. The important virulence factors could be identified in such strains and this information can then be used as a specific base to develop S. aureus mastitis control measures.     

牛源金黄色葡萄球菌粘附因子Efb与ClfA的免疫生物学特性比较

为评价与比较金黄色葡萄球菌的粘附因子Efb(Extracellular fibrinogen-binding protein)和Clf A(Clumping factor A)的抗原性、粘附特性及其抗血清对金黄色葡萄球菌菌体的识别能力、抗粘附能力及免疫保护力等免疫生物学特性,分别构建Efb和Clf A的重组表达载体,并用纯化的重组蛋白免疫实验动物,分别检测家兔抗血清对菌体的识别能力、抗粘附能力以及小鼠抗血清的抗体效价和免疫保护作用。结果显示,Efb和Clf A均有与纤维蛋白原(Fibrinogen,Fg)结合的能力,且Efb蛋白对纤连蛋白(Fibronectin,Fn)的结合能力优于Clf A(P0.01),Clf A免疫组抗血清对全细菌的识别能力优于Efb免疫组(P0.01)。与对照组相比Efb和Clf A免疫组的抗血清均能显著抑制金黄色葡萄球菌对Fg和Fn的粘附(P0.01),Efb免疫组对金黄色葡萄球菌的粘附抑制优于Clf A,两种粘附因子免疫小鼠后产生的血清抗体效价与对照组比较有显著的升高,抗体滴度可达1∶40 500,攻毒结果显示Efb与Clf A免疫组均对小鼠具有良好的保护效果。该研究结果对Efb在金黄色葡萄球菌的亚单位疫苗的应用奠定了基础。     

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.     

Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov, SEL and TSST-1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.     

RAPD-PCR analysis of Staphylococcus aureus strains isolated from bovine and human hosts

Staphylococcus aureus is one of the most important pathogens in humans and animals. In this study eighty strains were analyzed by RAPD-PCR to assess the genetic relationship between S. aureus isolates from bovine and human hosts. Results were compared with those obtained by biotyping. Fifty-two percent of the S. aureus isolates belonged to a host specific biotype (human, bovine and poultry). Bovine and human ecovars were the most prevalent. Dendrogram obtained by RAPD results showed that all the isolates clustered into eleven groups (A-K) at a relative genetic similarity of less than 30% when analyzed with the three primers. Group A clustered 95% of the human host isolates and the remaining groups (B-K) clustered the bovine host isolates. Principal coordinate analysis also showed that the isolates could be arbitrarily divided into two groups, bovine and human, by the second coordinate. Only 9 isolates (11%) were not clustered into these groups. The genetic diversity among the S. aureus isolates from bovine hosts is relatively low compared to that of isolates from human hosts. There were no statistically significant differences among isolated from bovine and human hosts. This study shows that RAPD-PCR assayed with three primers can be successfully applied to assess the genetic relationship of S. aureus isolates from different hosts.