Typing of methicillin-resistant Staphylococcus aureus with IS256

A simple one-step procedure is described for specifically amplifying and labelling insertion element IS256 which is associated with the gentamicin-resistance transposon Tn4001. The product has been used to probe DNA digests of methicillin-resistant Staphylococcus aureus. The resulting restriction fragment length polymorphisms were found to be able to distinguish isolates which were indistinguishable by other typing methods. The probe also hybridised with methicillin-resistant Staphylococcus aureus which were isolated before the emergence of gentamicin resistance, demonstrating its usefulness in typing species other than those that are gentamicin-resistant.     

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.     

金黄色葡萄球菌的耐药性分析及基因分型研究

目的通过分析上海地区院内分离金黄色葡萄球菌的药敏谱型及对耐甲氧西林的金黄色葡萄球菌（MRSA）进行基因谱型的研究,了解金黄色葡萄球菌的院内流行状况。方法对临床分离出的43株金黄色葡萄球菌进行药敏试验和SCCmec基因盒的多重PCR检测,并将结果整合后用MEGA3．1软件分析其进化相关关系。结果药敏结果显示43株金葡菌对青霉素和甲氧西林的耐药率最高。甲氧西林的耐药率达到62．8％。MecA阳性菌株SCCmec的分型显示均为Ⅱ型或Ⅲ型,且所占比例相近,未见Ⅰ型和Ⅳ型。进化树分析发现了在同一医院中亲缘关系相近的菌株,为院内感染流行株。结论MecA基因介导的MRSA在分离菌株中所占比例高,存在院内感染爆发性流行。     

Typing Staphylococcus aureus using the spa gene and novel distance measures

We developed an approach for identifying groups or families of Staphylococcus aureus bacteria based on genotype data. With the emergence of drug resistant strains, S. aureus represents a significant human health threat. Identifying the family types efficiently and quickly is crucial in community settings. Here, we develop a hybrid sequence algorithm approach to type this bacterium using only its spa gene. Two of the sequence algorithms we used are well established, while the third, the Best Common Gap-Weighted Sequence (BCGS), is novel. We combined the sequence algorithms with a weighted match/mismatch algorithm for the spa sequence ends. Normalized similarity scores and distances between the sequences were derived and used within unsupervised clustering methods. The resulting spa groupings correlated strongly with the groups defined by the well-established Multi locus sequence typing (MLST) method. Spa typing is preferable to MLST typing which types seven genes instead of just one. Furthermore, our spa clustering methods can be fine-tuned to be more discriminative than MLST, identifying new strains that the MLST method may not. Finally, we performed a multidimensional scaling of our distance matrices to visualize the relationship between isolates. The proposed methodology provides a promising new approach to molecular epidemiology.     

Determination of Genome Size of Selected Typing Bacteriophages of Staphylococcus aureus

The molecular weights of six representative typing bacteriophages were determined by sucrose gradient centrifugation. Genome size appeared to be related to the size of the phage head.     

Oxygen-dependent killing of Staphylococcus aureus by human neutrophils

Luminol-dependent chemiluminescence was used as a monitor of reactive oxidant generation during phagocytosis of Staphylococcus aureus by human neutrophils. Reactive oxidants play a crucial role in the killing of this organism because: (a) S. aureus was killed most rapidly when the rate of increase of chemiluminescence was greatest; (b) neutrophils which had been activated to generate reactive oxidants by re-aeration of anaerobic suspensions killed this bacterium more efficiently than control suspensions; and (c) neutrophils from a patient with chronic granulomatous disease could neither generate reactive oxidants nor kill S. aureus.     

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.     

Comparison of Capsular Typing of Staphylococcus aureus with Bacteriophage Typing: A Study in Gulbarga, India

Staphylococcus aureus (S. aureus) is important both as a nosocomial and community acquired pathogen causing various degrees of infections. Typing S. aureus has been a question that is still being addressed. Bacteriophage typing is still used as a golden standard for typing though molecular methods are investigated. In developing countries where neither molecular typing nor the bacteriophage typing methods can be routinely used, the recently developed capsular typing method can be considered as screening method. We compared capsular typing with bacteriophage typing of the strains isolated in Gulbarga, India. We observed that the typeability of capsular typing was significantly higher (96%) among the phage typed strains, and the predominant capsular type in the region was type-8. The data so generated can be used to group S. aureus based on capsules both as a screening prior to bacteriophage typing, and to identify capsular candidate for developing prophylactic and therapeutic alternatives.     

Base Ratio and Deoxyribonucleic Acid Homology Studies of Six Staphylococcus aureus Typing Bacteriophages

Genetic relatedness among Staphylococcus aureus typing bacteriophages 80, 47, 81, 71, 77, and 187 was investigated by using base ratio determinations and deoxyribonucleic acid (DNA)-DNA hybridization. Guanine/cytosine (G/C) content, as determined by thermal denaturation and chromatographic analysis of the purines released by acid hydrolysis of the DNA, was between 31 and 36%. No pattern correlating G/C content with serological or lytic group was discernible. DNA-DNA hybridization studies indicated high degrees of homology (43% or more) among the genomes of phages in the same serological group. Less homology (29% or less) was observed between the genomes of phages belonging to different serological groups. These findings implied a positive correlation between serological and genetic relatedness.     

A Cooling Modification to an Apparatus for Phage Typing of Staphylococcus aureus

Difficulty is experienced through overheating of the Tarr apparatus for phage typing. The provision of a cooling coil which overcomes this difficulty is described.     

Biotyping of Enterotoxigenic Staphylococcus aureus by Enterotoxin Gene Cluster (egc) Polymorphism and spa Typing Analyses

Thirty-five Staphylococcus aureus strains, including 10 reference strains and 25 strains recovered from clinical specimens and food samples, were analyzed by PCR REA (restriction endonucleases analysis) of the egc operon and spa typing. Nineteen spa types and seven different egc operons, including four putative new egc variants, were revealed. In 13 strains, allelic variants of sei and/or seg were found. By an analysis of their nucleotide sequence identities, a new homogeneous cluster of a sei variant, called the sei variant, was detected in six strains. In addition, the prototype sei was shown to be more polymorphic than assumed so far. Seven strains possessed the recently described seg variant, also exhibiting several nucleotide exchanges. spa typing was more effective than REA egc grouping as a typing technique. Since, in some cases, the REA typing method was able to discriminate strains showing the same spa type, it must be considered for PCR approaches involved in diagnostic procedures and may be useful for epidemiological studies. Hence, the polyphasic approach used in this study can be reliably and advantageously applied for typing egc-positive S. aureus strains.     

Development of a Single-Reaction Multiplex PCR Toxin Typing Assay for Staphylococcus aureus Strains

We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.     

Methicillin-resistant Staphylococcus aureus in human milk

We collected and analyzed 500 samples of human milk, from five Brazilian cities (100 from each) to detect methicillin-resistant strains of Staphylococcus aureus (MRSA) producing enterotoxins. We found 57 strains of MRSA, and the mecA gene, responsible for resistance, was detected in all of them using a specific molecular probe. We examined 40 strains for the presence of four enterotoxins, after selecting a subset that included all strains from each region, except for the largest sample, from which 10 were randomly selected. Among these two presented enterotoxin B, and growth in human colostrum and trypicase soy broth. After 5 h of incubation at 37 degrees C, population sizes were already higher than 9.4 x 10(5) UFC/ml and enterotoxin was released into culture medium and colostrum. Our results stress the importance of hygiene, sanitary measures, and appropriate preservation conditions to avoid the proliferation of S. aureus in human milk.     

Typing of Staphylococcus aureus by amplification of the 16S–23S rRNA intergenic spacer sequences

Abstract An acid phosphatase containing a 27-kDa polypeptide component has been identified in Escherichia coli by means of a zymogram technique. The enzyme is secreted in the periplasmic space and is able to hydrolyze several organic phosphate esters, but not diesters, showing preferential activity on p -nitrophenyl phosphate and other phenolic phosphate esters. Production of the enzyme apparently occurs only in cells growing on carbon sources other than glucose.     

Adhesive proteins of haemagglutinating Staphylococcus aureus isolated from bovine mastitis

Two proteins derived from the cell wall of Staphylococcus aureus, exhibiting apparent molecular masses of 116 kDa and 145 kDa, were found to bind to human buccal and bovine lactiferous sinus epithelial cells. By using antibodies specific for fibronectin-binding protein of S. aureus of human origin, the 116 kDa protein, but not the 145 kDa protein, was identified as a fibronectin-binding protein. The 145 kDa protein bound to bovine fat globule membranes, human buccal epithelial cells, bovine lactiferous sinus epithelial cells and sheep erythrocytes. The properties of the 145 kDa protein suggest that it is an adhesin with a possible role in the early stages of the development of bovine mastitis.     

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.     

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.     

Gamma interferon enhances the killing of Staphylococcus aureus by human neutrophils

The effect of purified human interferon-gamma on the responsiveness of human neutrophils was investigated. Pre-incubation of neutrophils with 100 U interferon ml-1 for 10 min at 37 degrees C resulted in a 2.5-fold increase in N-formylmethionyl-leucyl-phenylalanine-stimulated reactive oxygen metabolite generation (as assayed by luminol-dependent chemiluminescence). Pre-treatment of neutrophils with interferon also potentiated their ability to kill Staphylococcus aureus, and thus it is proposed that this lymphokine may also enhance neutrophil function in vivo under certain pathological conditions.     

Carotenoid Formation by Staphylococcus aureus

The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; zeta-carotene; delta-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-(14)C added to the culture was incorporated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The incorporation and turnover of (14)C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids.