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PubMed2002

Molecular epidemiology and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus in Taiwan.

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen and has increased frequency in the past 20 years. It is highly transmissible between wards, hospitals, and cities. In most major hospitals in Taiwan, MRSA accounts for more than 60% of the S. aureus isolates. Whether there is a predominant strain, which is spread over the whole of Taiwan has not yet been studied. We collected 208 sequential clinical isolates of MRSA from 22 hospitals: seven in northern Taiwan, seven in western Taiwan, five in southern Taiwan, and three in eastern Taiwan during a three-month period in 1998. Using pulsed-field gel electrophoresis, 112 MRSA isolates were shown to belong to one major type,--type C, and this type was shown to have spread widely across all of Taiwan. Ninety-six isolates belonged to 20 other minor types. Most MRSA isolates of this major type were multi-drug resistant and only susceptible to vancomycin and rifampin. We concluded that the high prevalence of MRSA in Taiwan was partly due to the spreading of a predominant strain and most of them were multi-drug resistant. This might imply that more effort should be made to control the spread of MRSA in Taiwan.

Bacterial Typing Techniques↗
PubMed2005

Methicillin-resistant Staphylococcus aureus, Western Australia.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a notable cause of hospital-acquired infections. A statewide screening and control policy was implemented in Western Australia (WA) after an outbreak of epidemic MRSA in a Perth hospital in 1982. We report on statutory notifications from 1998 to 2002 and review the 20-year period from 1983 to 2002. The rate of reporting of community-associated Western Australia MRSA (WAMRSA) escalated from 1998 to 2002 but may have peaked in 2001. Several outbreaks were halted, but they resulted in an increase in reports as a result of screening. A notable increase in ciprofloxacin resistance during the study period was observed as a result of more United Kingdom epidemic MRSA (EMRSA) -15 and -16. WA has seen a persistently low incidence of multidrug-resistant MRSA because of the screening and decolonization program. Non-multidrug-resistant, community-associated WAMRSA strains have not established in WA hospitals.

Adult↗
PubMed1997

Comparison of methods for the isolation of methicillin resistant Staphylococcus aureus.

The control of methicillin resistant Staphylococcus aureus (MRSA) relies on the rapid and sensitive detection of carriage. The roles of an enrichment broth, duration of incubation, and Baird-Parker medium containing ciprofloxacin (BPC) were evaluated in comparison with standard media in a centre where the prevalence of ciprofloxacin resistance among MRSA is over 98%. Screening swabs from 402 sites were plated onto BPC, mannitol salt agar (MSA), and MSA with methicillin (MMSA). The swabs were enriched in Tryptone-T broth with 6% salt for 24 hours and the broths subcultured onto BPC, MSA, and MMSA. MRSA was isolated from 134 swabs. Significantly more isolates were obtained by incubating culture plates for 42 hours rather than 18 hours, by the use of broth enrichment, and by addition of methicillin or ciprofloxacin to media. BPC was the most sensitive medium (107 isolates (80%) by direct culture at 42 hours), grew the fewest contaminants, and allowed provisional reporting of 73% of isolates at 18 hours by colonial appearance and use of Staphaurex Plus rapid latex reagent. This may allow the introduction of infection control measures a day earlier than when other established methods are used.

Bacteriological Techniques↗
PubMed1989

Protection of mice by a pseudodiffuse strain of Staphylococcus aureus possessing polyvalent capsular type antigen.

Staphylococcus aureus strain MC31 showed pseudodiffuse growth in serum-soft agar and reacted with immune rabbit sera to strains Smith diffuse (capsular type A), NS58D (type B) and NS41D (type C) but not with strain NS68D (type D) in serum-soft agar. Immunisation of mice with 300 micrograms of cell-surface polysaccharide extracted from strain MC31 protected against lethal infection by strain MC31 and the strains of capsular types A, B and C. Immune rabbit serum prepared against strain MC31 passively protected mice against challenge infection with the homologous strain, but approximately 30 times more anti-MC31 serum was required to protect against infection with the strains of capsular types A, B and C. Absorption of the passive protective activity of immune sera raised against the three capsular type strains required at least 10 times the quantity of MC31 cell-surface polysaccharide than the quantity of cell-surface polysaccharide from the homologous capsular strain. Electronmicrographs of strain MC31 treated with ferritin-labelled antisera to the three capsular strains showed only small amounts of ferritin granules around the cell wall.

Animals↗
PubMed1988

Oxacillin-induced inhibition of protein and RNA synthesis in a tolerant Staphylococcus aureus isolate.

A clinical isolate of Staphylococcus aureus was found to be tolerant (MBC much greater than MIC) to a number of beta-lactam antibiotics, including oxacillin. Biophotometric analysis showed that a number of concentrations of oxacillin were capable of stimulating rapid cellular lysis in this organism, but the extent of lysis was antibiotic concentration dependent and limited. Cell cultures treated with an antibiotic concentration yielding the maximum rate and extent of lysis were analyzed for protein and RNA synthesis by pulse-labeling techniques. RNA synthesis was initially stimulated and then severely inhibited. Protein synthesis was not inhibited initially; however, the increase in the rate of synthesis expected as the result of logarithmic growth was not observed. Instead, the antibiotic-treated culture maintained for approximately 50 min the rate of protein synthesis ongoing at the time of antibiotic addition. The rate of protein synthesis declined thereafter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein samples taken 1 and 3 h after antibiotic addition showed that the shutdown of protein synthesis was not coordinate but rather was suggestive of the operation of a stress regulon perhaps similar to those responsible for heat shock, SOS, and oxidation stress.

Bacterial Proteins↗
PubMed1998

The effect of culture conditions on the in-vitro adherence of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant isolates of Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behaviour into two subgroups, epidemic MRSA (EMRSA) and sporadic MRSA (SMRSA) strains. An existing adherence assay was modified to determine differences in adherence properties between these two groups of MRSA, and the influence of culture conditions on the adherence of SMRSA and EMRSA strains to plastic, human collagen I (HuCol I) and pharyngeal carcinoma Detroit 562 cells (D562) was determined. In-vitro parameters, such as culture medium, growth temperature and growth phase of the bacterium, influenced the adherence of MRSA strains to plastic significantly. Even more pronounced differences in adherence due to changes in growth conditions and growth phase of the bacteria were found for the adherence of MRSA strains to HuCol I. Growth phase had a significant effect on the adherence of MRSA strains to the pharyngeal carcinoma cells D562. However, the study did not find conditions which made it possible to distinguish EMRSA from SMRSA strains. These data show that extrapolation of in-vitro data concerning adherence of MRSA strains to in-vivo conditions should be treated with caution.

Bacterial Adhesion↗
PubMed2004

Origins of community strains of methicillin-resistant Staphylococcus aureus.

To characterize methicillin-resistant Staphylococcus aureus (MRSA) strains circulating in the community, we identified predictors of isolating community MRSA and genotyped a sample of MRSA collected from a community-based, high-risk population. Computerized databases of the Community Health Network of San Francisco and the Clinical Microbiology Laboratory were searched electronically for the years 1992-1999 to identify community-onset infections caused by MRSA. Sequential analyses were performed to identify predictors of MRSA strains. The majority (58%) of infections were caused by strains traceable to the hospital or to long-term care facilities. Injection drug use was associated with infections that were not associated with health care settings. Genotypes for 20 of 35 MRSA isolates recovered from injection drug users did not match any of >600 genotypes of clinical isolates. In a nonoutbreak setting, the hospital was the main source of community MRSA; however, the presence of genetically distinct and diverse MRSA strains indicates MRSA strains now also originate from the community.

Anti-Bacterial Agents↗
PubMed1992

Effect of specific antibody on adherence of Staphylococcus aureus to bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen in the bovine mammary gland. The ability of S. aureus to adhere to epithelial cells in the ductules and alveoli of the bovine mammary gland is believed to add greatly to its virulence and may be necessary for colonization. Two in vitro methods were developed for the purposes of quantifying adherence and of determining the effect which specific antibody may have in inhibiting the adherence of this organism. Both methods utilize bovine mammary epithelial primary cells as targets for labeled bacteria. In one assay, the bacteria are labeled with [methyl-3H]thymidine and incubated on the primary epithelial monolayers. The second assay involves labeling the bacteria with biotin. An enzyme-linked immunosorbent assay is then performed with streptavidin conjugated to horseradish peroxidase. Both methods have proven to be reliable and allow for the testing of many criteria in one assay. Cows were immunized with a whole-cell vaccine, and immune serum and milk were collected. The bacteria were then incubated in the presence of serum or milk as a test for antiadherent capability. By using the methods described, distinct antiadherent activity in both serum and milk was demonstrated.

Animals↗
PubMed1983

Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus.

pT181 is a naturally occurring Staphylococcus aureus plasmid, encoding inducible resistance to tetracycline. The plasmid has a copy number of about 20 per cell, and belongs to the incompatibility group inc3. The complete nucleotide sequence of pT181 has been determined and consists of 4437 bp. The nucleotide sequence contains 69.8% A-T and 30.2% G-C pairs. pT181 was found to contain four open reading frames capable of coding for polypeptides containing more than 50 amino acids. All the putative polypeptides are coded by one strand. The molecular weights of the four putative polypeptides are (in daltons): A, 37,500; B, 35,000; C, 23,000, and D, 18,000. Polypeptide A corresponds to the repC protein, earlier shown to be specifically required for pT181 replication. Polypeptide B (and possibly polypeptide D) are involved in tetracycline resistance. No role has yet been established for polypeptide C; deletion of the coding sequence for the C polypeptide has no detectable effect on any property of the pT181 plasmid. A region consisting of about 1200 bp contains information for the replication and copy number control of this plasmid. The sequencing results are discussed in relation to the replication properties and tetracycline resistance associated with the pT181 plasmid.

Base Sequence↗
PubMed1997

In vitro assay of Staphylococcus aureus enterotoxin A activity in food.

Staphylococcus aureus enterotoxin A (SEA) is a leading cause of food poisoning. The current test for functional activity of SEA requires monkeys or kittens. The major drawbacks of animal assays are lack of quantitation, poor reproducibility, low sensitivity, and high cost. In this report we describe and evaluate an alternative assay using T-cell proliferation to measure SEA activity in food. Human and rat lymphocytes proliferate in response to concentrations of SEA as low as 1 pg/ml, well below the pathogenic dose of 100 ng. This proliferation assay is highly sensitive, quantitative, and simple. Nonradioactive assays of T-cell proliferation were also suitable for detecting and measuring SEA, although with a 10-fold lower sensitivity. To evaluate the utility of this assay for food testing, four different food samples were mixed with SEA. In each sample, SEA was detected at a concentration of 1 ng/ml. Heat-inactivated SEA produced no detectable proliferation. These results demonstrate that an in vitro cell proliferation assay is an advantageous alternative to existing animal assays for measuring SEA activity in food.

Animals↗
PubMed2006

Use of oligoarrays for characterization of community-onset methicillin-resistant Staphylococcus aureus.

Until recently, methicillin-resistant Staphylococcus aureus (MRSA) was considered the prototype of a hospital-acquired bacterial pathogen. However, recent reports have shown that MRSA has now emerged in the community. Characterization of specific markers for distinguishing the origin of isolates could contribute to improved knowledge of MRSA epidemiology. The release of whole-genome sequences of hospital- and community-acquired S. aureus strains allowed the development of whole-genome content analysis techniques, including microarrays. We developed a microarray composed of 8,191 open reading frame-specific oligonucleotides covering >99% of the four sequenced S. aureus genomes (N315, Mu50, MW2, and COL) to evaluate gene contents of hospital- and community-onset S. aureus strains. In parallel, pulsed-field gel electrophoresis, variable number of tandem repeats, antibiogram, staphylococcal cassette chromosome-mec element typing, and presence of the Panton-Valentine leukocidin gene were evaluated in a collection of 15 clinical isolates. Clusters obtained with microarrays showed a high degree of similarity with those obtained by pulsed-field gel electrophoresis or variable number of tandem repeats. Clusters clearly segregated hospital-onset strains from community-onset strains. Moreover, the microarray approach allowed definition of novel marker genes and chromosomal regions specific for given groups of isolates, thus providing better discrimination and additional information compared to pulsed-field gel electrophoresis and variable number of tandem repeats. Finally, the comparative genome hybridization approach unraveled the occurrence of multiple horizontal transfer events leading to community-onset MRSA as well as the need for a specific genetic background in recipient strains for both the acquisition and the stability of the mec element.

Adult↗
PubMed1987

Prominent IgM rheumatoid factor production by human cord blood lymphocytes stimulated in vitro with Staphylococcus aureus Cowan I.

We previously reported that Staphylococcus aureus Cowan I (SAC) is a potent stimulant of IgM rheumatoid factor (RF) production by normal adult peripheral blood mononuclear cells. In the current study, we compared the capacity of normal adult peripheral blood mononuclear cells and cord blood mononuclear cells to produce IgM RF. Although both populations of cells consistently produced IgM RF in response to SAC, the quantity of RF produced by cord blood cells (128 +/- 18 ng/ml, mean +/- SEM) greatly exceeded that of adult cells (37 +/- 5 ng/ml, p less than 0.0005) even though both populations of cells demonstrated comparable total IgM responses. Remarkably, 16.7% of total IgM produced by cord blood cells in response to SAC showed RF activity compared to only 3.4% (p less than 0.0001) of the total IgM produced by SAC-stimulated adult cells. Thus, precursors of IgM RF-secreting cells are not only a consistent feature of the normal adult human B cell repertoire, but they are especially represented at the time of birth. These findings are consistent with the hypothesis that IgM RF originates from germ line genes and underscore the utility of SAC as a probe for analyzing the production of this autoantibody.

Cells, Cultured↗
PubMed2004

The N-terminal A domain of fibronectin-binding proteins A and B promotes adhesion of Staphylococcus aureus to elastin.

The ability of Staphylococcus aureus to adhere to components of the extracellular matrix is an important mechanism for colonization of host tissues during infection. We have previously shown that S. aureus binds elastin, a major component of the extracellular matrix. The integral membrane protein, elastin-binding protein (EbpS), binds soluble elastin peptides and tropoelastin via its surface-exposed N-terminal domain. In this study, we demonstrate that some strains of S. aureus adhere strongly to immobilized human elastin and that this interaction is independent of EbpS but instead is mediated by the fibronectin-binding proteins, FnBPA and FnBPB. Our results show that EbpS mutant cells adhere to elastin-coated plates, whereas the cells negative for FnBPA and FnBPB do not adhere to the plates. Furthermore, only wild-type cells from the exponential phase of growth adhered when FnBPs were expressed maximally. We show that adherence to elastin promoted by FnBPA was not affected by soluble fibronectin, suggesting that the elastin binding domain is distinct from the fibronectin binding regions. Recombinant FnBPA(37-544) (rFnBPA(37-544)) protein corresponding to the A region of FnBPA and anti-FnBPA(37-544) antibodies inhibited FnBPA-mediated bacterial adherence to immobilized elastin. Finally, recombinant A domain proteins, rFnBPA(37-544) and rFnBPB(37-540), bound immobilized elastin dose-dependently and saturably. This interaction was inhibited by soluble elastin peptides, suggesting a specific receptor-ligand interaction.

Adhesins, Bacterial↗
PubMed1988

Bactericidal efficacy of mupirocin in multi-antibiotic resistant Staphylococcus aureus burn wound infection.

Methicillin resistant Staphylococcus aureus strains (MRSA) have become increasingly prevalent as pathogenic organisms, especially in burn wounds, with an associated mortality of 20-40% among those clinically infected. Mupirocin ointment, a new topical antibiotic, has proved in vitro and in vivo to be highly effective in the treatment of MRSA infections. A modified Walker burn wound model was used to define the rate of trans-eschar penetration, biodynamic availability and bactericidal efficacy of 2% mupirocin ointment in established MRSA burn wound infection. In-vitro penetration trials confirmed the effective diffusion of mupirocin through 1.5 mm eschar within 2 h. A single topical application of mupirocin resulted in a 98.3% (5.67 x 10(8) cfu/g of tissue--1.0 x 10(7) cfu/g of tissue) reduction in intra-eschar viable organisms within 36 h post application. A second topical application of mupirocin at 24 h resulted in a total reduction of 99.6% in viable intra-eschar organisms (1.85 x 10(8) cfu/gram of tissue--6.76 x 10(5) cfu/g of tissue). It is concluded that mupirocin is highly effective in controlling MRSA burn wound infection and should be applied topically every 24 h.

Animals↗
PubMed1999

Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315.

In methicillin-resistant Staphylococcus aureus, the methicillin resistance gene mecA is localized within a large chromosomal region which is absent in the methicillin-susceptible S. aureus chromosome. The region, designated mec DNA, is speculated to have originated from the genome of another bacterial species and become integrated into the chromosome of the S. aureus cell in the past. We report here cloning and determination of the structure of the entire mec DNA sequence from a Japanese S. aureus strain, N315. The mec DNA was found to be 51,669 bp long, including terminal inverted repeats of 27 bp and a characteristic pair of direct repeat sequences of 15 bp each: one is situated in the right extremity of mec DNA, and the other is situated outside the mec DNA and abuts the left boundary of mec DNA. The integration site of mec DNA was found to be located in an open reading frame (ORF) of unknown function, designated orfX. Clusters of antibiotic resistance genes were noted in mec DNA carried by transposon Tn554 and an integrated copy of plasmid pUB110. Both the transposon and plasmid were integrated in the proximity of the mecA gene, the latter being flanked by a pair of insertion sequence IS431 elements. Many ORFs other than those encoding antibiotic resistance were considered nonfunctional because of the acquired mutations or partial deletions found in the ORFs. Two ORFs potentially encoding novel site-specific recombinases were found in mec DNA. However, there was no ORF that might encode mec DNA-specific transposase or integrase proteins, indicating that the mec DNA is not a transposon or a bacteriophage in nature.

Amino Acid Sequence↗
PubMed1999

The essential catalytic redox couple in arsenate reductase from Staphylococcus aureus.

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite), which is subsequently extruded from the cell. ArsC couples to thioredoxin, thioredoxin reductase, and NADPH to be enzymatically active. A novel purification method leads to high production levels of highly pure enzyme. A reverse phase method was introduced to systematically analyze and control the oxidation status of the enzyme. The essential cysteinyl residues and redox couple in arsenate reductase were identified by a combination of site-specific mutagenesis and endoprotease-digest mass spectroscopy analysis. The secondary structures, as determined with CD, of wild-type ArsC and its Cys mutants showed a relatively high helical content, independent of the redox status. Mutation of Cys 10, 82, and 89 led to redox-inactive enzymes. ArsC was oxidized in a single catalytic cycle and subsequently digested with endoproteinases ArgC, AspN, and GluC. From the peptide-mass profiles, cysteines 82 and 89 were identified as the redox couple of ArsC necessary to reduce arsenate to arsenite.

Adenosine Triphosphatases↗
PubMed2006

Mechanistic basis for the action of new cephalosporin antibiotics effective against methicillin- and vancomycin-resistant Staphylococcus aureus.

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created challenges in treatment of nosocomial infections. The recent clinical emergence of vancomycin-resistant MRSA is a new disconcerting chapter in the evolution of these strains. S. aureus normally produces four PBPs, which are susceptible to modification by beta-lactam antibiotics, an event that leads to bacterial death. The gene product of mecA from MRSA is a penicillin-binding protein (PBP) designated PBP 2a. PBP 2a is refractory to the action of all commercially available beta-lactam antibiotics. Furthermore, PBP 2a is capable of taking over the functions of the other PBPs of S. aureus in the face of the challenge by beta-lactam antibiotics. Three cephalosporins (compounds 1-3) have been studied herein, which show antibacterial activities against MRSA, including the clinically important vancomycin-resistant strains. These cephalosporins exhibit substantially smaller dissociation constants for the preacylation complex compared with the case of typical cephalosporins, but their pseudo-second-order rate constants for encounter with PBP 2a (k(2)/K(s)) are not very large (< or =200 m(-1) s(-1)). It is documented herein that these cephalosporins facilitate a conformational change in PBP 2a, a process that is enhanced in the presence of a synthetic surrogate for cell wall, resulting in increases in the k(2)/K(s) parameter and in more facile enzyme inhibition. These findings argue that the novel cephalosporins are able to co-opt interactions between PBP 2a and the cell wall in gaining access to the active site in the inhibition process, a set of events that leads to effective inhibition of PBP 2a and the attendant killing of the MRSA strains.

Anti-Bacterial Agents↗
PubMed2004

Atomic force microscopy of cell growth and division in Staphylococcus aureus.

The growth and division of Staphylococcus aureus was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol. 141:315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.

Cell Division↗