Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms.

Staphylocoagulase, a major phenotypic determinant of Staphylococcus aureus, exists in multiple allelic forms, in part because of the existence of gene variants within the 3'-end coding region. This region contains a series of repeating 81-bp DNA sequences which differ both in the number of tandem repeats and the location of AluI restriction sites among different isolates. Utilizing this finding, we developed a novel typing method for S. aureus based on polymerase chain reaction amplification of the variable region of the coagulase gene followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP). Among 30 S. aureus isolates studied initially, a total of 10 distinct RFLP patterns were observed. There was excellent correlation of the RFLP patterns with typing of these isolates by multilocus enzyme electrophoresis at 20 chromosomal loci. This coagulase RFLP method was used to analyze an additional 39 S. aureus isolates and successfully traced the source of an outbreak of methicillin-resistant S. aureus infections at a local hospital.     

Strategies for typing and properties of epidemic methicillin-resistantStaphylococcus aureus

Isolates of 17 strains of epidemic methicillin-resistant Staphylococcus aureus from outbreaks in ten hospitals in the UK were investigated with a variety of techniques both to explore their properties and to type them in order to confirm or refute known or suspected epidemiology. The techniques consisted of a biotyping system, peptidogylcan analysis, testing of antibiotic sensitivity to 21 agents, various phage-typing methods including heat shock, plasmid pattern analysis, and heat cure derivation of plasmid-less isogenic strains. All strains resembled those originally isolated in Australia, being in the possession of a large number of chromosomal resistance factors, pigmentation, ability to produce lipase and large molecular weight plasmids (c.15 Md to c.23 Md) which conferred resistance to gentamicin, propamidine, ethidium bromide, cetrimide and chlorhexidine. Some strains also had a c.3 Md plasmid conferring chloramphenicol resistance and others a c.1 Md cryptic plasmid. A large percentage of the population was resistant to 25 mg/l methicillin at 37 °C, an unusual feature. All the strategies, with the exception of peptidoglycan analysis, contributed to typing of the strains.     

Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism.

The X region of the protein A gene of Staphylococcus aureus contains a highly polymorphic sequence which is composed of repeats of 24 bp. We used amplification by PCR to investigate whether this region could be used to discriminate between epidemic and nonepidemic methicillin-resistant S. aureus (MRSA) strains. Most epidemic MRSA strains (24 of 33) harbored more than seven repeats, while most nonepidemic MRSA strains (10 of 14) contained seven or fewer repeats. It is conceivable that a longer X region results in a better exposition of the Fc-binding region of protein A, thereby facilitating colonization of host surfaces and contributing to the epidemic phenotype.     

A probe for the detection of methicillin-resistantStaphylococcus aureus

An approximately 300 base pair DNA fragment for use as a probe was isolated from methicillin-resistantStaphylococcus aureus DNA partially digested withSau3AI. This probe hybridized with 25 methicillin-resistant clinical isolates ofStaphylococcus aureus belonging to 18 different phage types, but not with 41 clinical isolates susceptible to methicillin.     

Discrimination of Staphylococcus aureus isolates on the basis of gene coding protein A using PCR-restriction enzyme analysis

In a comparative study, isolates of Staphylococcus aureus from bovine mastitis cases with known coa and aroA types were analyzed by molecular typing based on polymerase chain reaction and restriction enzyme analysis (REA) of protein A-encoding gene (spa) for assessment of its utility over coa and aroA typing in discrimination of the isolates. Fifty-eight isolates of S. aureus from nine dairy herds in two Iranian provinces were typed based on polymorphism characterizing the gene encoding for the X region of protein A (spa). Five differently sized amplicons of approximately 1,200?bp to 1,410?bp were observed. Spa gene REAs produced a total of eight distinct patterns, designated as S1–S8, after digestion with restriction endonuclease Hin6I. For the spa gene, the lack of amplification was also considered a distinct genotype (S9). The majority of isolates were classified into spa types S2 (24.14%) and S6 (24.14%). This study also showed that genetic analysis of the repeat region of protein A might help to understand the distribution of prevalent S. aureus clones among bovine mastitis isolates. Interestingly, based on spa, coa, and aroA typing, some isolates which were identified as dominant types by one method were classified as rare types by another and vice versa. Finally, spa typing has 62.1% concordance with coa typing from the standpoint of the assignment of the isolates to predominant and rare lineages. This study also demonstrates the importance of spa genotyping in the discrimination of S. aureus isolates, which were otherwise indistinguishable by coa and aroA gene typing.     

Usefulness of PCR-restriction fragment length polymorphism typing of the coagulase gene to discriminate arbekacin-resistant methicillin-resistant Staphylococcus aureus strains

We compared the results of two typing methods for 678 strains of methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus. PCR-restriction fragment length polymorphism typing of the coagulase gene was a more reliable method than coagulase serotyping from the viewpoint of arbekacin resistance.     

Comparison of screening methods to identify methicillin-resistantStaphylococcus aureus

Screening methods to identify methicillin-resistantStaphylococcus aureus (MRSA) were compared using 96 isolates representing 17 distinct clones. The sensitivity of four commercial agglutination tests was determined in comparison to the tube coagulation test, and the results related to the presence of the coagulase gene. The broth screening test, agar dilution test and disc diffusion test were carried out, and the results related to the presence of themecA gene. Mannitol salt agar and Iso-Sensitest agar with varying salt supplements were used. All agglutination tests had high rates of detection ofStaphylococcus aureus (95.8–99.0%). Resistance in mecA gene-positiveStaphylococcus aureus isolates was correctly detected by the oxacillin broth test, the agar dilution test and the disc diffusion test on mannitol salt agar, whereas on Iso-Sensitest agar detection rates were lower (between 68.5% and 94.4%, depending on the salt supplement). Incubation of the Iso-Sensitest plates for 48 hours significantly improved the rate of detection of resistance, but increased the major error rate up to 71.4%.MecA genepositiveStaphylococcus aureus isolates not detected by the disc diffusion test on Iso-Sensitest agar had significantly lower oxacillin minimal inhibitory concentration values and were significantly less resistant to a variety of antibiotics. Thus, mannitol salt agar might be a suitable medium for use in the disc diffusion and agar dilution test to detect resistance to oxacillin inStaphylococcus aureus.     

Control of methicillin-resistantStaphylococcus aureus bacteraemia in high-risk areas

In a 3,000-bed tertiary care hospital, 88 cases of methicillin-resistantStaphylococcus aureus (MRSA) bacteraemia were identified from 22,383 blood cultures (0.39 %) submitted to the microbiology laboratory over a one-year period. Two high-risk areas were identified: the paediatric oncology unit, in which 12 cases of MRSA bacteraemia were identified from 924 blood cultures (1.3 %), and the intensive care unit (ICU), in which 14 cases of MRSA bacteraemia were identified from 1,391 blood cultures (1.0 %). In a one-year targeted intervention programme in which staff and patients were screened for MRSA carriage, patient carriers isolated, and mupirocin and chlorhexidine treatment administered, the number of MRSA bacteraemia cases decreased in these areas to 0 and 4, respectively (p=0.000123 and 0.016), while the incidence of MRSA bacteraemia in non-targeted areas increased from 62 of 20,068 blood cultures (0.3 %) to 82 of 18,784 blood cultures (0.44 %) (p=0.047). In the year post intervention the incidence of MRSA bacteraemia increased to 3 of 815 cultures (0.37 %) in the paediatric oncology unit, 10 of 1,934 cultures (0.5 %) in the ICU, and 112 of 18,977 cultures (0.59 %) in the rest of the hospital (p=0.00004 versus preintervention period). This study demonstrates the efficacy of targeted MRSA control measures in a hospital in which MRSA is endemic.     

Diversity of methicillin-resistantStaphylococcus aureus isolated in a Canadian hospital

Three neonates and three other patients located elsewhere in the hospital became infected withStaphylococcus aureus. Initial automated microdilution susceptibility testing with oxacillin and disk diffusion testing with amoxicillin-clavulanic acid indicated the isolates had borderline oxacillin resistance (MICs 4 µg/ml), presumably due to hyperproduction of -lactamase. Chromosomal DNA restriction fingerprinting and phage typing revealed the neonatal isolates to be identical; whereas, the other patients were infected with three different strains. Further analysis of the four strains by Southern hybridization with amecA specific oligoprobe and a quantitative -lactamase assay demonstrated that two strains carried themecA gene (coding for low affinity penicillin-binding protein 2a), and two strains were hyperproducers of -lactamase, including one which wasmecA gene positive. One strain neither carried themecA gene nor hyperproduced -lactamase. The twomecA gene positive strains displayed oxacillin MICs of 16 µg/ml on dilution susceptibility testing in 4% NaCl supplemented Mueller-Hinton agar. Hence, they were considered intrinsically methicillin-resistantStaphylococcus aureus. Both oxacillin and amoxicillin-clavulanic acid MICs were increased on NaCI supplementation. Results of amoxicillin-clavulanic acid disk diffusion susceptibility testing did not correlate with quantitative -lactamase production. It is recommended that clinical laboratories do not use amoxicillin-clavulanic acid disk diffusion assays to differentiate suspected borderline resistance due to -lactamase hyperproduction frommecA gene expression of PBP-2a since additional mechanisms may account for resistance.     

Laboratory and epidemiologic experience with methicillin-resistantStaphylococcus aureus in the USA

Methicillin-resistant Staphylococcus aureus (MRSA) was a rare occurrence in US hospitals until the mid-1970s. Since that time outbreaks of MRSA infection have been reported in both large and small hospitals, in rehabilitation facilities, and in nursing homes. Transmission has been documented not only between hospitals, but between long-term care facilities and hospitals, and between the community and hospitals. Patient-to-patient spread within hospitals appears to result from transient colonization of the hands of health care workers, with colonized or infected patients being the intrahospital reservoir for the organisms. The best opportunity for control of outbreaks of MRSA infection within hospitals may depend on the rapid recognition of newly admitted patients who are colonized or infected. The laboratory plays a crucial role in this by providing prompt and accurate information indicating the presence of MRSA. Susceptibility test methods found to be most reliable for detecting MRSA in the USA include the broth microdilution MIC determination (performed in salt-supplemented broth), the Bauer-Kirby test with slight modification, or oxacillin-salt agar screening plates.     

Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR–restriction fragment length polymorphism

Human trichomoniasis, caused by the protozoan Trichomonas vaginalis , is a highly prevalent sexually transmitted infection. However, little is known about the degree of strain variability of T. vaginalis . A reliable classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, pathogenesis and transmission of T. vaginalis . A PCR–restriction fragment length polymorphism typing method was designed and evaluated using T. vaginalis isolates obtained after culture of vaginal specimens collected in the Democratic Republic of Congo and in Zambia. The variation of the actin gene of T. vaginalis was determined for three ATCC reference strains and 151 T. vaginalis isolates. Eight different types were identified, on the basis of the digestion patterns of the amplified actin gene, with each of the restriction enzymes Hin dII, Mse I and Rsa I . It was determined that the ATCC reference strains 30001, 30240 and 50141 were of actin genotypes G, H and E, respectively. The actin genotype type E was more common in the Democratic Republic of Congo, whereas type G was the commonest type in Zambia. Translation of the nucleotide sequence showed up to three amino acid substitutions. We developed a reproducible, sensitive and specific typing method for T. vaginalis , and were able to distinguish at least eight T. vaginalis actin genotypes. Further studies are needed to evaluate the method using clinical specimens and to determine the utility of the typing method for the genotypic characterization of T. vaginalis.     

Molecular typing of methicillin and gentamicin resistantStaphylococcus aureus in Dublin

The high incidence of infection in Dublin hospitals caused by non-typable strains of methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) has created an important epidemiological problem as conventional methods of sub-dividing these organisms have not been useful. This report describes a novel approach to the typing and analysis of MGRSA strains, particularly non-typable isolates, by (i) comparing restriction endonuclease Hin dIII digest patterns of total cellular DNA; and (ii) by using Southern hybridization analysis to detect size variations or polymorphisms in restriction endonuclease cleavage fragments within small regions of the chromosome. Non-typable MGRSA strains and isolates belonging to two phenotypically related groups of phage-type 77 and 77/84 strains were readily subdivided on the basis of molecular size differences in high molecular weight DNA fragments generated by the enzyme Hin dIII. Restriction endonuclease fragment size polymorphisms were readily detected in many of the non-typable strains tested in hybridization experiments, and these were used for strain sub-division. Both techniques were useful tools for the separation of closely related MGRSA strains.     

Comparison of DNA sequencing of the protein A gene polymorphic region with other molecular typing techniques for typing two epidemiologically diverse collections of methicillin-resistant Staphylococcus aureus

The aim of this study was to compare the recently developed typing approach for methicillin-resistant Staphylococcus aureus (MRSA) based on the DNA sequencing of the protein A gene polymorphic region (spaA typing) with a combination of three well-established molecular typing techniques: ClaI-mecA vicinity polymorphisms, ClaI-Tn554 insertion patterns, and SmaI pulsed-field gel electrophoresis (PFGE) profiles. In order to evaluate the applicability of this typing technique in different types of studies, two groups of MRSA clinical isolates were analyzed: a collection of 185 MRSA isolates circulating in Hungary recovered from 17 hospitals in seven cities during a 3-year period (1994 through 1996), and a selection of 53 MRSA strains isolated in a single hospital in Hungary between 1997 and 1998. The 238 MRSA clinical strains from Hungary were first classified in clonal types (defined as ClaI-mecA::ClaI-Tn554::SmaI-PFGE patterns), and 65 of the 238 strains, representing major MRSA clones and some sporadic clones, were further analyzed by spaA typing. Our results showed that the lineages most recently introduced in the hospital setting showed little variability in spaA types, whereas the MRSA clones circulating for a longer period of time and spread among several hospitals showed a higher degree of variability. The implementation of the spaA typing method was straightforward, and the results obtained were reproducible, unambiguous, and easily interpreted. This method seems to be adequate for outbreak investigations but should be complemented with other techniques in long-term surveillance or in studies comparing distant clonal lineages.     

Long-term efficacy of a program to control methicillin-resistantStaphylococcus aureus

The long-term efficacy of a program to control methicillin-resistantStaphylococcus aureus (MRSA) was evaluated in a 350-bed university hospital. Three periods were monitored: pre-epidemic (January 1989–November 1989), outbreak (December 1989–June 1990) and control program (July 1990–December 1992) periods. Control measures included cohort isolation, patient care measures and therapy (oral cotrimoxazole plus fusidic acid ointment) of MRSA carriage in patients, roommates and personnel. A total of 117 MRSA-infected patients were detected. For each period respectively, MRSA incidence (number of cases per 1,000 patient-days) was 3.2, 8.2 and 2.0 in the intensive care unit (ICU) and 0.08, 0.23 and 0.26 in the general wards. During the outbreak there was a 2.7-fold overall increase of baseline MRSA incidence (p<0.02). The crude mortality was 68 % and the attributable mortality was estimated to be 50 %. The program was estimated to have prevented 76 % (CI95 28–91, p<0.0001) of expected MRSA cases and 85 % (CI95 62–94, p<0.0001) of expected fatalities due to MRSA in the ICU, but it had no significant effect in the general wards. The program did not control vancomycin consumption.     

Control of epidemic methicillin-resistantStaphylococcus aureus in a Dutch University Hospital

Between 1986 and 1989 a single strain of a methicillin- and multiply-resistantStaphylococcus aureus caused three distinct outbreaks at Utrecht University Hospital, involving 11, 19 and 32 patients, respectively. In all three episodes, members of staff were screened for MRSA carriage, and 58 persons were found to have positive nose cultures. In each outbreak it became necessary to isolate colonized and infected patients on a separate isolation ward. Staff carriers were also treated. Over the 18 months since the last outbreak, no new acquisitions of this epidemic MRSA strain have occurred. Between 1986 and 1989, the strain which caused the three outbreaks was not the only MRSA strain which was introduced into the hospital. Six other strains, which differed from the epidemic strain as shown by phage typing and antimicrobial susceptibility pattern, were found in single patients. The experience at Utrecht University Hospital illustrates the need for strict measures to eradicate epidemic strains of MRSA as well as the differences in epidemicity among various strains of MRSA.     

Molecular typing of Staphylococcus aureus isolated from food samples in Iran

Staphylococcus aureus is the third most common cause of confirmed food poisoning in the world and is the predominant species involved in staphylococcal food poisoning outbreaks. Considerable genetic heterogeneity has been shown in natural populations of S. aureus isolates. Coagulase gene typing is one of the numerous molecular techniques to identify and compare S. aureus genotypes. The present study was conducted to type the coagulase gene in 25 S. aureus isolates isolated from food samples. All isolates were identified by routine biochemical tests and then confirmed by species-specific PCR and yielded products with the expected molecular size of 1.3 kb. PCR amplification of DNA with the primers COAG2 and COAG3 yielded single-banded PCR products in 24 isolates with the molecular size of approximately 500 bp (n?=?2, 8 %), 750 bp (n?=?1, 4 %), 850 bp (n?=?12, 48 %), and 950 bp (n?=?9, 36 %), while one isolate produced no band in PCR amplification of coagulase gene. Since human and bovine reservoirs of S. aureus represent two subpopulations that rarely cross-infect, detection of single bands by coagulase PCR in S. aureus isolates suggests that these isolates may be of bovine origin not human one, and contamination of food samples may initiate from the animal source not the food handlers. Digestion of coagulase PCR products with restriction endonuclease enzymes AluI and Hin6I yielded four different restriction profiles that indicate presence of heterogeneity in the coagulase gene of the isolates. This work showed that restriction analysis of the coagulase gene can be considered as a reliable and fast method for determining the origin of S. aureus in food samples.     

Molecular and phenotypic typing of Staphylococcus aureus isolates from rabbit meat

Twenty-six Staphylococcus aureus isolates were recovered from rabbit carcasses and cuts during a period of seven months. Samples from 51 different animals, flocks and farms were obtained from five establishments in four Spanish provinces. To determine their diversity and possible origin, isolates were typed by three molecular and three phenotypic methods. PFGE, with the highest discrimination index (D=0.966), identified 19 patterns (more than one band difference) and 10 types (more than three band differences). Based on > or = 90% similarity, RAPD (D=0.877) produced nine patterns while ribotyping (D=0.786) produced seven types. On the basis of biotyping (D=0.644), 11 isolates belonged to human ecovars and 15 to the non-host-specific crystal violet type C (NHS CV:C) biotypes. By direct phage typing (D=0.761), 17 isolates were lysed by human phages into groups II (8 isolates), III (5 isolates), I/III (2 isolates) and V (2 isolates). The overall resistance to antimicrobials (D=0.783) was 76.9%, with most isolates being resistant to tetracycline (61.5%) and penicillin G (26.9%). PFGE showed that samples from each processing plant carried different S. aureus types, some of them persisting over time. There also was evidence of interestablishment dissemination of genetically related clones, most of them belonging to the PFGE type A and phenotype "NHS CV:C biotypes-3A/3C/55/71 phage type", which is highly virulent for European commercial rabbitries. The ubiquity of the virulent phenotype, as well as the high incidence of resistance to antibiotics with application in human medicine, is a matter of concern in public and animal health.