We used a sample of Staphylococcus aureus strains that are carried by humans and that are representative of the natural population of S. aureus strains in order to assess the value of multilocus sequence typing (MLST), pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing as epidemiological tools. Only MLST was able to define clonal complexes unambiguously. All DNA-based typing approaches achieved a high degree of agreement, implying phylogenetic concordance, but predicted epidemiological associations with variable accuracy.Staphylococcus aureus is one of the most important pathogens in clinical settings. It is also one of the leading causes of nosocomial infections and the dissemination of multipledrug-resistant strains, mainly methicillin-resistant S. aureus (MRSA), and the recent emergence of a vancomycin-resistant MRSA is of concern to hospitals worldwide. Naturally, an understanding of the dynamics of spread and an identification of transmissions or outbreaks are of interest not only to public health epidemiologists but also to clinical microbiologists and indeed clinicians involved in patient management on a daily basis. Most of the current epidemiological typing schemes compare fragment patterns generated by restriction or amplification of chromosomal DNA or by differences in lysis patterns based on susceptibilities to lytic bacteriophages. All of these approaches are able to distinguish between unrelated strains (albeit with different precision), but the probability with which two indistinguishable patterns predict an epidemiological relationship, i.e., transmission, cannot be determined without knowledge of the underlying genetic structure of the naturally occurring population. In order to describe the genetic structure of bacterial populations and the abundance of certain clones in a given environment, typing tools such as multilocus sequence typing (MLST) provide a comprehensive genetic framework (1, 6). We used MLST and three other conventional epidemiological typing approaches to describe the genetic population structure of strains that were systematically collected from nonhospitalized individuals in the community. This way we were able to objectively assess the value of typing techniques deployed for epidemiological purposes.

A multiplex polymerase chain reaction (PCR), involving detection of the mecA and femB genes, was combined with a novel immunoassay system capable of detecting specific PCR products. The resulting PCR-immunoassay was evaluated in comparison with conventional microbiological techniques used in the routine diagnostic laboratory for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA), either in pure culture or in overnight broth cultures obtained following enrichment of patient screening swabs. Among the 480 purified isolates of staphylococci and 246 enrichment broths examined, only one 'false-negative' result was obtained by PCR, compared with 18 'false-negative' results obtained by conventional methodology and demonstrated by further conventional examination. Five demonstrable 'false-positive' results were obtained by conventional methodology, compared with a possible 10 by the PCRimmunoassay, although it was not certain that these 10 PCR results were true 'false positives' as, by definition, MRSA could not be isolated by conventional methodology. The results indicated that the routine diagnostic laboratory was encountering difficulties in identifying MRSA correctly, and that the conventional microbiological techniques lacked sensitivity. Overall, the PCR technique was more accurate and sensitive than conventional methodology in detecting MRSA, and results were available within 24 h of screening swabs arriving in the laboratory, compared with a minimum of 48-72 h by conventional techniques. The immunoassay system added to the usefulness of the method by allowing the detection of specific PCR products within 5 min of completing the PCR, without the normal additional step of agarose gel electrophoresis.

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The relationships between isolates suggested by a novel DNA typing method (RAPD-ALFA) that combines randomly amplified polymorphic DNA with automated on-line laser fluorescence analysis of DNA fragments were compared with those suggested by four other computer-assisted typing strategies (biotyping, antibiogram typing, pulsedfield gel analysis of chromosomal fingerprints and arbitrarily-primed DNA amplification with three different primers) for 25 isolates of Acinetobacter baumannii obtained from 12 different hospitals in four countries over a period of 12 years. The results obtained by cluster analysis with two different software packages confirmed that the relationships suggested by RAPD-ALFA were robust and essentially similar to those suggested by the other more laborious computer-assisted typing methods. The technique of RAPD-ALFA appears to offer the possibility of routine on-line molecular identification and typing of isolates from particular hospital wards or units (e.g., intensive care units), and could, therefore, play a key role in the early recognition and prevention of outbreaks of infection.

A total of 1572 isolates of Escherichia coli obtained from the faeces of young farm animals with diarrhoea over the period 1980-1983 were screened for resistance to trimethoprim (Tp). Resistance to Tp was detected in 263/954 (28%) of bovine isolates, 59/441 (13%) of porcine isolates and 15/177 (9%) of ovine isolates. Seventy-five resistant isolates from separate outbreaks of infection on farms within a 25 mile radius of Nottingham were examined in detail. Sixty-eight (91%) of the 75 isolates were resistant to greater than 1024 mg Tp/l and 34 (50%) of these 'highly resistant' isolates (45% of total resistant isolates) transferred their Tp resistance to E. coli K12. A further 13 (17%) isolates were demonstrated to carry non-self-transferable plasmids which were capable of being mobilized to E. coli K12 by the broad host range plasmid RP4. Thirty-one self-transferable Tp R plasmids were divided between the following incompatibility groups: IncB (14 plasmids), IncFII (4 plasmids), IncH2 (1 plasmid), IncI alpha (10 plasmids), IncI delta (1 plasmid) and IncP (1 plasmid). In terms of antibiotic resistance patterns and incompatibility properties, many of these plasmids closely resembled those isolated from human patients in the same area, suggesting that there may be a common pool of Tp R plasmids.

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The aim of this study was to compare the molecular relationships and antibiograms of nosocomial isolates of Acinetobacter spp. from two acute-care hospitals in Nottingham, UK, and Soweto, South Africa, with different hospital infection control problems and procedures. In contrast to Nottingham, where randomly amplified polymorphic DNA fingerprinting demonstrated that a single multiresistant strain of Acinetobacter baumannii has predominated in the hospital intensive care unit over an 11-year period, the Soweto isolates formed a heterogeneous group of unrelated molecular clusters of different antibiograms, with numerous different strains of Acinetobacter baumannii, Acinetobacter sp. 3 and Acinetobacter sp. 13TU apparently being endemic throughout the Soweto hospital. The contrasting results illustrate the need to maintain exemplary infection control procedures in hospitals where high standards have been achieved and warn of what might result if such measures are diminished.

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