Identification of MRSA incidents in hospitals.
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Biomedical subjects
Publications and source records attributed to B Cookson.
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A polyphasic taxonomic study that included DNA-rRNA hybridizations, DNA-DNA hybridizations, DNA base ratio determinations, whole-cell protein and fatty acid analyses, and an examination of classical phenotypic characteristics was performed in order to classify human and veterinary isolates that resemble Bordetella avium. Twelve poultry isolates and two human isolates were assigned to a new species, for which we propose the name Bordetella hinzii. The position of this organism in the family Alcaligenaceae and various genotypic, phenotypic, and chemotaxonomic characteristics are described.
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Eight clinical isolates of Enterococcus faecium highly resistant to gentamicin (MIC, > 1,000 mg/liter) from patients in six hospitals on three continents were investigated for evidence of spread of either a clone of high-level gentamicin-resistant (HLGR) E. faecium or wide dissemination of a gentamicin resistance (Gmr) plasmid. A combination of ribotypes, plasmid profiles, and extended antimicrobial susceptibilities enabled us to distinguish all but two of the isolates and did not suggest clonal dissemination of a single strain. Two isolates from hospitals situated close together appeared identical by these methods. All of the isolates carried Gmr plasmids which appeared to be closely related following digestion with restriction endonucleases. Cross-hybridization studies confirmed extensive DNA homology between these plasmids. The fragments of these plasmids which hybridized with a probe specific for the aac6'aph2" resistance gene did not resemble those seen in the Gmr transposon Tn5281, which was characterized previously in E. faecalis HH22. This study suggests that there has been widespread dissemination of a single Gmr plasmid and its derivatives amongst isolates of HLGR E. faecium, although a Gmr plasmid from an HLGR E. faecium isolated in the United States showed little homology with the other Gmr plasmids studied.
DNA probes specific for genes encoding rRNA and the glycopeptide resistance gene vanA were used to investigate a cluster of vancomycin-resistant (MICs, > 512 mg/liter) Enterococcus faecalis and Enterococcus faecium isolated from separate patients in a renal unit in a London hospital. When digested with BamHI, 12 of 13 vancomycin-resistant E. faecalis isolates exhibited a common restriction fragment length polymorphism pattern of rRNA genes (ribotype). A vanA probe hybridized with chromosomal DNA in these 12 isolates. The other isolate of vancomycin-resistant E. faecalis had a different ribotype and the vanA gene was located on plasmid DNA. These data suggest that cross-infection with a single strain of vancomycin-resistant E. faecalis occurred in most instances. In contrast, 23 vancomycin-resistant E. faecium isolates showed greater heterogeneity, comprising 8 ribotypes, suggesting that multiple strains were present in the unit. Twenty-one of these 23 isolates harbored a 24-MDa plasmid which hybridized with the vanA probe, implying that interstrain dissemination of a vancomycin resistance plasmid may have occurred among E. faecium isolates in the renal unit.
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One hundred and forty-two coagulase-negative staphylococci (CNS) isolated from dialysate effluent or skin of patients receiving continuous ambulatory peritoneal dialysis (CAPD) were typed by extended antibiogram (16 antibiotics) and biotype (26 reactions). These isolates were then typed by supplementary methods to determine the most suitable typing method for an epidemiological study of antibiotic resistance. These included phage typing, reverse phage typing, plasmid typing, whole-cell protein typing by SDS-PAGE with analysis by densitometry, and immunoblotting. The percentage of isolates typed successfully by the supplementary methods were: phage typing 20%, reverse phage typing 0%, plasmid typing 66%, SDS-PAGE 100%, immunoblotting 100%. The discrimination of each method was: phage typing 20%, plasmid typing 37%, SDS-PAGE 69%, immunoblotting 57%. Reproducibility was 88% for phage typing and 97% for plasmid typing. The reproducibility of the whole-cell protein typing was 83% if the same extracts were used but only 43% when separate protein extracts were analysed on separate occasions. However, strain relatedness was highly reproducible. The determination of an antibiogram-biotype profile was not a sufficiently accurate typing method for an epidemiological study of antibiotic resistance. Whole-cell protein typing by SDS-PAGE or immunoblotting was technically demanding but was the most effective of the supplementary methods for detecting erroneous discrimination and false matching produced by antibiogram-biotype combinations.
Nine methicillin-sensitive (MSSA) and 37 methicillin-resistant (MRSA) Staphylococcus aureus isolates of various phage types and resistotypes from seven countries were investigated. Chromosomal DNA was restricted with HindIII or EcoRI, Southern blotted and hybridised with a cDNA probe to 16S+23S rRNA derived from MRSA NCTC 10442. Resulting rDNA profiles could be differentiated on the basis of 22 patterns which were unaffected by changes in plasmid, transposon, enterotoxin A or phage content. Percentage similarity values were calculated using the Dice coefficient and UPGA clustering. Australian and epidemic (EMRSA-1) isolates from the UK showed a high degree of similarity, but the pattern was not unique and was also found in MSSA and other MRSA, e.g. NCTC 10442. An MSSA of phage group II was the most distinct isolate. The method shows potential as an additional tool in a complex typing system, types non-phage typable strains and may provide clues to the clonal evolution of MRSA and MSSA.
Bordetella pertussis infection probably involves attachment to and destruction of ciliated epithelial cells, but most previous studies have used animal tissue. During an epidemic, nasal epithelial biopsy specimens of 15 children (aged 1 month to 3 1/2 years) with whooping cough were examined for ciliary beat frequency, percent ciliation of the epithelium, and ciliary and epithelial cell ultrastructure. In addition, the in vitro effects of filtrates from a 24-h broth culture and of tracheal cytotoxin derived from B. pertussis on human nasal tissue organ culture were measured. B. pertussis was cultured from nasal swabs from 12 children. The mean ciliary beat frequency of their nasal biopsy specimens, 11.3 Hz (range, 10.4 to 13.0 Hz) was similar to that found in biopsy specimens from 10 normal children (mean, 12.5 Hz; range, 11.8 to 13.5 Hz). The abnormalities of the epithelium observed in 14 of 15 patients were a reduction in the number of ciliated cells, an increase in the number of cells with sparse ciliation, an increase in the number of dead cells, and extrusion of cells from the epithelial surface. In vitro, neither culture filtrate nor tracheal cytotoxin had any acute effect on ciliary function, but culture filtrate and tracheal cytotoxin (1 and 5 microM, respectively) caused extrusion of cells from the epithelial surface of turbinate tissue, loss of ciliated cells, an increased frequency of sparsely ciliated cells, and toxic changes in some cells. These changes were dose dependent and progressive, and between 36 and 90 h ciliary beating ceased. The observations made with patient tissue confirm that B. pertussis infection damages ciliated epithelium, and the in vitro experiments suggest that tracheal cytotoxin may be responsible for the abnormalities observed in vivo.
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Plasmids mediating high-level resistance to mupirocin (MIC greater than 1000 mg/L) in staphylococci from various sources were studied by restriction endonuclease cleavage. Several patterns were obtained but six plasmids isolated from various Staphylococcus aureus and S. epidermidis strains were indistinguishable. The diversity and spread of these plasmids is illustrated.
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Isolates from four UK centres, comprising four Staphylococcus aureus strains and one Staphylococcus epidermidis strain with high-level mupirocin resistance, and one strain each of Staphylococcus aureus and Staphylococcus epidermidis with low-level mupirocin resistance, were assessed for intracellular or extracellular enzymatic alteration of the antibiotic. No change was seen in a number of assays, which included comparative disk diffusion, well diffusion and high-pressure liquid chromatography. Other mechanisms of resistance need to be explored in these isolates.
The spread of two strains of Staphylococcus aureus with high level resistance to mupirocin is described. The resistance proved to be easily transferred to other S. aureus strains by filter mating experiments and on the skin of mice. No plasmid band corresponding to the resistance could be demonstrated by agarose gel electrophoresis or by caesium chloride gradient centrifugation but cleavage of 'chromosomal' DNA from resistant recipients showed bright bands of DNA absent from sensitive controls.
Fatty acid analysis was done with GC and GC-MS on 21 strains of 'Streptococcus milleri', representative of the various proposed species. Although no qualitative differences were found in the fatty acid profiles, discriminant analysis of the quantitative data revealed three groups. Streptococcus anginosus and Streptococcus constellatus were indistinguishable but separated from the other two groups which comprised Streptococcus intermedius, with a wide fermentation pattern and Streptococcus intermedius with a narrow fermentation pattern. Three of the strains could be distinguished from the others by a 'fingerprint' of a particularly prominent fatty acid peak. The results support the suggestion that there is more than one species in this group of organisms and that the technique might be of value in epidemiological investigations of 'S. milleri'.
Twenty-six nurses were repeatedly screened for carriage of epidemic methicillin-resistant Staphylococcus aureus (EMRSA) immediately before and after duty periods in which they solely attended six patients widely colonized with two EMRSA strains distinguishable by plasmid analysis. EMRSA carriage was detected in 13 nurses. Three EMRSA carriage patterns emerged: transient carriage in 12 nurses, when the EMRSA was isolated from noses or fingers of nurses after duty but was gone before their next day's duty; short-term nasal carriage, seen on occasion in 4 of these 12 nurses, when EMRSA carriage was detected on two consecutive screens; and persistent nasal carriage, seen in 1 nurse only, when the EMRSA was seen on more than two consecutive occasions. All but one of these incidents of carriage could be explained by close patient, rather than environmental, exposure and occurred despite an intensive control programme. Transient or short-term carriage in nurses probably resulted in transfer of the EMRSA between patients. Staff decontamination should be considered following a period of cohort nursing of EMRSA patients, especially if staff members are shortly to nurse unaffected patients. Our findings may explain some of the difficulties in controlling EMRSA.
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