Emergence, spread, and characterization of phage variants of epidemic methicillin-resistant Staphylococcus aureus 16 in England and Wales.

Epidemic methicillin-resistant Staphylococcus aureus 16 (EMRSA-16) and EMRSA-15 are the two most important and prevalent EMRSA strains found in the United Kingdom and have also been found in a number of European countries and the United States. We describe for the first time the spread of an EMRSA strain (EMRSA-16) from its point of origin in one hospital to the surrounding hospitals and regions over the following 2 years. In the first 18 months after its original appearance, 136 hospitals referred EMRSA-16 isolates for typing, and interhospital and intraregional spread were reported: it was more prevalent in males between 60 and 80 years old and was isolated from sputum and throat more often than EMRSA-15. Important characteristics, e.g., carriage of the enterotoxin A (sea) and toxic shock syndrome toxin (tst) genes and production of urease, are described. Phage-variant strains of EMRSA-16 which share some of the characteristics of the classical strain, including toxin carriage and urease production, emerged, but without genotypic investigations, their relationship could only be inferred. A total of 129 clinical isolates from 52 hospitals, collected between March 1998 and April 1999 and representing classical EMRSA-16 (49 isolates) or phage variants (80 isolates), were compared by phage typing, pulsed-field gel electrophoresis (PFGE) following SmaI macrorestriction, antimicrobial susceptibility testing, urease production, and PCR detection of toxin gene carriage. PFGE analysis revealed 29 profiles, A1 to A29, with A1 representing the prototypic strain, NCTC 13143. All other profiles differed from A1 by 1 to 6 bands, but some differed from each other by up to 10 bands.

Identification and characterization of phage variants of a strain of epidemic methicillin-resistant Staphylococcus aureus (EMRSA-15).

EMRSA-15 is one of the most important strains of epidemic methicillin-resistant Staphylococcus aureus (EMRSA) found in the United Kingdom. It was originally characterized by weak lysis with phage 75 and production of enterotoxin C but not urease. Two variant strains of EMRSA-15 which show a broader phage pattern than the progenitor strain have emerged. A total of 153 recent clinical isolates representing classical EMRSA-15 (55 isolates) or these phage variants (98 isolates) were compared by SmaI macrorestriction profiles in pulsed-field gel electrophoresis (PFGE) as well as by urease and enterotoxin C production. Eight of the 98 isolates were shown to be other unrelated strains by both PFGE and their production of urease, a misidentification rate of 8% by phage typing. Seventy-one EMRSA-15 isolates were enterotoxin C negative, and the majority of these were sensitive to phage 81. Examination of PFGE profiles and Southern blotting studies suggest that the enterotoxin C gene locus is encoded on a potentially mobile DNA segment of ca. 15 kb. After elimination of the eight non-EMRSA-15 isolates, the remaining 145 were characterized by PFGE, yielding 22 profiles. All profiles were within five band differences of at least one other profile. Classical EMRSA-15 isolates showed nine PFGE profiles, with the majority of isolates (68%) in profile B1. Six of these nine PFGE profiles were unique to the classical EMRSA-15 isolates. Among the phage variants of EMRSA-15, 16 profiles were seen, but the majority of isolates (83%) fell into 1 of 4 profiles (B2, B3, B4, and B7) which correlated well with phage patterns. The most divergent PFGE profiles among the EMRSA-15 isolates had as many as 12 band differences from one another, suggesting that in examining isolates belonging to such a temporally and geographically disseminated epidemic strain, the range of PFGE profiles must be regarded as a continuum and analyzed by relating the profiles back to the most common or progenitor profile.

Large-scale survey of Campylobacter species in human gastroenteritis by PCR and PCR-enzyme-linked immunosorbent assay.

A PCR-based study of the incidence of enteropathogenic campylobacter infection in humans was done on the basis of a detection and identification algorithm consisting of screening PCRs and species identification by PCR-enzyme-linked immunosorbent assay. This was applied to DNA extracted from 3,738 fecal samples from patients with sporadic cases of acute gastroenteritis, submitted by seven regional Public Health Laboratories in England and Wales over a 2-year period. The sending laboratories had cultured "Campylobacter spp." from 464 samples. The PCR methodologies detected 492 Campylobacter-positive samples, and the combination of culture and PCR yielded 543 Campylobacter-positive samples. There was identity (overlap) for 413 samples, but 79 PCR-positive samples were culture negative, and 51 culture-positive samples were PCR negative. While there was no statistically significant difference between PCR and culture in detection of C. jejuni-C. coli (PCR, 478 samples; culture, 461 samples), PCR provided unique data about mixed infections and non-C. jejuni and non- C. coli campylobacters. Mixed infections with C. jejuni and C. coli were found in 19 samples, and mixed infection with C. jejuni and C. upsaliensis was found in one sample; this was not apparent from culture. Eleven cases of gastroenteritis were attributed to C. upsaliensis by PCR, three cases were attributed to C. hyointestinalis, and one case was attributed to C. lari. This represents the highest incidence of C. hyointestinalis yet reported from human gastroenteritis, while the low incidence of C. lari suggests that it is less important in this context.

Development of amplified 16S ribosomal DNA restriction analysis for identification of Actinomyces species and comparison with pyrolysis-mass spectrometry and conventional biochemical tests.

Identification of Actinomyces spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (n = 27) and clinical isolates (n = 36) of Actinomyces spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings obtained by ARDRA were also compared with those obtained by pyrolysis-mass spectrometry. The ARDRA profiles generated by the combination of HaeIII and HpaII endonuclease digestion differentiated all reference strains to the species or subspecies level. The profiles correlated well with the findings obtained by pyrolysis-mass spectrometry and by conventional tests and enabled the identification of 31 of 36 clinical isolates to the species level. ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for routine identification of Actinomyces spp. of clinical origin.

PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes.

A reference library of types of Clostridium difficile has been constructed by PCR ribotyping isolates (n = 2,030) from environmental (n = 89), hospital (n = 1,386), community practitioner (n = 395), veterinary (n = 27), and reference (n = 133) sources. The library consists of 116 distinct types identified on the basis of differences in profiles generated with PCR primers designed to amplify the 16S-23S rRNA gene intergenic spacer region. Isolates from 55% of infections in hospitals in the United Kingdom belonged to one ribotype (type 1), but this type was responsible for only 7. 5% of community infections.

PCR-ribotyping and pyrolysis mass spectrometry fingerprinting of environmental and hospital isolates of Clostridium difficile.

The relationships between environmental isolates of Clostridium difficile were examined by two typing methods, PCR ribotyping and pyrolysis mass spectrometry (PyMS). The 184 isolates were divided into 23 different PCR ribotypes, 13 of which were producers of toxins A and B; the remaining 10 types did not produce either toxin A or B. PyMS analysis resolved 31 groups with 60 (32.5%) isolates in one group (group 9). In both methods most of the isolates showed similar clustering. PCR ribotypes of the environmental isolates were compared with those of clinical isolates that had been typed previously. Seventeen PCR types (13 toxigenic PCR types and four non-toxigenic types) were found in both sets of isolates.

Clostridium difficile-associated diarrhoea: epidemiological data from Western Australia.

The incidence of Clostridium difficile-associated diarrhoea (CDAD) was investigated retrospectively at a 690-bed teaching hospital for the period 1983-92. Our aims were to determine: (i) the distribution by age and sex of patients with CDAD, (ii) the possibility of a seasonal trend and, (iii) the influence of infection control procedures, contamination of the hospital environment and the use of third-generation cephalosporins. The laboratory diagnosis of CDAD was based on demonstration of the organism by stool culture and/or detection of specific cytotoxin in stool filtrates. C. difficile was detected in 917 patients who were being investigated for diarrhoeal illness. Yearly isolations varied from a low of 49 in 1983 to a high of 120 in 1990 (Chi square for linear trend 128.8; P < 0.005). Most patients were elderly, with 63% aged 60 years or more; the majority (59%) were female. The relationship between culture of C. difficile and detection of cytotoxin in faecal extracts was also examined. Sixty percent of a sample of 132 isolates from patients in whom faecal cytotoxin was not detected produced cytotoxin in vitro, suggesting that culture is a more sensitive indicator of infection with C. difficile than cytotoxin detection. When the total number of faecal specimens received in the laboratory was used as a denominator there was an increase in the number of incident cases of CDAD between 1983 and 1990, apart from 1986. When occupied bed days was used as the denominator a similar trend was observed with a peak in 1990.(ABSTRACT TRUNCATED AT 250 WORDS)

Ciprofloxacin and Clostridium difficile-associated diarrhoea.

Recent reports have implicated ciprofloxacin as a cause of Clostridium difficile-associated diarrhoea. This problem was examined in three ways. First, the MIC of ciprofloxacin for C. difficile was determined. The MIC range was 8-32 mg/L, with C. difficile were 'treated' with ciprofloxacin and clindamycin in a test-tube, and the growth of C. difficile monitored. The clindamycin-treated emulsions supported growth of C. difficile, while the ciprofloxacin-treated and control emulsions did not differ significantly and failed to support the growth of C. difficile. Finally, 213 patients on ciprofloxacin monotherapy were investigated. Twenty-nine patients were given ciprofloxacin as treatment for diarrhoea, while a further 15 patients developed diarrhoea while being treated. None of these 44 patients harboured C. difficile. Faecal samples from 73 of the remaining 169 patients who did not have or develop diarrhoea were investigated for C. difficile, but none was positive. It was concluded that ciprofloxacin is unlikely to promote C. difficile-associated diarrhoea.

Relapse versus reinfection with Clostridium difficile.

Relapse of Clostridium difficile-associated diarrhoea occurs in 15-20% of patients; however, whether relapse is due to an endogenous source of the organism or reinfection from the environment remains unclear. Restriction enzyme analysis (REA) of chromosomal DNA was used to type multiple isolates from ten patients who had experienced apparent relapses. More than half the relapses were due to infection with a new strain of C. difficile. The remaining patients were infected with the same strain, but whether this strain was acquired from the environment or from endogenous sources could not be determined. Relapses with a different strain of C. difficile could occur if an individual harboured more than one strain in their gastrointestinal tract. To investigate this possibility ten other patients were assessed for carriage of multiple strains. Ten colonies from a primary culture plate from each patient were typed by REA and tested for their ability to produce cytotoxin. All isolates from the same patient were identical by both methods, indicating that multiple carriage of strains may be a rare event.

Gastrointestinal carriage of Clostridium difficile in cats and dogs attending veterinary clinics.

Cats and dogs being treated at two veterinary clinics were investigated for gastrointestinal carriage of Clostridium difficile using selective solid and enrichment media. Thirty-two (39.5%) of 81 stool samples yielded C. difficile. There were significant differences in isolation rates between clinics, 61.0% of animals being positive at one clinic compared to 17.5% at the other (Chi-square, P less than 0.005). Of 29 animals receiving antibiotics, 15 (52.0%) harboured C. difficile while 11 (23.9%) of 46 animals not receiving antibiotics were positive (Chi-square, P less than 0.01). There was no difference in carriage rate between cats (38.1%) and dogs (40.0%). The environment at both veterinary clinics was surveyed for the presence of C. difficile. Fifteen of 20 sites at one clinic were positive compared to 6 of 14 sites at the other clinic. Both cytotoxigenic and noncytotoxigenic isolates of C. difficile were recovered from animals and environmental sites. These findings suggest that household pets may be a potentially significant reservoir of infection with C. difficile.

Non-radioactive restriction fragment length polymorphism (RFLP) typing of Clostridium difficile.

A typing method for Clostridium difficile based on restriction fragment length polymorphisms (RFLP) is described. The technique utilizes commercially available Escherichia coli ribosomal ribonucleic acid (rRNA) as probe material. Probe labelling, hybridization and detection was performed using the Enhanced Chemiluminescence (ECL) gene detection system. The probe labelling procedure was easy to perform, taking only 20 min. The complete typing method was comparatively simple, reproducible and readily adaptable to most bacterial genera.