Irish-1 and Irish-2: UK epidemic meticillin-resistant Staphylococcus aureus strains associated with Northern Ireland.

Since 1998, an increasing number of meticillin-resistant Staphylococcus aureus (MRSA) isolates with one of two characteristic phage patterns have been referred to the authors' laboratory from Northern Ireland. These strains were designated 'Irish-1' and 'Irish-2'. Analysis of 956 submitted isolates classified as Irish-1 or Irish-2 showed that 97% of the former and 95% of the latter were from Northern Ireland. Only 0.2% and 3%, respectively, were from England. Eleven Irish-2 isolates had been referred from Western Australia as representatives of an epidemic strain originally isolated there in 1994. Ninety isolates with the Irish-1 phage pattern and 91 isolates with the Irish-2 phage pattern, from numerous hospitals, were characterized by SmaI pulsed-field gel electrophoresis (PFGE), toxin gene carriage and antibiotic susceptibility. PFGE showed that, within each collection, a few isolates represented unrelated strains, but the majority were within six band differences of the most common profiles. Half of the Irish-1 isolates were homogeneous, with 22 DNA profiles among the remainder. Irish-2 isolates had two common profiles, D1 and D2, equally divided between one-third of the isolates and differing from each other by two bands; the remaining isolates shared 31 DNA profiles. Cluster analysis showed some overlap in DNA profiles between the Irish-1 and Irish-2 strains, but clear separation from other epidemic MRSA strains. There was no obvious correlation between PFGE profile and either antibiotic resistance pattern or toxin gene possession. All but three Irish-1 isolates possessed only the staphylococcal enterotoxin A (sea) gene, whereas almost all Irish-2 isolates were negative for all 12 enterotoxin genes. Sixty-nine percent of Irish-2 isolates were resistant to ciprofloxacin, erythromycin, kanamycin, neomycin and streptomycin, while 90% of Irish-1 isolates were resistant to all these plus gentamicin and mupirocin. All isolates were sensitive to quinupristin/dalfopristin, teicoplanin and vancomycin. Urease production was negative in both strains. The results suggest that Irish-1 and Irish-2 are distinct epidemic strains, identifiable by phage typing, DNA profiles, antibiotic resistance and toxin gene carriage.

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.

Increased serum resistance in Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases.

The aim of this study was to determine whether there is an association between serum resistance, O serotypes, and the production of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae. Ninety ESBL-producing and 178 non-ESBL-producing K. pneumoniae isolates gathered in five European countries were O serotyped and tested for sensitivity to the serum's bactericidal effect. The frequency of serum-resistant isolates was higher among ESBL-producing strains (30%; 27/90 isolates) than among non-ESBL-producing strains (17.9%; 32/178 isolates) (P = 0.037; odds ratio [OR] = 1.96; 95% confidence interval [95% CI] = 1.08 to 3.53). Although O1 was the most common O serotype in both Klebsiella groups, its frequency among ESBL-producing strains was significantly higher (59%; 53/90 isolates) than among non-ESBL producers (36%; 64/178 isolates) (P = 0.0006; OR = 2.5; 95% CI = 1.52 to 4.29). Furthermore, the prevalence of the O1 serotype was higher among serum-resistant strains of both ESBL-producing (74%; 20/27isolates) and non-ESBL producers (75%; 24/32 isolates) than among serum-sensitive ESBL producers (52.4%; 33/63 isolates) and non-ESBL producers (27.4%; 40/146 isolates). Serum resistance among ESBL-producing strains (36%; 17/47 isolates) versus non-ESBL-producing strains (16%; 27/166 isolates) was also significantly higher after the exclusion of clonal strains (P = 0.0056; OR = 2.9; 95% CI = 1.41 to 6.01). Sixteen ESBL types were detected, among which the frequency of serum resistance was significantly lower among the SHV-producing strains (9/48 isolates) than among the TEM producers (16/35 isolates) (P = 0.016; OR = 3.65; CI = 1.3 to 9.7). Curing ESBL-coding plasmids did not influence the serum resistance of the bacteria; all six plasmid-cured derivatives maintained serum resistance. The present findings suggest that ESBL-producing strains have a greater pathogenic potential than non-ESBL-producing strains, but the linkage between O serotypes, serum resistance, and ESBL production remains unclear at this stage.

Impairment of respiratory burst in polymorphonuclear leukocytes by extended-spectrum beta-lactamase-producing strains of Klebsiella pneumoniae.

The ability of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing Klebsiella pneumoniae strains to induce a respiratory burst in polymorphonuclear leukocytes (PMNLs) was investigated. Ninety ESBL-producing and 178 non-ESBL-producing Klebsiella pneumoniae isolates were serotyped and their ability to induce a respiratory burst in PMNLs tested by monitoring the cells' chemiluminescence (CL) response. The percentage of isolates inducing high levels of CL response (CL>75%) was significantly higher among non-ESBL producers (52%) than among ESBL producers (32.2%) ( P<0.0001; OR=3.396; 95%CI=2.036-5.664). The median CL response was significantly higher among the non-ESBL producers (76.9%) than among the ESBL producers (52.6%) ( P=0.034). The two groups did not differ in their ability to resist intracellular killing by PMNLs ( P>0.05), with strains inducing high levels of CL response having significantly lower survival rates (31.8% vs. 42.4%) than strains inducing low levels of CL response (164% vs. 200%) ( P<0.01). The frequencies of the K2 and the K25 serotypes were significantly higher among ESBL-producing strains (17.8% and 22.2%, respectively) than among the non-ESBL producers (6.2% and 1.7%, respectively) ( P=0.0057 and P<0.0001). Of the 77 Klebsiella K serotypes, 71 were detectable among the non-ESBL producers, but only 24 were detectable among the ESBL producers. ESBL-producing Klebsiella pneumoniae strains might have a greater pathogenic potential by virtue of their ability to escape the phagocytic activity of PMNLs.

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.

Rate of carriage of Serratia marcescens in patients with and without evidence of infection.

The epidemiology of Serratia marcescens is poorly understood. We designed a study to investigate carriage sites of the organism, and possible modes of transmission of infection. Using Sorbitol-MacConkey agar with colistin 200 IU/ml and MacConkey agar with a 10 microg colistin disc we performed cultures from various sites in patients already infected with S. marcescens. Over the same period of time we also investigated all patients in the intensive care unit (ICU) for colonization with the agent. Environmental screening was performed in the ICU only. Of 37 infected patients, 65% demonstrated carriage at a second site and 43% at multiple sites. Throat carriage was found in 59%, faecal carriage in 42%, nasal carriage in 31% and urinary carriage in 22%. Carriage over several weeks was found in 22%. Of 40 ICU patients, 10% demonstrated nasal and/or throat carriage. Environmental screening yielded 4 isolates. All ICU patient strains and a strain from the ICU bedpan macerator were O14:K14 with similar random amplified polymorphic DNA types. These results show that patients with S. marcescens infection are likely to carry the organism at multiple sites and that carriage may be prolonged. A significant level of carriage was also found in non-infected patients in a unit where the organism was prevalent.

Interpretation of band differences to distinguish strains of Serratia marcescens by pulsed-field gel electrophoresis of XbaI DNA digests.

The number of band differences in DNA macrorestriction profiles required to distinguish unrelated strains from an index strain varies in an outbreak with the species and restriction enzyme used. In order to define this difference for epidemiological studies of Serratia marcescens, we produced DNA fingerprints from 57 isolates of the organism using the restriction enzyme XbaI and pulsed-field gel electrophoresis (PFGE). The isolates were selected on the basis of their epidemiology, serotype and phage-typing patterns to include 28 unrelated strains and 29 representatives from 2 distinct outbreaks. One of the outbreaks was prolonged. lasting for several years. Electrophoretic profiles consisting of 20 or more clearly resolved bands were obtained for all isolates. Twenty-six of the unrelated strains had unique profiles with over 10 band differences from all other strains, while 27 of the outbreak representatives could be assigned to the appropriate outbreak with confidence. The majority of the outbreak isolates had none or 2 band differences from the index profile, although 3 isolates differed by 5-7 bands. The 2 exceptions among the unrelated strains differed by 4 bands, and 3 phage typing reactions, and were isolated from London and Berlin 3 years apart, while the 2 exceptions among the outbreak collection had clearly unique profiles with over 20 band differences from each other and the outbreak profiles. Cluster analysis using Dice coefficient and UPGMA gave cut-off values of 75-78% similarity overall for related isolates, while the closest similarity for unrelated strains was 70%. The results of this study together with those of the 6 previous reports of PFGE for S. marcescens (which used either enzymes XbaI or SpeI) confirm that this technique is of value for this species and that with XbaI at least, most epidemiologically related strains will only differ by 3-4 bands. However, on occasion up to 7 band differences can be found within an apparent outbreak, which may be suggestive of genetic drift.

Epidemiology and susceptibility of serratia marcescens in a large general hospital over an 8-year period.

Over the 8 year period 1988-1995, 1367 isolates of Serratia marcescens were isolated from 582 patients on 12 different wards of a large Dublin hospital and were particularly associated with the surgical intensive care unit. The annual incidence was over 200 isolates from 1990 to 1992 but fell to below 100 following the opening in April 1992 of a replacement surgical hospital incorporating a new intensive care unit on the same site. The most common source of S. marcescens was sputum from patients. Strain identities were determined by serotyping and phage typing at least one isolate from each of 311 of the 582 patients. The results showed that a single epidemic strain of serotype O14:K14 was present in 69% of these patients, and persisted throughout the hospital for the whole of the eight-year period. This strain was recovered from a variety of clinical specimens, including blood cultures. A minor outbreak involving a serotype O16:K28 strain also occurred and this strain also persisted from at least 1989 to 1994. Extensive surveillance failed to reveal an environmental source or faecal carriage. The likely mode of transmission appears to have been via staff hands from both symptomatic and asymptomatic patients acting as reservoirs of the organism, as has commonly been reported for this species.

Isolation of Acinetobacter spp. including A. baumannii from vegetables: implications for hospital-acquired infections.

A. baumannii is rarely recovered from the skin of patients or healthy European subjects as other genospecies predominate, but it isa significant nosocomial pathogen. The natural reservoir of this organism is therefore uncertain. We determined the isolation rates of Acinetobacter spp. from vegetables (as an indicator of the natural environment) using a selective technique and classified the genospecies by amplified ribosomal DNA restriction analysis (ARDRA). Of the 177 samples of vegetables examined, 30 yielded Acinetobacter, with genospecies 2 and 11 being the most common, each with a frequency of 27%. MIC assays showed that strains of genospecies 1, 2, 3, and 13TU (the A. calcoaceticus-A. baumannii complex) were significantly more resistant than other genospecies to ciprofloxacin and gentamicin. Vegetables may therefore be a natural habitat of A. baumannii and provide a route by which these bacteria are introduced into hospitals with obvious implications for infection control.

Distribution of Acinetobacter species on skin of healthy humans.

The distribution of the 19 currently known genospecies of Acinetobacter on human skin, i.e. forehead, forearm and toe webs, was determined. Three selective media were compared for their specificity for all genospecies of Acinetobacter. A minimal-salts agar supplemented with 1% acetate proved to be more efficient than the Leeds medium for the isolation of most genospecies in mixed culture with other bacterial species. Acinetobacter isolates were provisionally identified using biochemical tests and the DNA transformation assay of Juni. Genospecies identification was performed using amplified ribosomal DNA restriction analysis, and duplicate isolates of the same genospecies from individuals were ruled out by random amplified polymorphic DNA analysis. Over 40% of 192 healthy volunteers carried Acinetobacter spp. at one or more body sites, and the frequencies of colonisation were as follows: forearm (51%), forehead (47%) and toe web (34%). Genospecies 8/9 (Acinetobacter lwoffii) was the most common (61%), followed by genospecies 15BJ and 12 (Acinetobacter radioresistens) at 12.5% and 8%, respectively. The Acinetobacter baumannii-Acinetobacter calcoaceticus group (genospecies 1, 2, 3 and 13TU) that predominates in hospital-acquired infections was found in only one individual.

Different O and K serotype distributions among clinical and environmental strains of Serratia marcescens.

Recent revision of the O serotyping scheme for Serratia marcescens has allowed the definitive serological identification of a collection of 511 epidemiologically distinct strains in terms of both lipopolysaccharide (O) antigens and capsular (K) antigens. High levels of typability were achieved, 88% and 91% respectively, with only 2% failing to type with either method. In most cases, non-typability was due to a lack of antigen, i.e., the strains produced only rough LPS or were acapsular, suggesting that typability would be little improved by the discovery of additional serotypes. The distribution of the 58 O:K serotypes was very uneven, with O14:K14 accounting for 30% of the 423 clinical strains in the collection, but only 5% of the 88 non-clinical, environmental strains. Thus, the prevalence of O14:K14 strains in hospitals is not reflected in the environment. Similar conclusions were valid for O27:K14, O21:K3 and O21:K14 strains, as well as those with rough lipopolysaccharide. Conversely, the proportions of O6:K3, O6:K14, O8:K14 or O28:K28 strains were significantly lower among the clinical collection than among their environmental counterparts (12% in total rather than 65%). This suggests that O14:K14 may have a selective advantage in colonising or infecting hospitalised patients and, therefore, that the O14 and K14 polysaccharides themselves may contribute towards the apparent pathogenicity of these serotypes.

Antibiotic resistance and putative virulence factors of Serratia marcescens with respect to O and K serotypes.

Serratia marcescens serotypes O6:K14, O8:K14 and O28:K28 are common in the natural environment, but rare in hospitals. Serotypes O14:K14 and O27:K14 predominate among clinical strains, but not in the environment, suggesting that the latter serotypes may be more suited for survival in the clinical setting. Consequently, 469 epidemiologically distinct strains of S. marcescens were tested for various putative virulence factors and analysed for associations with serotype. The factors positively associated with serotype O14:K14 were agglutination of five different species of red blood cells and expression of type 1 fimbriae. These were found in 63% and 53% of O14:K14 strains, respectively, compared with 7% and 12% of the three 'environmental serotypes'. Almost a quarter of the collection expressed the mannose-resistant haemagglutinin indicative of type 3 fimbriae, but this was not associated with any serotype. The production of DNAase, haemolysin, lipase, lecithinase, proteases and siderophores was almost universal and showed no serotype correlations. Almost half of the strains (46%) were resistant to serum and serotypes O27:K14 and O6:K14 were strongly associated with this characteristic. Serotype O27:K14 was also associated with higher proportions of antibiotic-resistant strains than other serotypes, but the same was not true of serotype O14:K14. All three 'environmental serotypes' were associated with low frequencies of antibiotic resistance; <12% were resistant to gentamicin, carbenicillin or piperacillin, or any combination of these three, compared with 20-25% of O14:K14 strains and >42-51% of O27:K14 strains. Pigment production was strongly associated with serotype. None of the O14:K14 or O27:K14 strains produced prodigiosin, but frequencies for the three 'environmental serotypes' ranged from 31% of O28:K28 strains to 85% of O6:K14 strains. The results of this study suggest that the adherence capability of S. marcescens strains may play a role in the colonisation of hospital patients, while the production of prodigiosin is a marker of environmental origin.

Persistence of Pseudomonas aeruginosa strains in respiratory infection in AIDS patients.

OBJECTIVES: To establish the clinical pattern of Pseudomonas aeruginosa respiratory infections in HIV-seropositive patients and to determine whether repeated isolation of the organism represents reinfection or recurrence and to assess whether common source, nosocomial infection occurred. DESIGN AND METHODS: Evaluation of the clinical pattern of P. aeruginosa respiratory infections by case note review and epidemiological characterization of P. aeruginosa by serotype determination and Xbal DNA macrorestriction analysis. Serum sensitivity testing of strains was performed to further define phenotypic characteristics of the isolated organisms. RESULTS: Seventy-three per cent (29 out of 40) of individuals had P. aeruginosa isolated on two or more occasions in the setting of clinical respiratory infection. Overall, 85% had evidence of P. aeruginosa to within 2 months of study completion or death. Epidemiological characterization revealed persistence of unique single strains in 93% of individuals where multiple isolates were available for testing, whereas only two patients harboured a common strain. The serotype distribution of strains was similar to that reported from non-HIV-positive patients. CONCLUSIONS: Once established, eradication of P. aeruginosa from the respiratory tract of HIV-seropositive individuals with advanced immunosuppression is problematic and a chronic infective state appears common. There was no evidence of nosocomial transmission. Serotype loss and development of sensitivity to normal human serum were both observed and were highly correlated. This represents truncation of O-antigenic lipopolysaccharide on the cell surface of P. aeruginosa and may reflect progression to phenotypes commonly associated with chronic infection in other clinical settings such as cystic fibrosis.

Epidemiological typing of klebsiellae with extended-spectrum beta-lactamases from European intensive care units.

Extended-spectrum beta-lactamases (ESBLs) are an increasing cause of resistance to oxyimino-aminothiazolyl cephalosporins, especially in klebsiellae. In a recent survey we detected ESBLs in 220 (23%) of 966 consecutive klebsiellae from 35 intensive care units (ICUs) in southern and western Europe. The present study examined the extent to which this distribution reflected epidemic strain spread, as against the distribution of ESBL genes into unrelated strains. All 220 ESBL producers were subjected to capsular serotyping and pulsed-field gel DNA electrophoresis (PFGE). Beta-Lactamases were typed for strains isolated on three or more occasions, with the emphasis on SHV enzymes, as these were commoner than TEM variants. Serotyping and PFGE typing defined 85 distinct strains, from 23 of the 35 participating centres. Of 14 centres that contributed five or more ESBL producers, all sent representatives of more than one strain, and two centres sent members of ten or more different strains in contributions of 17-21 ESBL-producing isolates. Nevertheless, epidemic strains-defined as those represented by three or more isolates-accounted for a majority (61%) of the collection. Fifty-two isolates of the same serotype K25 (occasionally acapsular) strain with SHV-4 beta-lactamase were recovered at two French hospitals and one in Belgium. This strain has been found by others in France, and has become particularly widespread. Another single strain was found in two separate Portuguese centres, and many individual hospitals had one or more epidemic strain(s), as well as a scatter of diverse ESBL producers. Major variation in antibiogram and plasmid profile was apparent within strains, with some intra-strain variation in beta-lactamase type. These data imply a fluid situation, with resistance determinants being gained, modified or lost. The endemicity of ESBL producers is disturbing since it limits the potential for control by blocking strain spread, while the diversity within strains is disturbing because it complicates the design of antibiotic policies even during 'single strain' outbreaks.

Identification of capsular antigens in Serratia marcescens.

Previous studies with 31 strains of Serratia marcescens, including 28 reference O-serotype strains, have indicated that 19 of them have an acidic polysaccharide which copurifies with lipopolysaccharide during phenol-water extraction. Polysaccharide in crude extracts from 18 of the 19 strains was precipitated with Cetavlon (hexadecyltrimethyl ammonium bromide), and capsules were demonstrated around these 18 strains by Indian ink exclusion zones. Capsule-antibody binding by the Quellung reaction suggested that the acidic polysaccharide formed the capsule around the bacterial cells. Anticapsular (anti-K) antibody was detected in reference O antisera which had been prepared against boiled whole cells. Cross-titration and absorption studies revealed 14 different K antigens among these strains.