Evaluation of a nine-locus variable-number tandem-repeat scheme for typing of Pseudomonas aeruginosa.

Repeat numbers at nine variable-number tandem-repeat (VNTR) loci were determined for 177 isolates of Pseudomonas aeruginosa representing 77 strains distinguished by pulsed-field gel electrophoresis (PFGE). Eight loci provided for discrimination similar to that provided by PFGE, with variation at the ninth locus (ms61) sometimes allowing discrimination within a PFGE-defined type. The Liverpool and Midlands 1 strains, which are common among patients with cystic fibrosis in the UK, could be unambiguously identified by their characteristic VNTR profiles. In rare cases, the repeat number at the ninth locus alone provided discrimination among isolates that were distinct according to PFGE. In each case, the two isolates shared the same bla(OXA-50-like) allele and belonged to the same oprD sequence type group, supporting the VNTR results in suggesting that they are similar.

Rapid identification of uropathogenic Escherichia coli of the O25:H4-ST131 clonal lineage using the DiversiLab repetitive sequence-based PCR system.

Several recent studies have highlighted the emergence of a globally disseminated clone of uropathogenic and invasive Escherichia coli isolates of serotype O25:H4 and sequence type 131. The ability to characterize rapidly E. coli isolates of this lineage would facilitate enhanced surveillance for this pathogen. We have used the semi-automated DiversiLab repetitive PCR-based system to analyse a collection of 35 clinical isolates of uropathogenic E. coli from across the UK, with particular focus on the O25:H4-ST131 lineage. All isolates had been characterized using multilocus sequence typing (MLST), and 14 had previously been typed using pulsed-field gel electrophoresis (PFGE). The DiversiLab system allowed discrimination of O25:H4-ST131 isolates from those of other E. coli lineages. It was slightly more discriminatory than MLST, but was less discriminatory than PFGE. With an analysis time of <4 h between receipt of a cultured organism and provision of a typing result, the system offers information on a real-time basis, a major advantage over current practice. We suggest that introduction of the DiversiLab system would be useful for rapid exclusion of E. coli isolates during outbreak investigations, and that the approach could be employed for surveillance for pathogenic or antibiotic-resistant clones of this organism.

Variable number tandem repeat loci providing discrimination within widespread genotypes of Acinetobacter baumannii.

Some genotypes of Acinetobacter baumannii, defined by pulsed-field gel electrophoresis (PFGE), have been found in many hospitals. Our aim was to find variable number tandem repeat (VNTR) loci capable of providing discrimination among isolates with highly similar or identical PFGE profiles, to gain insights into the epidemiology. Thirteen loci identified in A. baumannii ATCC 17978 were tested using a panel of isolates that included multiple representatives of genotypes belonging to the three European clonal lineages. Two loci, with repeat units of 9 and 6 bp respectively were selected. Repeat numbers varied between 3 and 29, and 9 and 26 respectively at the two loci. The repeat numbers of representatives of each genotype often differed between hospitals, providing a means of tracking patient transfers and possible transmissions between patients. The results suggest that this analysis accurately reflects the known epidemiological information, and provides a valuable tool for cross-infection studies.

Use of sequence-based typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii.

Representatives (n = 31) of outbreak strains of Acinetobacter baumannii from five countries fell into three clear groups, designated Groups 1-3, based on their ompA (outer-membrane protein A), csuE (part of a pilus assembly system required for biofilm formation) and bla(OXA-51-like) (the intrinsic carbapenemase gene in A. baumannii) gene sequences. With the exception of the closely related alleles within the Group 1 clonal complex, alleles at each locus were highly distinct from each other, with a minimum of 14 nucleotide differences between any two alleles. Isolates within a group shared the same combination of alleles at the three loci, providing compelling evidence that the outbreak strains investigated belonged to three clonal lineages. These corresponded to the previously identified European clones I-III. Sequence differences among the alleles were used to design multiplex PCRs to rapidly assign isolates belonging to particular genotypes to sequence groups. In the UK, genotypes belonging to the Group 1 clonal complex have been particularly successful, accounting for the vast majority of isolates referred from hospitals experiencing problems with Acinetobacter.

An outbreak of wound infection in cardiac surgery patients caused by Enterobacter cloacae arising from cardioplegia ice.

This paper describes an outbreak of postoperative sternal wound infections. A cardiac surgeon noted a cluster of serious infections leading to wound dehiscence, despite the fact that none of his colleagues had noticed a rise in infection rates. The infections were predominantly with Enterobacter cloacae, and molecular typing and serotyping showed these isolates to be indistinguishable. Observation of the surgeon's practice revealed nothing untoward, and there were no infections among his patients operated on in another hospital. There appeared to be no significant difference between the modes of operation of the different surgeons. The operating theatres were screened to exclude an environmental source, with samples cultured on CHROMagar Orientation, a selective/differential medium designed for urine samples. Further questioning revealed one difference between the practices of the different surgeons; this surgeon used semi-frozen Hartmann's solution to achieve cardioplegia. The freezer used for this was swabbed and yielded E. cloacae, indistinguishable from the clinical isolates. It is hypothesized that this organism contaminated the freezer, and that the contamination was passed on to the ice/slush solution, thus infecting the patients. There have been no more cases since the freezer was replaced, a rigorous cleaning schedule instituted, and steps taken to reduce the possibility of any further contamination.

Epidemiology of infections caused by extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp.: a nested case-control study from a tertiary hospital in London.

Information on risk factors for acquisition of extended-spectrum ss-lactamase (ESBL)-producing organisms and their outcomes in patients with invasive infections is scant. The objectives of this study were to evaluate risk factors and all-cause mortality associated with infection due to ESBL-producing organisms using a nested case-control design, and to document transmission within a hospital employing molecular and conventional epidemiological methods. From December 2003 to April 2005, 50 patients with bloodstream infections (BSIs) due to ESBL-producing E. coli and Klebsiella spp. were recruited. Controls (N=50) were chosen, within the same period, from patients with non-ESBL-producing BSIs by simple random sampling; account was taken of potential confounding factors. Cases and controls were followed-up until November 2005, and outcomes were recorded as discharged or deceased. Molecular methods, supported by conventional epidemiology, were used to study the transmission of organisms. Logistic regression analyses showed prior ss-lactam antibiotics [odds ratio (OR) 11.57; 95% confidence intervals (CI) 2.31-51.15; P=0.003], hospital stay >15 days (OR 2.63; 95% CI 1.01-6.89; P=0.04) and prior admission to the intensive care unit (OR 13.98; 95% CI 1.88-19.15; P=0.006) to be independent risk factors for the acquisition of ESBL-producing organisms. In the first 15 days of follow-up, a significant proportion of patients with ESBL-producing organisms died; however, there was no difference in mortality between cases and controls at the end of the follow-up period. Molecular epidemiology identified five clusters amongst the ESBL-producing isolates. Conventional epidemiological analyses supported the evidence of transmission in three of these clusters.

Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-8, a novel metallo-beta-lactamase, in a tertiary care center in Cali, Colombia.

The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates at a 195-bed tertiary care medical center in Cali, Colombia, rose from 2% in 1996 to 28% in 1997 and to over 40% in 2003. Many isolates showed high-level multiresistance, and phenotypic characterization suggested the spread of a predominant strain with minor variants. Sixty-six resistant isolates collected between February 1999 and July 2003 from hospitalized patients (n = 54) and environmental samples (n = 12) were subjected to a fuller analysis. Genetic fingerprints were compared by pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA, and bla(IMP) and bla(VIM) genes were sought by PCR. PFGE and serotyping indicated that 52 of the 66 isolates belonged to a single strain, with 82% similarity; the PFGE pattern for this organism was designated pattern A. Two further pairs of isolates represented single strains; the remaining nine isolates were unique, and in the case of one isolate, no satisfactory PFGE profile could be obtained. The pattern A isolates were mostly of serotype O12 and were highly resistant to imipenem (MICs, 32 to >256 microg/ml), with this resistance decreased eightfold or more in the presence of EDTA. They yielded amplicons with bla(VIM)-specific primers, and sequencing of DNA from a representative isolate revealed bla(VIM-8), a novel allele with three polymorphisms compared with the sequence of bla(VIM-2). Two of these nucleotide changes were silent, but the third determined a Thr139Ala substitution. Only 4 of 13 resistant isolates (2 clinical isolates and 2 environmental isolates) assigned to other PFGE types carried bla(VIM) alleles, whereas the others were less multiresistant and mostly had lower levels of imipenem resistance (MICs, < or =32 microg/ml) which was not significantly reduced by EDTA. No bla(IMP) alleles were detected. During 2003, when the environmental study was undertaken, serotype O12 isolates with bla(VIM) were recovered from sinks and stethoscopes in the most-affected units, although not from the hands of staff; the problem declined once these reservoirs were disinfected and hygienic precautions were reinforced.

A prevalent, multiresistant clone of Acinetobacter baumannii in Southeast England.

A multiresistant clone of Acinetobacter baumannii was identified in 24 hospitals in the UK, predominantly in the London area, over a period of three years. Isolates were characterized by distinctive ApaI macrorestriction profiles, as resolved by pulsed-field gel electrophoresis (PFGE), which all clustered within 80% similarity using a 1% band position tolerance setting. The first isolates identified were received by the reference laboratories in April 2000, and by June 2003, a total of 375 isolates with similar PFGE profiles from 310 patients from 24 hospitals had been received. The isolates originated mainly from sputum and wound specimens, with the majority from patients in intensive care units. Amplified fragment length polymorphism analysis of a subset of isolates showed that they clustered closely, supporting the PFGE results. All the isolates tested were highly resistant to ampicillin, piperacillin, piperacillin/tazobactam, ceftazidime, cefotaxime, gentamicin and ciprofloxacin, and most isolates were carbapenem resistant. Amikacin sensitivity varied from susceptible [minimum inhibitory concentration (MIC) 256 mg/L).

Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum beta-lactamases in the UK.

OBJECTIVES: During 2003, the Health Protection Agency's Antibiotic Resistance Monitoring and Reference Laboratory began to receive isolates of Escherichia coli for confirmation of extended-spectrum beta-lactamase production with a phenotype implying a CTX-M-type beta-lactamase, i.e. MICs of cefotaxime > or = 8-fold higher than MICs of ceftazidime. Many were referred as being from community patients. We examined 291 CTX-M-producing isolates from the UK and investigated the genetic basis of their phenotype. METHODS: PCR was used to detect alleles encoding CTX-M enzymes and to assign these to their blaCTX-M phylogenetic groups. Selected alleles were sequenced. Producers were compared by analysis of banding patterns generated by pulsed-field gel electrophoresis of XbaI-digested genomic DNA. MICs were determined by an agar dilution method or by Etest. RESULTS: Of 291 CTX-M-producing E. coli isolates studied from 42 UK centres, 70 (24%) were reportedly from community patients, many of whom had only limited recent hospital contact. Community isolates were referred by 12 centres. Two hundred and seventy-nine (95.9%) producers contained genes encoding group 1 CTX-M enzymes and 12 contained blaCTX-M-9-like alleles. An epidemic CTX-M-15-producing strain was identified, with 110 community and inpatient isolates referred from six centres. Representatives of four other major strains also produced CTX-M-15, as did several sporadic isolates examined. Most producers were multi-resistant to fluoroquinolones, trimethoprim, tetracycline and aminoglycosides as well as to non-carbapenem beta-lactams. CONCLUSIONS: CTX-M-producing E. coli are a rapidly developing problem in the UK, with CTX-M-15 particularly common. The diversity of producers and geographical scatter of referring laboratories indicates wide dissemination of blaCTX-M genes. Because of the public health implications, including for the treatment of community-acquired urinary tract infections, the spread of these strains--and CTX-M-15 beta-lactamase in particular--merits close monitoring.

Emerging linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolated from two Austrian patients in the same intensive care unit.

Of two patients in the same intensive care unit who were treated with linezolid, one yielded linezolid-resistant Enterococcus faecalis, whereas the other yielded linezolid-resistant Enterococcus faecium. In each case, molecular typing indicated that the resistant isolates were related to linezolid-susceptible isolates from the same patient, but differed from them by the same G2576U ribosomal RNA mutation. This is the first clinical case report of emerging resistance to linezolid among Enterococcus faecalis and also the first report of resistance involving vancomycin-susceptible rather than vancomycin-resistant enterococci. The linezolid-resistant isolates showed cross-resistance to the experimental oxazolidinone AZD2563, suggesting that oxazolidinone resistance might be a class effect.

The epidemiology of glycopeptide-resistant enterococci on a haematology unit--analysis by pulsed-field gel electrophoresis.

As part of an interventional study to determine glycopeptide-resistant enterococci (GRE) acquisition on a three-ward haematology unit, rectal swabs were taken weekly from 293 patients recruited to the study between June 1995 and December 1996. The GRE isolates obtained from the first positive rectal swab from 120 colonized patients, the isolates from 7 patients with clinical infection and 43 isolates obtained from the ward environment were compared by pulsed-field gel electrophoresis (PFGE). Sixty-three of 120 patients were colonized by one of strains A-H, while 49 were colonized by unique strains. The first 18 weeks were associated with the highest prevalence of GRE by rectal swab, with a single strain A responsible for 52% of acquisitions on ward 2, 22% on ward 3 and 36% on ward 4. Other smaller ward associated clusters were evident. Environmental sampling of ward 2 during this time showed that all but 2 of 30 isolates were indistinguishable from strain A. As the GRE prevalence fell, rectal swab and environmental isolates became more heterogeneous, and strain A disappeared after week 55. GRE prevalence rose again in the final 15 weeks of the study, and a new predominant strain B emerged on ward 2 responsible for 50% of new acquisitions. In the seven patients with clinical infection with GRE, the clinical isolates were compared with the contemporaneous rectal swab isolate, and were found to be the same in only two cases. An analysis of five long-term carriers colonized for a median of 19 weeks (range 11-34) showed colonization with at least two and in one case six distinct strains, raising the question of how many strains may be colonizing a patient at any one time, and suggesting that multiple colonies should be analysed. These data suggest that cross-infection was an important factor in the spread of GRE when the colonization rate was high.

Rapid and accurate identification of coagulase-negative staphylococci by real-time PCR.

Biprobe identification assays based on real-time PCR were designed for 15 species of coagulase-negative staphylococci (CNS). Three sets of primers and four biprobes were designed from two variable regions of the 16S rRNA gene. An identification scheme was developed based on the pattern of melting peaks observed with the four biprobes that had been tested on 24 type strains. This scheme was then tested on 100 previously identified clinical isolates and 42 blindly tested isolates. For 125 of the 142 clinical isolates there was a perfect correlation between the biprobe identification and the result of the ID 32 Staph phenotypic tests and PCR. For 12 of the other isolates a 300-bp portion of the 16S rRNA gene was sequenced to determine identity. The remaining five isolates could not be fully identified. LightCycler real-time PCR allowed rapid and accurate identification of the important CNS implicated in infection.

Characterisation of VanA and VanB elements from glycopeptide-resistant Enterococcus faecium from Greece.

Ten glycopeptide-resistant Enterococcus faecium isolates from separate patients in Laikon General Hospital, Athens were studied. Eight isolates had the VanA phenotype and represented variants of three strains based on SmaI macrorestriction banding patterns. Their VanA elements were compared with the prototype element, Tn1546, by an overlapping PCR method. Three related isolates contained resistance elements indistinguishable from Tn1546 (designated Greek type I). The other five isolates all contained identical elements that differed from Tn1546 by the presence of IS1251 between vanS and vanH, by a point mutation (G --> T) at nucleotide position 8234 within vanX and by a partial loss of transposition gene orf1 (designated Greek type II). Two distinct strains of E. faecium with the VanB phenotype were obtained. HhaI digestion of an amplified fragment of the vanB gene indicated that both strains contained the vanB2 allele, and further PCR assays confirmed that the vanB2 gene cluster was located within a Tn5382-like element.

Chryseobacterium (Flavobacterium) meningosepticum outbreak associated with colonization of water taps in a neonatal intensive care unit.

From September 1994 to May 1996, a strain of multi-resistant Chryseobacterium (Flavobacterium) meningosepticum was isolated from eight neonates on a neonatal intensive care unit. The strain was resistant to ampicillin, ceftazidime, imipenem, gentamicin, ciprofloxacin and trimethoprim-sulphamethoxazole, susceptible to piperacillin and amikacin, and had variable susceptibility to rifampicin and vancomycin. Two neonates were infected (one had pneumonia and one septicaemia and meningitis); the remaining six neonates were colonized in the respiratory secretions. Two cases occurred that could not be explained by cross-infection during the outbreak. Environmental screening recovered C. meningosepticum from sink taps. Pulsed-field gel electrophoresis of chromosomal macrorestriction digests of patient and environmental isolates showed them to be representatives of a single strain. The outbreak was controlled after staff were required to use an alcoholic handrub after washing hands, and toiletting of babies was done with sterile water instead of tap-water. Repair and chlorination of the water-tanks and changing the sink-taps resolves the outbreak.

Typing of Acinetobacter baumannii isolated from hospital-acquired respiratory infections in a tertiary care centre in southern India.

In an attempt to define the epidemiology of Acinetobacter baumannii infection, 27 isolates, obtained from hospital-acquired respiratory infections, were typed using random amplified polymorphic DNA (RAPD) profile and antimicrobial susceptibility patterns. Ten different patterns were obtained with ERIC2 primer: 14 isolates had a similar profile representing a single strain. Within RAPD types, isolates could be further classified based on their antibiogram; however, strains of different types had similar antibiograms. This study showed that many different genetic types of A. baumannii are prevalent in our hospital. While antibiograms alone are not sufficiently discriminatory, RAPD typing helps in identifying outbreaks and in assessing infection control procedures within a hospital.