Prevalence and role of efflux pump activity in ciprofloxacin resistance in clinical isolates of Klebsiella pneumoniae.

We investigated the prevalence and role of efflux pump activity and possible drug influx resistance in ciprofloxacin susceptibility amongst 26 distinct clinical isolates of Klebsiella pneumoniae of varying ciprofloxacin susceptibilities and known quinolone resistance-determining region (QRDR) genotypes. Cellular [(14)C]ciprofloxacin accumulation patterns and the amount of cell-associated [(14)C]ciprofloxacin of mid-logarithmic phase cells were determined before and after challenging with the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Most isolates (24/26), and all with ciprofloxacin minimum inhibitory concentrations (MICs) >1 µg/ml, had efflux activity that could extrude up to 90% of cell-associated [(14)C]ciprofloxacin; none had significant influx resistance. In isolates with no QRDR mutations, efflux alone reduced ciprofloxacin susceptibility. In isolates with QRDR mutations, the efflux activity varied: in one isolate with no efflux activity, the most common fluoroquinolone resistance-causing QRDR mutation did not bring about clinically significant ciprofloxacin resistance; isolates with multiple mutations had high MICs and, usually, high levels of efflux activity. Fluoroquinolone efflux activity is much more common in clinical isolates of K. pneumoniae than previously reported and it can contribute to decreased ciprofloxacin susceptibility.

Hypermutability in clinical isolates of Klebsiella pneumoniae is uncommon and is unrelated to ciprofloxacin resistance.

We investigated hypermutability in Klebsiella pneumoniae and its association with ciprofloxacin resistance and mutations in the quinolone resistance-determining region (QRDR). Sixty-four strains of K. pneumoniae isolated in London, UK, between 1995 and 2002 with widely differing ciprofloxacin minimum inhibitory concentrations (MICs) and known gyrA and parC sequences were tested for mutation frequencies by selection with rifampicin. Only three hypermutable (frequency >or=10(-6)) strains were identified, with ciprofloxacin MICs of 0.25 microg/mL, 8 microg/mL and 64 microg/mL. There was no relationship between hypermutation and the ciprofloxacin MIC or QRDR mutations. Screening selected strains with streptomycin did not reveal any hypermutators, and screening with ciprofloxacin identified only two of the three hypermutators identified by rifampicin. Hypermutation in K. pneumoniae is uncommon and does not contribute to accumulation of QRDR mutations or directly to ciprofloxacin resistance.

Diagnosis of bacteriuria by detection of volatile organic compounds in urine using an automated headspace analyzer with multiple conducting polymer sensors.

The Osmetech Microbial Analyzer (OMA) is an automated headspace analyzer fitted with a novel detector system consisting of an array of polymer sensors, each of which responds to different volatile organic compounds. The system can be used for screening clinical urine specimens for significant bacteriuria by sampling urine headspace and subjecting the output of the multiple-detector response to principal component analysis. The OMA readily distinguished artificially infected urine samples from sterile controls. The OMA was then used to analyze 534 unselected clinical urine specimens, of which 21.5% had significant bacteriuria (containing >10(5) CFU of bacteria/ml). The sensitivity and specificity of the OMA compared with conventional culture were 83.5 and 87.6%, respectively. The OMA is a promising automated system for the rapid routine screening of urine specimens, and further clinical trials are in progress.

Highly epidemic strains of methicillin-resistant Staphylococcus aureus not distinguished by capsule formation, protein A content or adherence to HEp-2 cells.

In order to investigate whether highly epidemic methicillin-resistant Staphylococcus aureus (EMRSA) strains possess special properties that favour their dissemination and survival, a study was undertaken that examined methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus isolated in the UK. Included in the study were EMRSA types 1, 2, 3, 15 and 16. Phage types EMRSA-15 and -16, in particular, have emerged as significant hospital pathogens in the UK, resisting standard methods of control and spreading widely, while the incidence of other epidemic types has either declined or not changed. All of the strains included in the study were examined for capsule formation, the amount of bound protein A produced, and quantitative adherence to the human continuous epithelial cell line HEp-2. Although all of these properties varied amongst the strains examined, there was no relationship between any of them and methicillin resistance or epidemic type and, incidentally, no relationship between cell wall-bound protein A content and adherence.

A novel plasmid pIJB1 possessing a putative 2,4-dichlorophenoxyacetate degradative transposon Tn5530 in Burkholderia cepacia strain 2a.

A 102-kb plasmid, pIJB1, was isolated from Burkholderia cepacia strain 2a, which is able to use 2,4-dichlorophenoxyacetate (2,4-D) as a sole carbon source, and a physical map of the plasmid has been established. It was observed that spontaneous loss of a 37-kb fragment of the plasmid after growth in nonselective medium occurred, generating a plasmid of diminished size, pIJB2. The deletion event is concomitant with the loss of the 2,4-D dissimilatory phenotype, indicating that at least some of the 2,4-D degradative genes are on the missing fragment. The missing fragment is flanked by two identical 4.3-kb insertion sequences (IS) and shows a typical composite transposon structure of 41-kb in size, designated Tn5530. The mutant plasmid pIJB2 possesses a single copy of the IS element.

vanA genes in vancomycin-resistant clinical isolates of Oerskovia turbata and Arcanobacterium (Corynebacterium) haemolyticum.

We report the cloning and sequencing of vanA genes present in the high-level vancomycin- and teicoplanin-resistant clinical isolates Oerskovia turbata 892 and Arcanobacterium (Corynebacterium) haemolyticum 872. The presence of vanA was detected by Southern blotting and PCR and confirmed by DNA sequencing. vanA-like sequences were encoded on plasmids of 15 and 20 kb respectively. The A. haemolyticum 872 DNA sequence was identical to the published vanA sequence of vancomycin-resistant Enterococcus faecium BM4147, but the O. turbata 892 sequence showed three coding changes. Induction experiments indicated that vancomycin resistance in A. haemolyticum 872 and O. turbata 892 was constitutive. SDS-PAGE analysis of membrane proteins showed the presence of a c. 39 kD protein in both clinical isolates whose expression was unaltered in the presence of vancomycin, while a similar protein in E. faecium BM4147 was inducible. Since A. haemolyticum and O. turbata are naturally susceptible to vancomycin, the high-level constitutive resistance seen in these isolates appears to be mediated by vanA. This is the first report confirming the presence of vanA in genera other than Enterococcus.