Molecular epidemiology of extended-spectrum beta-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to 2006.

The genetic characteristics and molecular epidemiology of extended-spectrum beta-lactamases (ESBLs) among Escherichia coli isolates were investigated at a general hospital and its associated health care facilities in Stockholm, Sweden, during the period from 2001 to 2006. Of 87 consecutive nonduplicate ESBL-positive isolates, 80 isolates encoded CTX-M-type ESBLs, 64 of which were group 1 enzymes. TEM-type and OXA-type beta-lactamases were encoded in 63 and 59% of the ESBL isolates, respectively. Pulsed-field gel electrophoresis (PFGE) analysis revealed 40 different pulsotypes, consisting of 11 clones accounting for 66% of all isolates, and 29 unique patterns. Moreover, of the 11 clones, clones 1 and 4 comprised half of the clonally related isolates (28 of 57). Clone 1 was a persistent endemic clone in the area throughout the years, and clone 4 emerged in 2003. However, in recent years, clone 1 isolates were no longer predominant and were gradually replaced by new emerging strains. Concerning beta-lactamase gene profiles in relation to PFGE pulsotypes, clone-related bla profiles were observed in certain clones, while in most cases different bla profiles could be observed in the same clone, and the same bla profile could be present in different clones. The molecular epidemiology of ESBL-positive E. coli in the area shows shifts in predominant strains and increased clonal diversity over time. The study also indicated that both clonal spread of epidemic strains and transfer of transposable genetic elements might contribute to the proliferation of ESBLs.

Verification of clinical samples, positive in AMPLICOR Neisseria gonorrhoeae polymerase chain reaction, by 16S rRNA and gyrA compared with culture.

We compared 956 samples for AMPLICOR Neisseria gonorrhoeae polymerase chain reaction (PCR) (Roche) with species verification using the 16S rRNA gene to verification using gyrA gene. Control was the culture method. The gyrA verification uses pyrosequencing of the quinolone resistance-determining region of gyrA. Of 52 samples with optical density >/=0.2 in PCR, 27 were negative in culture, two samples from pharynx were false negative in culture and four samples from pharynx were false positives in verification with 16S rRNA. Twenty-five samples showed growth of gonococci, 18 of the corresponding PCR samples were verified by both methods; three urine samples were positive only in gyrA ; and one pharynx specimen was positive only in 16S rRNA. Three samples were lost. We conclude that AMPLICOR N. gonorrhoeae PCR with verification in gyrA gene can be considered as a diagnostic tool in populations with low prevalence of gonorrhoea and that pharynx specimens should not be analysed by PCR.

DNA gyrase gene in Neisseria gonorrhoeae as indicator for resistance to ciprofloxacin and species verification.

We have identified a unique region of eight amino acids in the quinolone resistance-determining region in the gyrA gene in Neisseria gonorrhoeae as an indicator of resistance to fluoroquinolones. We sequenced that region by the Pyrosequencing technology in 46 N. gonorrhoeae strains and 11 urine samples positive in AMPLICOR N. gonorrhoeae polymerase chain reaction (Roche Diagnostics), with corresponding isolates of N. gonorrhoeae. The results showed that 28 samples with minimum inhibitory concentration (MIC) of ciprofloxacin >1 mg/L had mutations in positions 91 and 95. Fifteen samples with MIC 0.125-1.0 mg/L had either one or both of the mutations. The 14 susceptible samples had no mutations. The target region also discriminates N. gonorrhoeae from other species of Neisseria. Our conclusion is that gyrA is an indicator of resistance to ciprofloxacin in N. gonorrhoeae and sequencing by Pyrosequencing technology is a suitable tool for analysis of DNA in urine samples.