J. Van Eldere

The possibilities and limitations of nucleic acid amplification technology in diagnostic microbiology

Nucleic acid amplification technology is examined from the critical viewpoint of a clinical microbiologist working in a routine diagnostic bacteriology laboratory. Widely recognised limitations of amplification technology include those of false-positive and false-negative results, the difficulty of obtaining quantitative results, the problem of using this technology for susceptibility testing, and the difficulty of detecting routinely the wide range of possible pathogens contained in a clinical sample. On the positive side, amplification technology brings welcome new possibilities for rapid detection of specific pathogens in a sample, including viruses, slowly growing bacteria, fastidious or uncultivable bacteria, fungi and protozoa. Other possible applications include screening normally sterile clinical samples for non-specific bacterial contamination and the use of amplification-based DNA fingerprinting methods for identification and typing of microorganisms. Nevertheless, it is predicted that-in contrast to research and reference facilities-routine bacteriology laboratories will continue to rely on culture as the preferred amplification method for most diagnostic applications.

Detection and characterization of class A extended-spectrum-β-lactamase-producing Pseudomonas aeruginosa isolates in Belgian hospitals

OBJECTIVESnTo investigate the presence of extended-spectrum beta-lactamases (ESBLs) among Pseudomonas aeruginosa clinical isolates referred to two Belgian reference laboratories.nnnMETHODSnAntibiograms were analysed for P. aeruginosa isolates referred between 2004 and 2008. Isolates resistant to ceftazidime (MIC > 8 mg/L) and with a positive double-disc synergy test between ceftazidime and clavulanate were serotyped and screened for the presence of ESBL-encoding genes. Genes encoding metallo-beta-lactamases (bla(MBL)) were sought by PCR in ESBL-producing isolates with positive imipenem/EDTA synergy tests. PFGE of SpeI-digested genomic DNA was used to compare isolates and selected strains were characterized by multilocus sequence typing.nnnRESULTSnForty-eight (2.2%) of 2150 P. aeruginosa isolates were confirmed as class A ESBL-producing isolates by molecular testing. bla(BEL) and bla(PER) alleles were detected, respectively, in 39 and 10 P. aeruginosa isolates originating from 16 hospitals (two isolates were simultaneously positive for BEL and PER). Fifteen of the isolates were found to co-produce ESBLs and VIM carbapenemases. These strains were pan-resistant and remained susceptible only to colistin (MICs <or= 2 mg/L). The majority of the ESBL-producing isolates belonged to the same PFGE clone and were identified as ST235; serotype O11.nnnCONCLUSIONSnBEL enzymes were produced by 80% of P. aeruginosa isolates with phenotypic evidence of ESBL production. BEL or PER ESBLs co-existed with VIM carbapenemases in 15 isolates and caused outbreaks in four hospitals. Our data further highlight the epidemic potential of the international clone ST235, which may have acquired bla(BEL-1) gene cassettes from a yet unidentified local gene reservoir.

Polyclonal Staphylococcus Endocarditis

Coagulase-negative staphylococcus (CNS) is the most frequent cause of nosocomial bacteremia and prosthetic valve endocarditis. CNS bacteremia can be polyclonal. No data exist on the clonality of CNS causing endocarditis. We present a case of CNS aortic homograft endocarditis in which at least five different genotypes of CNS were identified in initial blood-culture isolates by genomic macrorestriction enzyme analysis and pulsed field gel electrophoresis. Since the polyclonality was accompanied by differences in antibiotic susceptibility, this observation may have important consequences for the treatment of CNS endocarditis. Because of the parallels in the pathogenesis of CNS prosthetic valve endocarditis and CNS infections of a variety of other prosthetic devices, it might also have consequences for CNS prosthetic device infections in general. We suggest that antibiotic susceptibility testing of just one blood-culture isolate may be insufficient.

Influence of a cecal volume-reducing intestinal microflora on the excretion and entero-hepatic circulation of steroids and bile acids

From mouse fecal material we have isolated four strictly anaerobic bacteria which, when associated with germfree mice or rats, reduced the cecal volume by 80 and 60%, respectively. This cecal volume-reducing flora did not metabolize estrone-3-sulfate, taurolithocholate-3-sulfate or taurolithocholate but gnotobiotic rats associated with this particular flora (CRF-rats) excreted these compounds faster in feces plus urine than did germfree rats. The time needed for 50% excretion (t1/2) of orally administered estrone-3-sulfate was 32 h in germfree rats versus 13 h in CRF rats; for intraperitoneally injected taurolithocholate-3-sulfate the t1/2 was 63 h in germfree versus 17 h in CRF rats and for taurolithocholate the t1/2 was 199 h in germfree and 96 h in CRF rats. Association of germfree rats with the cecal volume-reducing flora did not change the cecal absorption rate of estrone-3-sulfate, but shortened the 50% small intestinal transit time of [14C]PEG from 10 to 3 h; a value also found in conventional rats. These results stress the important influence of the intestinal microflora on the absorption and excretion of steroids via its effect on the physiology of the whole intestinal tract and point to the deficiencies inherent to the use of germfree animals in excretion studies.