Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa tend to be geographically scattered, such as GES-2, which partially compromises the efficacy of imipenem. The G170N mutation, ascribed to a CC to AA base pair substitution on positions 493-494 of the blaGES-2 coding region, distinguishes this ESBL from blaGES-1 and the blaIBC-type genes, making it an ideal target for developing a novel sequence-specific, peptide nucleic acid (PNA)-based, multiplex-PCR detection method. Utilizing two primer pairs in conjunction with a PNA probe, this novel method delivered accurate identification of blaGES-2 compared to standard PCR and gene sequencing techniques, when tested against one hundred (n = 100) P. aeruginosa clinical isolates as well as previously published, well-described control strains. This method has the potential to be used in large-scale, cost-effective screening programmes for specific or geographically restricted ESBLs. To date, in addition to being only described in South Africa, GES-2 is notoriously difficult to identify in P. aeruginosa, using standard methodology. A real-time PCR method using the LightCycler™ was compared to a two-step nested-PCR assay for the detection of blaGES and blaIBC genes from one hundred P. aeruginosa clinical isolates collected over a four-year period from two teaching hospitals in Pretoria, South Africa. Real-time PCR amplification was monitored through hybridisation of fluorescently labelled probes followed by melting curve analysis to detect the relevant G170N mutation occurring in the omega loop region of blaGES-2. Nested-PCR products were subjected to automated DNA sequencing and compared to melting point (Tm) analyses results obtained from the LightCycler assay. Real time and nested-PCR assays detected a blaIBC gene product from 83 and 88 clinical isolates respectively, with the LightCycler thus exhibiting a sensitivity of 94.3% compared to the nested-PCR assay. Comparison of Tm and gene sequencing data however revealed 100% specificity for sequence specific detection of blaGES-2 with the LightCycler. One clinical isolate was found to harbour a blaGES-1 gene, making this the first report of this specific ESBL from South Africa. Selective antibiotic pressure has recently been implicated as a possible driving force behind point mutations observed in blaGES–type genes. This part of the study subjected two well-characterized clinical isolates with class 1 integron-borne blaGES-type genes to five days incubation in the presence of sub-inhibitory concentrations of 15 different antibiotics, including beta-lactams, aminoglycosides and quinolones. Restriction enzyme analysis and DNA sequencing of blaGES-1, blaGES-2 and their immediate upstream genetic environments failed to demonstrate any changes compared to non-exposed controls. Short-term exposure to a sub-inhibitory level of a single antimicrobial agent is thus unlikely to select significant mutations in these beta-lactamase genes or their regulatory mechanisms.Thesis (PhD (Medical Microbiology))--University of Pretoria, 2004.Medical Microbiologyunrestricte

Salmonella typhi central nervous system infection

This report aims to document Salmonella typhi as a cause of central nervous system infection

Random Amplified Polymorphic DNA Typing of Clinical and Environmental Aeromonas hydrophila Strains from Limpopo Province, South Africa

The aim of the present study was to determine the genetic relatedness of strains isolated from diarrhoeal stool and water specimens collected from water-storage containers from different geographical areas in the Limpopo province. In total, 32 Aeromonas strains isolated from stool specimens collected from HIV/AIDS patients suffering from gastroenteritis and their household drinking-water stored in 20-L and 25-L containers were analyzed by random amplified polymorphic DNA PCR (RAPD). The RAPD fingerprints obtained proved reproducible when repeated on three different occasions using whole-cell DNA isolated from the Aeromonas strains. In total, 12 unique RAPD fingerprints were found. The results revealed a tendency of the isolates to cluster according to their origin of isolation (best-cut test 0.80 and bootstrap values >50%). However, a certain degree of similarity was also observed between isolates of water sources and clinical sources which indicated genetic relatedness. There were also genetic similarities between the clinical and the environmental strains of Aeromonas spp. isolated from different geographical areas. This study has demonstrated the genetic relatedness of Aeromonas hydrophila isolates from household drinking-water and clinical sources in South Africa, which may be due to cross-contamination from water to patients or vice-versa. This observation is of public-health significance, particularly in the era of HIV/AIDS. This study points to the importance of monitoring and evaluating infection-control measures for improved hygiene and to prevent cross-contaminations