Proteomic profiling of clinical and environmental strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous bacterium, which is able to change its physiological characteristics in response to different habitats. Environmental strains are presumably less pathogenic than clinical strains and whether or not the clinical strains originate from the environment or through inter-host transmission remains poorly understood. To minimize the risk of infection, a better understanding of proteomic profiling of P. aeruginosa is necessary for elucidating the correlation between environmental and clinical strains. Based on antimicrobial susceptibility and patterns of virulence, we selected 12 clinical and environmental strains: (i) environmental, (ii) multidrug resistant (MDR) clinical and (iii) susceptible clinical strains. Whole-cell protein was extracted from each strain and subjected to two-dimensional differential gel electrophoresis (2-D DIGE) and liquid chromatography tandem mass spectrometry quadrupole time-of-flight (LC-MS QTOF). All 12 strains were clustered into 3 distinct groups based on their variance in protein expression. A total of 526 matched spots were detected and four differentially expressed protein spots (p < 0.05) were identified and all differential spots were downregulated in MDR strain J3. Upregulation of chitin binding and BON domain proteins was present in the environmental and some MDR strains, whereas the clinical strains exhibited distinct proteomic profiles with increased expression of serine protein kinase and arginine/ornithine transport ATP-binding proteins. Significant difference in expression was observed between susceptible clinical and MDR strains, as well as susceptible clinical and environmental strains. Transition from an environmental saprophyte to a clinical strain could alter its physiological characteristics to further increase its adaptation.

Comparison of Clinical Isolates of Aeromonas from Singapore and Malaysia with Regard to Molecular Identification, Virulence, and Antimicrobial Profiles.

OBJECTIVE: The objective of this study was to examine the species distribution, genetic relatedness, virulence gene profiles, antimicrobial sensitivities, and resistance gene distribution of clinical Aeromonas strains from Singapore and Malaysia. METHODS: A total of 210 Aeromonas clinical isolates were investigated: 116 from Singapore General Hospital and 94 archived clinical isolates from University of Malaya Medical Center, Malaysia. The isolates were genetically identified based on the gcat gene screening and the partial sequences of the rpoD housekeeping gene. Genetic relatedness, distribution of 15 virulence genes and 4 beta-lactamase resistance genes, and susceptibility patterns to 11 antimicrobial agents were compared. RESULTS: Of the 210 Aeromonas isolates, A. dhakensis-94 (45%) was the dominant species in Singapore and Malaysia. Species composition was similar and enterobacterial repetitive intergenic consensus-PCR did not show genetic relatedness between strains from the two countries. Of the 15 virulence genes, A. dhakensis and A. hydrophila harbored the most compared with other species. Different combinations of 9 virulence genes (exu, fla, lip, eno, alt, dam, hlyA, aexU, and ascV) were present in A. dhakensis, A. hydrophila, and A. veronii from both the countries. Distribution of virulence genes was species and anatomic site related. Majority (>80%) of the strains were susceptible to all antimicrobial agents tested, except amoxicillin and cephalothin. A. dhakensis strains from Malaysia significantly harbored the cphA gene compared with A. dhakensis from Singapore. Multidrug resistance was mostly detected in strains from peritoneal fluids of dialysis patients. CONCLUSION: This study revealed A. dhakensis as the dominant species isolated in both geographic regions, and that it carried a high number of virulence genes. It also highlights the geographic-related differences of virulence gene distribution and antimicrobial resistance profiles of clinical Aeromonas strains from Singapore and Malaysia.

Enzymatic and molecular characterisation of leucine aminopeptidase of Burkholderia pseudomallei.

BACKGROUND: Leucine aminopeptidase (LAP) has been known to be a housekeeping protease, DNA-binding protein and repressor or activator in the operon regulation of virulence-associated genes in several bacterial species. LAP activity was consistently detected in overnight cultures of Burkholderia pseudomallei, the causative agent of melioidosis and this enzyme was partially purified and characterised in this study. The intra- and inter-species nucleotide and deduced amino acid sequence variation of LAP encoding gene (pepA) was determined. A pepA/PCR-RFLP assay was designed to facilitate the identification of major LAP sequence types amongst clinical and environmental isolates of B. pseudomallei. RESULTS: LAP activity was detected in B. pseudomallei culture supernantants by zymographic analysis. Optimum activity was at pH 9 and stable at 50[degree sign]C. Enhanced enzymatic activity was observed in the presence of metallic ions Mg2+, Ca2+, Na+ and K+. LAP activity was inhibited by EDTA, 1,10-phenanthroline, amastatin, Mn2+ and Zn2+. Sequence analysis of the complete nucleotide and deduced amino acid sequences of LAP-encoding (pepA) gene showed close genetic relatedness to B. mallei (similarity 99.7%/99.6%), but not with B. thailandensis (96.4%/96.4%). Eight pepA sequence types were identified by comparison with a 596 bp DNA fragment encompassing central regions of the pepA gene. A pepA/PCR-RFLP was designed to differentiate pepA sequence types. Based on restriction analysis with StuI and HincII enzymes of the amplified pepA gene, clinical and environmental isolates showed different predominant RFLP types. Type I was the most predominant type amongst 71.4% (65/91) of the clinical isolates, while Type II was predominant in 55.6% (5/9) of the environmental isolates. CONCLUSIONS: This study showed that LAP is a secretory product of B. pseudomallei with features similar to LAP of other organisms. Identification of major LAP sequence types of B. pseudomallei was made possible based on RFLP analysis of the pepA gene. The high LAP activity detected in both B. pseudomallei and B. thailandensis, suggests that LAP is probably a housekeeping enzyme rather than a virulence determinant.

Sequence polymorphism and PCR-restriction fragment length polymorphism analysis of the flagellin gene of Burkholderia pseudomallei.

Four flagellin allelic types (I to IV) of Burkholderia pseudomallei were identified based on their sequence variation and restriction fragment length polymorphism (RFLP) analysis of the amplified flagellin gene. Flagellin allelic type I was the most predominantly (75.0%) found among the 100 clinical isolates of B. pseudomallei investigated in this study.

Validation and comparison of an extrapolysaccharide (EPS)-based in-house ELISA and the PanBio melioidosis rapid cassette test-kits for serodiagnosis of melioidosis in a non-endemic area.

Detection of anti-Burkholderia pseudomallei antibodies in sera from melioidosis patients still represents a keystone in the confirmation of the clinical diagnosis, especially in non-endemic areas. An in-house assay was compared to lateral flow assays for the rapid detection of melioidosis-specific IgG or IgM. Employing 50 positive sera from patients and 200 negative sera from blood donors, sensitivity of the ELISA, the IgG and IgM assay were 84.0%, 90.0% and 84.0%, respectively. Specificity ranged from 98.0% (ELISA) to 99.5% (IgM assay). The application of the described diagnostic assays is a suitable method for the serodiagnosis of melioidosis in a non-endemic area.