Pseudomonas aeruginosa is the third most common pathogen causing nosocomial infections. The objective of this study was to investigate the antimicrobial resistance profiles and genetic diversity of hospital isolates of P. aeruginosa and to investigate the presence of several resistance genes and integrons.
Chronic bacterial infections occur as a result of the infecting pathogen's ability to live within a biofilm, hence escaping the detrimental effects of antibiotics and the immune defense system. Burkholderia pseudomallei, a gram-negative facultative pathogen, is distinctive in its ability to survive within phagocytic and non-phagocytic cells, to persist in vivo for many years and subsequently leading to relapse as well as the development of chronic disease. The capacity to persist has been attributed to the pathogen's ability to form biofilm. However, the underlying biology of B. pseudomallei biofilm development remains unresolved.
Subtyping of Salmonella Paratyphi A isolates from India, Pakistan, Indonesia and Malaysia was carried out by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these isolates from different endemic countries.
The prevalence of ceftriaxone resistance and the associated genes encoding extended-spectrum β-lactamase (ESBL) was determined in 149 non-duplicate non-typhoidal Salmonella isolated in 2008-2009 from patients in a tertiary care hospital in Kuala Lumpur, Malaysia. The resistance rate to ceftriaxone was 2.7% (2/74) in 2008, 4.0% (3/75) in 2009, and 3.4% (5/149) overall. CTX-M ESBL genes were detected in 2 of the 5 ceftriaxone-resistant isolates. The prevalence of ceftriaxone resistance, although low, is a concern because it limits therapeutic options. Continued surveillance of ceftriaxone resistance is important to monitor its trends.
Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions--exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.
Aeromonas hydrophila is a quorum-sensing (QS) bacterium that causes diarrhea in humans upon infection. Here, we report the genome of pathogenic Aeromonas hydrophila strain 187, which possesses a QS gene responsible for signaling molecule N-acyl homoserine lactone (AHL) synthesis and has been found to be located at contig 36.
The predictors of severe disease or death were determined for 85 melioidosis patients in Kuala Lumpur, Malaysia. Most of the patients were male, > 40 years old, and diabetic. Severe disease or death occurred in 28 (32.9%) cases. Lower lymphocyte counts and positive blood cultures were significant independent predictors of severe disease, but age, presentations with pneumonia, inappropriate empirical antibiotics, or flagellin types of the infecting isolates were not. Knowledge of local predictors of severe disease is useful for clinical management.
The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.
Burkholderia pseudomallei is the causative agent of melioidosis. We initiated this investigation with a virulent and an attenuated strain of B. pseudomallei. Pulsed-field gel electrophoresis was carried out initially for macrogenomic comparison of both strains of B. pseudomallei. However, the pulsotypes obtained were identical and therefore we applied a subtractive hybridization technique to distinguish and determine the possible differences between the two strains. Six virulence strain-specific DNA fragments were obtained and the encoding homolog proteins were identified as a xenobiotic-responsive element family of transcriptional regulator, a hypothetical protein, an unknown protein, a plasmid recombination enzyme, a regulatory protein and a putative hemolysin activator protein. A combination of at least three of these determinants was identified in 45 clinical isolates when screening was carried out with self-designed multiplex PCR targeting the six putative virulent determinants. Our data demonstrated that different combinations of the six putative virulence genes were present in the clinical isolates indicating their probable role in the pathogenesis of B. pseudomallei infections.
We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.
Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C(6). Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.
Rhodococcus equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized hosts. We describe four immunocompromized patients who had serious R. equi infections with an overall mortality of 75%. The natural habitat of R. equi is soil, particularly soil contaminated with animal manure. Necrotizing pneumonia is the commonest form of infection but extrapulmonary infections, such as wound infections and subcutaneous abscess, have also been described in humans. R. equi is cultured easily in ordinary non-selective media. Large, smooth, irregular colonies appear within 48 hours. It is a facultative, intracellular, nonmotile, non-spore forming, gram-positive coccobacillus, which is weakly acid-fast staining and bears a similarity to diphtheroids. It forms a salmon-colored pigment usually after 48 hours incubation. A particular characteristic of this organism is that it undergoes synergistic hemolysis with some bacteria on sheep blood agar. R. equi may be misidentified as diphtheroids, Mycobacterium species, or Nocardia. In vitro R. equi is usually susceptible to erythromycin, ciprofloxacin, vancomycin, aminoglycosides, rifampin, imipenem and meropenem. The organism can be difficult to eradicate, making treatment challenging. Increased awareness of the infection may help with early diagnosis and timely treatment.
Persons infected with human immunodeficiency virus (HIV) have an increased risk of salmonellosis when compared to the general population. We describe seven such patients with Salmonella bacteremia, of whom two had recurrent salmonellosis. In the latter two cases the infection was unusually severe, characterized by widespread infection, bacteremia and relapse, despite standard antimicrobial therapy. HIV-infected individuals will benefit from education on the source of Salmonella, mode of acquisition and prevention through safe food handling and food preparation practices. Because of the difficulty of eradicating Salmonella infection in patients with acquired immunodeficiency syndrome, long-term suppressive treatment with antimicrobials is warranted.
The study was undertaken to determine clonal relationship and genetic diversity of the human strains of Salmonella enterica serovar Enteritidis isolated from 1995 to 2002 from different parts of Malaysia.
An indirect ELISA was used to detect antibodies against outer membrane protein preparations (OMPs) from Salmonella typhi. Sera from patients with a definitive diagnosis of typhoid fever (TF) gave a mean absorbance reading, at 414 nm, of 1.52 +/- 0.23 as compared to 0.30 +/- 0.11 for sera from healthy individuals. This gave a positive to negative ratio of absorbance readings of approximately 5.1. Suspected TF patients (no isolation of S. typhi), with positive and negative Widal titers had mean absorbance readings of 1.282 +/00.46 and 0.25 +/- 0.19, respectively. Sera from patients with leptospirosis, rickettsial typhus, dengue fever, and other infections gave mean absorbances of 0.20 +/- 0.08, 0.24 +/- 0.08, 0.27 +/- 0.08, and 0.31 +/- 0.16, respectively. The sensitivity, specificity, positive and negative predictive values were 100%, 94%, 80% and 100%, respectively. The antibody response detected in the definitive TF cases was predominantly IgG in nature and no cross-reactivity was seen with OMP preparations extracted from E. coli. Variable reactivity was noted with OMP preparations obtained from other Salmonella spp. Three major OMPs are presented in the antigen preparation and strong binding of positive sera was detected to all three bands.
Forty-nine isolates of Burkholderia pseudomallei from sporadic cases of melioidosis in Malaysia over the past 18 years were examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of total deoxyribonucleic acid (DNA). Twenty-four patients had septicaemic melioidosis with a mortality of 70%; mortality in the non-septicaemic disease was 16%. Five ribotype patterns were identified, 2 of which accounted for 90% of all isolates. PFGE revealed a number of different strains within these ribotypes, but some pairs of isolates from unrelated cases gave closely similar DNA profiles. These results are in agreement with Australian studies which showed a high prevalence of a few ribotypes of B. pseudomallei which are further divisible by genotyping, in areas where melioidosis is endemic.
A study was undertaken to determine the role of Helicobacter pylori in non-ulcer dyspepsia (NUD) and to determine the efficacy of colloidal bismuth subcitrate (CBS) in the treatment of NUD. Seventy-one patients were randomly allocated (double blind) to CBS or placebo, two tablets twice daily for 4 weeks. The severity of dyspepsia was scored and endoscopies performed before and after treatment, and antral biopsy specimens were taken for bacteriologic and histologic examination. Forty patients had H. pylori infection, and all had changes of chronic active gastritis. H. pylori was cleared from 17 to 21 patients (81%) treated with CBS, whereas none of the 19 patients treated with placebo cleared the bacteria. Improvement in histology was noted in 15 of 21 patients (71.4%) treated with CBS, whereas no improvement was noted in any of the placebo controls. Thirty-one patients were negative for H. pylori. All had either normal gastric histology or minor degrees of inflammation. Seventeen of these patients received CBS, and 14 received placebo. All groups reported improvement in the symptom score; however, the H. pylori-positive, CBS-treated group recorded a significantly higher improvement than the other groups (p less than 0.001). Relapse of H. pylori infection after initial clearance of the bacteria was high. Twelve of 16 patients evaluated relapsed 1 month after withdrawal of CBS.
The membrane-filter assay, GM1-ELISA, and DNA-DNA hybridization assay, were used to detect enterotoxigenic Escherichia coli (ETEC) in samples of water, weaning food, food preparation surface swabs, fingerprints of mothers, and the fingerprints and stools of children under 5 years of age, in 20 households in a Malaysian village. Weaning food and environmental samples were frequently contaminated by faecal coliforms, including ETEC. The membrane-filter assay detected and enumerated faecal coliforms and LT-ETEC in all types of water and weaning food samples. Highest concentrations of faecal coliforms and LT-ETEC were found in weaning food, followed by well-water, stored water and stored drinking water. The GM1-ELISA detected LT-ETEC in weaning food, food preparation surfaces, fingerprints and stool samples. The DNA-DNA hybridization assay detected a larger proportion of STa2-ETEC than the other toxotypes, either singly or in combination. All the assays in combination detected the presence of ETEC in all types of samples on at least one occasion in each household. It was not possible to classify households as consistently more or less contaminated with ETEC. On individual occasions it was possible to show a significant association of the presence of LT-ETEC between the fingerprints of children and their stools, fingerprints of mothers and children, and weaning food and the stools of the child consuming the food.