Salmonella enterica serovar Paratyphi A is a causative agent of paratyphoid fever. The clinical syndrome caused by paratyphoid fever overlaps with other febrile illnesses and cannot be distinguished from typhoid fever. Conventional methods used for diagnosis are time consuming, costly, and labor-intensive. We evaluated the specificity, sensitivity, and application of a multiplex polymerase chain reaction (PCR) previously developed by the method (Ou, H.Y., Teh, C.S.J., Thong, K.L., et al., J. Mol. Diagn., 9, 624-630, 2007) using 6 S. Paratyphi A, 22 S. Typhi, and 85 other Salmonella serovars as well as 36 non-Salmonella strains. The detection limit of the multiplex PCR was 4 x 10(4) cfu ml(-1). In a blind test of the other 50 strains, this multiplex PCR correctly identified the only S. Paratyphi A in the panel of strains. The sensitivity of this PCR using spiked blood and stool samples was 1 x 10(5) cfu ml(-1) and 2 x 10(5) cfu ml(-1), respectively, but increased to 1 x 10(4) cfu ml(-1) and 2 x 10(3) cfu ml(-1) after 5-h enrichment. We believe that this multiplex PCR is a promising technique for the specific and sensitive detection of S. Paratyphi A in clinical, environmental, and food samples.
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 incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.
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.
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.
Aeromonas dhakensis is an emergent human pathogen with medical importance. This study was aimed to determine the sequence types (STs), genetic diversity, and phylogenetic relationships of different clinical sources of 47 A. dhakensis from Malaysia using multilocus sequence typing (MLST), goeBURST, and phylogenetic analyses. The analysis of a concatenated six-gene tree with a nucleotide length of 2994 bp based on six housekeeping genes (gyrB, groL, gltA, metG, ppsA, and recA) and independent analyses of single gene fragments was performed. MLST was able to group 47 A. dhakensis from our collection into 36 STs in which 34 STs are novel STs. The most abundant ST521 consisted of five strains from peritoneal fluid and two strains from stools. Comparison of 62 global A. dhakensis was carried out via goeBURST; 94.4% (34/36) of the identified STs are novel and unique in Malaysia. Two STs (111 and 541) were grouped into clonal complexes among our strains and 32 STs occurred as singletons. Single-gene phylogenetic trees showed varying topologies; groL and rpoD grouped all A. dhakensis into a tight-cluster with bootstrap values of 100% and 99%, respectively. A poor phylogenetic resolution encountered in single-gene analyses was buffered by the multilocus phylogenetic tree that offered high discriminatory power (bootstrap value = 100%) in resolving all A. dhakensis from A. hydrophila and delineating the relationship among other taxa. Genetic diversity analysis showed groL as the most conserved gene and ppsA as the most variable gene. This study revealed novel STs and high genetic diversity among clinical A. dhakensis from Malaysia.
We performed genome size estimation of 17 recent human isolates of Salmonella typhi from geographically diverse regions using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with restriction endonucleases XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3') and SpeI (5'-ACTAGT-3'), and summation of the sizes of restriction fragments obtained. All 17 isolates had circular chromosomes, and genome sizes differed by as much as 959 kb, ranging from 3,964 to 4,923 kb (mean genome size = 4,528 kb). The data obtained confirm the usefulness of PFGE in studies of bacterial genome size and are in agreement with recent results indicating considerable genetic diversity and genomic plasticity of S. typhi. The variation in genome sizes noted may be relevant to the observed biological properties of this important human pathogen, including its virulence.
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.
Group B Streptococcus (GBS) infection was studied in 49 patients collected at convenience (convenience sampling), excluding infants and women with genital tract- and pregnancy-related isolates, according to the availability of stocked isolates and easy accessibility to epidemiological data. The data were examined both prospectively and retrospectively from 2003-2005 at a tertiary-level multidisciplinary hospital in Kuala Lumpur, Malaysia. Skin and soft-tissue infections in 35 patients (71.4%) were the most common clinical presentation, while diabetes mellitus was the most common underlying condition (35 patients, 71.4%). All GBS isolates were sensitive to penicillin, and most isolates tested were sensitive to erythromycin (97.7%). Serotyping of 45 GBS isolates using a commercial serotyping kit revealed that the most common serotype was Ia (22.2%), followed by VI (17.8%), III and V (13.3% each). Others included Ib, II, IV, VIII, and VII; 13.3% were nontypeable. The findings of this pilot study are limited by the small sample size, the sampling method and the possibility that the cases are not wholly representative of the University Malaya Medical Centre population. Further studies from our hospital with larger numbers and using probabilistic sampling techniques are required to confirm the relatively high occurrence of serotype VI (the second most common serotype) in the population studied.
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.
Forty clinical isolates of Vibrio parahaemolyticus were studied for the production of the thermostable direct hemolysin (TDH), and the TDH-related hemolysin (TRH) including the respective encoding genes, tdh and trh. The presence of TDH and its encoding genes were found amongst 95% of the strains, whereas the TRH was absent amongst these isolates. Thirty-two isolates were found to be plasmid-free, whereas eight isolates possessed plasmids with sizes ranging from 2.4 > or = 23 kb. Using a DNA probe coding for the homologous region of the tdh and trh, it was found that the tdh genes were present on the chromosomal DNA.
170 clinical isolates of Pseudomonas aeruginosa were tested for in vitro susceptibility to gentamicin, amikacin, tobramycin, netilmicin, kanamycin, streptomycin, cefotaxime, ceftriaxone, cefoperazone, ceftazidime, moxalactam, azlocillin, piperacillin and ticarcillin. Against 93 gentamicin-sensitive strains, the most active antibiotics were in descending order, ceftazidime, tobramycin, gentamicin, amikacin, and the ureidopenicillins. Against 77 gentamicin-resistant strains, only ceftazidime, amikacin and moxalactam had mode minimum inhibitory concentrations within achievable peak serum levels after standard therapeutic dosage. There was no correlation between cephalosporin resistance and aminoglycoside resistance except for cefoperazone, which, together with the ureidopenicillins and ticarcillin, showed marked decrease in activity against gentamicin-resistant strains.
Studies were performed on a cytotoxin (CT) from human strains of Campylobacter jejuni isolated in Malaysia. CT was detected by cytopathic effect (CPE) on HeLa cells at titres from 8 to 32, in culture filtrates from 14 (48%) of 29 human isolates. The CPE correlated well with a quantitative 51Cr-release assay where a specific release of 54-68% was noted. CT production was lost after 5-7 subcultures. CT activity was also detected in 5 (26%) of 19 faecal filtrates from which CT-producing isolates were subsequently obtained. The mol. wt of CT was estimated by Sephadex G-50 chromatography to be greater than 30,000. In a suckling-mouse assay, CT consistently failed to demonstrate fluid accumulation after intragastric inoculation of culture filtrate. The Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) assay was also used. Rabbits given CT-producing strains of C. jejuni developed bacteraemia and severe watery mucus-containing diarrhoea for the duration of the experiment with death of some animals. Rabbits given CT non-producing strains had less severe disease and none died. Rabbits given partially-purified CT had diarrhoea for 3 days but none died.
Drug abuse is a major problem in Malaysia. Serious complications of intravenous drug addiction include septicaemia and infective endocarditis. We present nine cases of endocarditis occuring amongst drug abusers. The tricuspid valve was most frequently involved and the common aetiological organisms were S. aureus and Str. faecalis. There was a high mortality rate of 67% in our study, inspite of appropriate therapy. Early recognition of the disease and aggressive treatment is required to improve the associated mortality.
The oxidative response of Burkholderia pseudomallei and Escherichia coli infected macrophages from normal and melioidosis subjects was determined by measuring the production of nitric oxide which is one of the reactive nitrogen intermediates, and the activation state of these macrophages was determined by measuring the generation of 8-iso-PGF(2alpha), a bioactive product of free radical induced lipid peroxidation. Macrophages obtained from the melioidosis patients generated significantly lower levels of nitric oxide and 8-iso-PGF(2alpha) compared to macrophages obtained from the normal subjects (P<0.001). The reduced efficiency of the oxygen dependent microbicidal mechanism in macrophages of melioidosis patients may be one of the survival strategies developed by B. pseudomallei to remain viable intracellularly.
Melioidosis is an important cause of sepsis in the tropics, is caused by an environmental saprophyte--B. pseudomallei. It affects mainly adults with underlying predisposing condition such as diabetes. The range of symptoms varies from benign and localized abscesses, to severe community-acquired pneumonia to acute fulminating septicaemia with multiple abscesses often leading to death. B. pseudomallei is an intracellular pathogen and some of the virulence mechanisms that govern the complex interaction between the organism and the host have been elucidated. Isolation of B. pseudomallei from bodily fluids of patients remains the "gold standard" in diagnosis but a sensitive and specific serological test can lend support to the diagnosis of melioidosis. Ceftazidime is the treatment of choice for severe melioidosis, but the response is slow. Maintenance or eradication therapy for a prolonged period is necessary to prevent relapse and recurrence. Monitoring IgG antibody levels may be useful as a guideline to determine the duration of eradication therapy.
There are few data on paediatric melioidosis in endemic areas outside rural north-eastern Thailand and northern Australia. This study reports 16 culture-confirmed cases of melioidosis in children aged < or = 15 years seen between 1976 and 2005 at an urban teaching hospital in Kuala Lumpur, Malaysia. Seven (43.8%) patients had septicaemic melioidosis (with three known deaths) and nine (56.2%) had localised disease (one death). Eleven (68.8%) patients had underlying diseases, including five with haematological malignancies. Skin, soft tissue and lymph nodes were most commonly affected. There were no cases of parotitis or pharyngocervical disease (seen in Thailand), or encephalomyelitis (seen in Australia). The differences in disease seen in this study compared with the mostly rural patients described in previous studies might be owing to a different patient population in an urban environment. Septicaemic melioidosis has a high mortality, but localised disease has a good prognosis, and selected cases may be cured without the full recommended treatment regimen.