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.
A patient with a clonal infection of Burkholderia pseudomallei had subpopulations with ceftazidime and amoxicillin-clavulanate susceptibilities that differed among the clinical specimens. Resistance was associated with a novel Cys69Tyr substitution in the Ambler class A beta-lactamase. Susceptibility testing of multiple colony variants from different sites should be performed for patients with culture-confirmed melioidosis.
Typhoid fever, which is endemic in Malaysia, affects all age groups and it has been stated that classical features described in textbooks were absent in children. The aim of this study was to find out whether this was true in the local setting and hence a retrospective study was undertaken.
With the increasing recognition of the importance of H. pylori in gastrointestinal disease, there is a need for a reliable, efficient and yet inexpensive diagnostic test. The performance of the rapid urease test (RUT) as an endoscopy suite diagnostic test was compared to the established methods of culture, histology and Gram stain of tissue smear, in 274 gastric biopsy samples. Histology had the highest sensitivity of 99.3% followed by the RUT (96.6%). Culture and Gram stain of tissue smear had 100% specificity, while the rapid urease test had 99.2% specificity. The RUT had a positive predictive value of 99.3% and a negative predictive value of 96.2%. The RUT is an inexpensive, rapid and reliable diagnostic test of H. pylori infection.
We examined human tissues infected by Burkholderia (Pseudomonas) pseudomallei which is endemic in Malaysia to study the types of inflammation invoked, and to look for histopathological clues to its diagnosis. The lesions which varied from acute to chronic granulomatous inflammation were not tissue-specific. In five autopsy cases, the inflammation was usually a focal or diffuse, acute necrotising inflammation with varying numbers of neutrophils, macrophages, lymphocytes and 'giant cells'. The 'giant cells' probably represent giant macrophages with phagocytosed leukocytes. There were numerous gram-negative, non-acid-fast, intra- and extracellular bacilli, occurring either singly or in chains. Intracellular bacteria within macrophages and 'giant cells' were so numerous as to resemble globi. This feature has not been previously reported and may be a useful diagnostic clue in melioidosis. In 14 surgical cases biopsies showed acute inflammatory lesions that appeared no different from acute inflammation due to other causes. In many biopsies, however, the inflammation was either an acute-on-chronic inflammation with a focal granulomatous component, or was purely granulomatous in character. Bacilli were difficult to demonstrate in surgical biopsies even with the gram strain.
31 cases of intracranial abscess seen over a period of 10 years showed a peak incidence in the second and third decades of life with a male preponderance. Tetralogy of Fallot and other congenital cyanotic heart diseases were the predominant associated factors (32%). The commonest site of infection was the frontal lobe. Gram-stained smears of pus proved to be extremely useful. The majority of the organisms (82%) were either microaerophilic or anaerobic bacteria with Streptococcus milleri being the most frequent isolate. With the exception of Corynebacterium species, all isolates were susceptible to penicillin or chloramphenicol, most being susceptible to both.
Fifty seven strains of Pseudomonas pseudomallei were tested for in vitro susceptibility to 15 antimicrobial agents. Amongst the generally recommended antibiotics for therapy of melioidosis, only 86%, 84% and 58% of the strains were found to be sensitive to trimethoprim-sulphamethoxazole, chloramphenicol and tetracycline respectively. Of the newer B-Iactams, in descending order of activity were, ceftazidime, ceftriaxone, cefotaxime, cefoperazone and cefuroxime. But on a weight for weight basis, ceftazidime was the most active agent and as such, may be considered in the therapy of acute septicaemic melioidosis."
Pulsed field gel electrophoresis (PFGE), the gold standard of molecular typing methods, has a major disadvantage of an unusually long electrophoretic time. From the original protocol of 6 days, it was modified to 3 days and subsequently to a single day. We describe the procedure of stacking five to six gels one on top of another in order to increase and maximize the output in a shorter time without compromising the resolution and reproducibility. All the variables that affect pulsed field gels during electrophoresis were taken into consideration. We firstly optimized the parameters to be used and secondly determined whether stacking of five to six gels had any effect on the molecular separation during electrophoresis in comparison with a single gel run. DNA preparation, restriction, electrophoresis, staining and gel documentation was carried out based on previously published methods. Gels were analysed using BioNumerics and dice coefficient and unweighted pair group methods were used to generate dendrograms based on 1.5% tolerance values. Identical band profiles and band resolution-separation were seen in the PFGE patterns with single gel and multiple stacking gels. Cluster analysis further strengthened the fact that results from stacking gels were reproducible and comparable with a single gel run. This method of stacking gels saves time and maximizes the output at the same time. The run time for a single gel was about 28 hours, but with six stacked gels the run time was 54 hours compared with 28 x 6 = 168 hours if they were run separately as single gels thus saving time of 67.86%. Beside the big factor of saving time, stacking gels save resources (electricity, reagents, water, chemicals and working time) by increasing the sample throughput in a shorter time without compromising on quality of data. But optimization of working parameters is vital depending on the PFGE system used.
The constancy of strain genotypes of multiple isolates of Burkholderia pseudomallei from 13 patients with melioidosis was examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of DNA. Seven of 8 patients with single episodes of melioidosis each yielded genetically identical isolates and only one of five patients with recurrent episodes was infected with a new strain clearly distinct from the original primary strain. Variation was observed in PFGE patterns of primary and relapse isolates of another patient but this was insufficient to define genetically distinct strains. We conclude that most patients with single or multiple episodes of melioidosis retain a single strain.
Burkholderia pseudomallei infection of the central nervous system (CNS) is rare with less than 50 cases reported over the last 30 years. The retrospective melioidosis study at University Malaya Medical Centre has documented three cases of CNS melioidosis out of more than 160 cases of melioidosis since 1978. There were two patients with brain abscess and one with spinal epidural abscess. The predisposing factors were: one patient was an aboriginal farmer and the other two were diabetic. Their age ranged from 17 to 45 years. Prominent neurological features were limb weakness, cranial nerve palsy (6th and 7th) and visual disturbance. CT brain scan and MRI spine showed abscess formation, subdural collection, and spinal epidural collection, osteomyelitis of vertebra and occipital bone and also sagital sinus thrombosis. All these patients underwent surgical drainage leading to bacteriological diagnosis as well as appropriate long-term antibiotic therapy. All had good recovery at 6 months after completion of treatment.
There is an increasing trend for Shigella isolates worldwide to be resistant to commonly prescribed antibiotics. The species distribution and antibiotic resistance of Shigella species isolated from children in Kuala Lumpur, Malaysia from 1978 to 1997 was reviewed. Three hundred and eighty six isolates were positive for Shigella species, representing 1.4% (95% CI: 1.3%-1.6%) of the 26320 total stool specimens and 13% (95% CI: 11.8%-14.2%) of 2986 isolates positive for bacterial pathogens. Shigella flexneri, constituting 74% of all isolates in the first five years of the study, decreased by 40% during the last five years (95% CI of decrease: 22.1%-57.9%), p-value < 0.0001) to 34%. There was a significant reduction (chi2 for linear trend = 77.6, p-value < 0.001) in the number of Shigella isolates as a percentage of total stool isolates obtained. 58% of the 241 isolates tested for antibiotic sensitivity were resistant to at least one antibiotic, and 42% wEre multi-resistant to three or more antibiotics. Shigella species was not a common pathogen among children admitted with diarrhoea in Kuala Lumpur, and was more likely to be resistant to commonly prescribed antibiotics.
Restriction enzymes SpeI and XbaI were used in a pulsed-field gel electrophoresis (PFGE) study for molecular characterization of 146 clinical Burkholderia pseudomallei isolates. The PFGE parameters were optimized to enable comparable, reproducible, and robust results. The optimized parameters for both SpeI and XbaI restriction enzymes used in this study were 200 V and a pulse time of 5 to 65 s for a 28-h runtime. Using SpeI, 9 different clusters were identified, whereas 6 clusters were identified by XbaI digestion, which exhibited 85% similarity to SpeI. SpeI (discrimination index [D]=0.854) showed higher discriminatory power than XbaI did (D=0.464).
In Malysia, the proportion of children fully immunized againest diphtheria is generally low (20%). On the other hand, the Schick conversion rate rises with age and reaches 90% by 11 years of age. It is noted that asymptomatic carriers are an important epidemiological factor in diphtheria and that carrier rates for school children are high (prevalence of 7.5% while the rate of coloization with C. diphtheriae over a period of one year was 30%). Although immunization protects against clinical diphtheria, it does not prevent the carrier state. Thus, for the control of diphtheria, one should aim for 100% compliance. Some suggestions as to how higher levels of immunity may be achieved are described.
The suitability of a PCR procedure using a pair of primers targeting the hilA gene was evaluated as a means of detecting Salmonella species. A total of 33 Salmonella strains from 27 serovars and 15 non-Salmonella strains from eight different genera were included. PCR with all the Salmonella strains produced a 784 bp DNA fragment that was absent from all the non-Salmonella strains tested. The detection limit of the PCR was 100 pg with genomic DNA and 3 x 10(4) c.f.u. ml(-1) with serial dilutions of bacterial culture. An enrichment-PCR method was further developed to test the sensitivity of the hilA primers for the detection of Salmonella in faecal samples spiked with different concentrations of Salmonella choleraesuis subsp. choleraesuis serovar Typhimurium. The method described allowed the detection of Salmonella Typhimurium in faecal samples at a concentration of 3 x 10(2) c.f.u. ml(-1). In conclusion, the hilA primers are specific for Salmonella species and the PCR method presented may be suitable for the detection of Salmonella in faeces.
The diagnostic value of the Widal test was assessed in an endemic area. The test was done on 300 normal individuals, 297 non-typhoidal fevers and 275 bacteriologically proven cases of typhoid. Of 300 normal individuals, 2% had an H agglutinin titre of 1/160 and 5% had an O agglutinin titre of 1/160. On the basis of these criteria a significant H and/or O agglutinin titre of 1/320 or more was observed in 93-97% of typhoid cases and in only 3% of patients with non-typhoidal fever. Of the sera from typhoid cases which gave a significant Widal reaction, the majority (79.9%) showed increases in both H and O agglutinins and 51 of 234 (21.8%) of these sera were collected in the first week of illness. The significance and implications of these findings are discussed.
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 occurrence of latency and relapse in human melioidosis suggests adaptations by Burkholderia pseudomallei that help to avoid the human immune response. Ruthenium red-stained preparations of bacterial cultures viewed by electron microscopy revealed three morphologically distinct variants; one with a very marked and another with a less electron-dense layer surrounding the cell wall, and a third variety devoid of such a structure. This structure may be attributable to a layer of polysaccharide, suggesting the presence of a glycocalyx that may aid in the survival of the organism during latency.
Group B streptococcus (GBS) is a leading cause of neonatal sepsis and is usually acquired via the woman's birth canal. GBS serotypes isolated from 200 pregnant women were determined. Serotypes V (19%) and VI (17%) were the most frequent followed by serotypes III (12%), Ia (11.5%) and IV (10%); 17% of the strains were non-typable. All isolates were susceptible to penicillin, 96% to erythromycin and 97.5% to clindamycin. The emergence of new GBS serotypes has important implications for vaccine prevention strategies.