Genetic variations in Aeromonas hydrophila isolates from clinical and environmental sources in Thailand.

Aeromonas hydrophila, a widely distributed human pathogen causing a variety of diseases, can be isolated from clinical and environmental sources. Analysis in Thailand of 110 isolates of Aeromonas hydrophila by randomly amplified polymorphic DNA-PCR (RAPD-PCR) revealed one specific RAPD pattern group (G) that was associated only with strains from environmental sources. Cytotoxic activity, adhesion to epithelial cells and exoenzyme secretions of A. hydrophila were also investigated. A comparison of isolates with pattern group G with a set of isolates derived from human blood showed low induction of cytotoxicity from those with RAPD pattern group G suggesting low virulence of these strains.

Ribotyping of Burkholderia pseudomallei from clinical and soil isolates in Thailand.

Burkholderia pseudomallei is a soil saprophyte that causes melioidosis in humans and animals. Restriction fragment length polymorphism of the ribosomal DNA regions (ribotyping) were analyzed in 577 isolates comprising 371 clinical and 206 soil isolates collected throughout Thailand in 1997. A total of 77 distinct ribotype patterns consisting of 2-9 bands with sizes ranging from 2.8 to >23 kb were found. Twelve major ribotypes were identified of which types 3, 8 and 23 were commonly found (278/577, 48.2%) in both clinical (217/371, 58.5%) and environmental isolates (61/206, 29.6%). Three unique environmental types were found whereas a unique clinical type was not observed. Even though ribotypes show high heterogeneity in the rDNA region, the unique environmental patterns were clearly different from the clinical patterns as clearly seen by UPGMA dendrogram. Moreover, the three major types (types 3, 8 and 23) were discovered in nearly half of B. pseudomallei isolates. Subtyping of these major ribotypes in correlation with clinical profiles may help researchers to identify the virulence factor of the organism.

Bacterial infections in hospitalized patients in Thailand in 1997 and 2000.

Two surveys to determine the patterns of bacterial infections and trends in resistance to antibiotics of bacteria causing infections in patients admitted to hospitals in Thailand were conducted in 36 and 37 hospitals throughout Thailand in June 1997 and February 2000. Approximately 50 per cent of infections in hospitalized patients in Thailand were hospital-acquired infections. Urinary tract and lower respiratory tract were the most common sites of infections. Eighty per cent of infections were caused by gram negative bacteria. Gram negative bacteria causing infections in 2000 were more resistant to most commonly used antibiotics when compared with those in 1997. The prevalence of gram positive bacteria causing hospital-acquired infections significantly increased during this period. The trend of increase in resistance in most gram positive bacteria in 2000 was not clearly observed.

Differentiation between non-virulent and virulent Burkholderia pseudomallei with monoclonal antibodies to the Ara+ or Ara- biotypes.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia. Environmental isolates of B. pseudomallei have two distinctive biotypes. Some soil isolates are arabinose-assimilators (Ara+ biotype) and are non-virulent in experimental animals. The others cannot assimilate arabinose (Ara- biotype) and are virulent in experimental animals. The Ara- biotype is found in almost all B. pseudomallei clinical isolates. In the present study, a panel of eight monoclonal antibodies that agglutinate the bacteria were produced and tested. The first group, Bps-D2, -D3, -D5, -L1, and -L2 agglutinated 100% of Ara+ clinical and soil isolates of B. pseudomallei. Another group Bps-A1, -A2, and -D1 agglutinated 92.9% and 90.9% of Ara- clinical and soil isolates, respectively. This panel of monoclonal antibodies may be useful for rapid differentiation between non-virulent Ara+ and virulent Ara- B. pseudomallei.

First report of methicillin-resistant Staphylococcus aureus with reduced susceptibility to vancomycin in Thailand.

To investigate whether there are methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin in Thailand, a total of 155 MRSA strains isolated from patients hospitalized between 1988 and 1999 in university hospitals in Thailand were tested for glycopeptide susceptibility. All the strains were classified as susceptible to vancomycin and teicoplanin when judged by NCCLS criteria for glycopeptide susceptibility using the agar dilution MIC determination. Vancomycin MICs at which 50 and 90% of the isolates tested were inhibited (MIC50 and MIC(90), respectively) were 0.5 and 1 microg/ml, respectively, with a range of 0.25 to 2 microg/ml. For teicoplanin, MIC50 and MIC90 were 2 microg/ml, with a range of 0.5 to 4 microg/ml. However, one-point population analysis identified three MRSA strains, MR135, MR187, and MR209, which contained subpopulations of cells that could grow in 4 microg of vancomycin per ml. The proportions of the subpopulations were 2 x 10(-4), 1.5 x 10(-6), and 4 x 10(-7), respectively. The subsequent performance of a complete population analysis and testing for the emergence of mutants with reduced susceptibility to vancomycin (MIC > or = 8 microg/ml) confirmed that these strains were heterogeneously resistant to vancomycin. Two of these strains caused infection that was refractory to vancomycin therapy. Pulsed-field gel electrophoresis showed that the two strains had identical SmaI macrorestriction patterns and that they were one of the common types of MRSA isolated in the hospital. This is the first report of heterogeneous resistance to vancomycin in Thailand and an early warning for the possible emergence of vancomycin resistance in S. aureus in Southeast Asia.

Microbial killing activity of peracetic acid.

In vitro killing activity of peracetic acid (Perasafe) at a concentration of 0.26 per cent w/v was tested against Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi A, Acinetobacter baumannii, Sternotrophomonas maltophilia, Enterococcus faecium, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis spore, Mycobacterium tuberculosis and human immuno-deficiency virus type I. Exposure to Peracetic acid (0.26% w/v) for 10 minutes resulted in massive killing of all the aforementioned organisms and spore.

Genotype analysis of Burkholderia pseudomallei using randomly amplified polymorphic DNA (RAPD): indicative of genetic differences amongst environmental and clinical isolates.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease common in the tropics. Melioidosis is most prevalent in the northeastern part of Thailand. The diseases has diverse clinical manifestations ranging from mild localized to fatal septicemic forms. The bacterial genetic factors contributing to the severity of melioidosis have not been completely identified. We have developed a genotyping method based upon randomly amplified polymorphic DNA (RAPD) analysis. Eighteen deca-oligo nucleotide primers with 70% GC content, eight previously published 60%GC RAPD primers, and four random deca oligomers were tested on nine strains of B. pseudomallei isolated from five patients with localized and four with septicemic melioidosis. The RAPD patterns were analyzed by polyacrylamide gel electrophoresis using a laser based automated fragment analyzer, GS2000. Based upon the pattern complexity, seven pairs consisting of eight primers were chosen for further analysis. Six hundred and thirty-two samples, including duplicates/triplicates, of B. pseudomallei isolated from melioidosis patients and the environment were analyzed. Two controls were included in each run of the test samples. All the samples were tested and patterns analyzed by blinded technical staff. Apparently, the method is reproducible. This is indicated by the RAPD patterns of the two controls of between run assay. Interestingly, some RAPD patterns were more prevalent in the clinical isolates than the environmental specimens and vice versa. For example, Q162KKU4-0 and Q162KKU1-0 were found 3. 5 and 3.3 times more often in the clinical specimens (P<0.025). Likewise, Q162KKU1-1 and Q162KKU4-1 were found 18 and 37 times more often in the environment (P<0.0000001). In addition, there was a bias in the distribution of arabinose positive strains and particular RAPD patterns; RAPD patterns of B. pseudomallei that were found frequently in septicemic patients were less likely to be arabinose positive. The data suggest the existence of bacterial genetic differences between the clinical and environmental isolates of B. pseudomallei. Further analysis of the RAPD patterns searching for common polymorphic DNA fragments and systemic comparative genomic analysis of B. pseudomallei in accordance with the clinical data should reveal genetic factors involved in severity and bacterial pathogenesis of B. pseudomallei in melioidosis.

Molecular phylogeny of Burkholderia pseudomallei.

In terms of population structure, the species Burkholderia pseudomallei contains both clonal and non-clonal elements. By indexing variation in rRNA loci using the restriction endonuclease BamHI, we found that two ribotypes (types 1 and 3) are predominant in nature. Ribotype 3 is prevalent in Asian countries while ribotype 1 is more widespread. Some disease association was suggested for 4 ribotypes and strains of ribotype 4 were markedly associated with a fatal outcome. DNA macrorestriction (XbaI) profiles resolved by pulsed-field gel electrophoresis revealed great heterogeneity within the prevalent ribotypes and these profiles appeared to be reliable strain markers. Arabinose environmental strains were characterised by BamHI ribotypes that were markedly distinct form clinical and environmental isolates of the arabinose negative phenotype.

Phylogenetic analysis of Ara+ and Ara- Burkholderia pseudomallei isolates and development of a multiplex PCR procedure for rapid discrimination between the two biotypes.

A Burkholderia pseudomallei-like organism has recently been identified among some soil isolates of B. pseudomallei in an area with endemic melioidosis. This organism is almost identical to B. pseudomallei in terms of morphological and biochemical profiles, except that it differs in ability to assimilate L-arabinose. These Ara+ isolates are also less virulent than the Ara- isolates in animal models. In addition, clinical isolates of B. pseudomallei available to date are almost exclusively Ara-. These features suggested that these two organisms may belong to distinctive species. In this study, the 16S rRNA-encoding genes from five clinical (four Ara- and one Ara+) and nine soil isolates (five Ara- and four Ara+) of B. pseudomallei were sequenced. The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara+ biotype was similar to but distinctively different from that of the Ara- soil isolates, which were identical to the classical clinical isolates of B. pseudomallei. The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara+ or Ara- biotypes. The differences were, however, not sufficient for classification into a new species within the genus Burkholderia. A simple and rapid multiplex PCR procedure was developed to discriminate between Ara- and Ara+ B. pseudomallei isolates. This new method could also be incorporated into our previously reported nested PCR system for detecting B. pseudomallei in clinical specimens.

Epidemiology of arabinose assimilation in burkholderia pseudomallei isolated from patients and soil in Thailand.

Burkholderia pseudomallei is an environmental saprophyte that has been isolated widely from soil in Southeast Asia and the relationship between environmental contamination and clinical melioidosis has been established. It has been shown that the arabinose assimilation property of B. pseudonrallei is probably one of the determinants indicating virulence of this organism. Therefore, the distribution of arabinose assimilation biotypes of B. pseudomallei collected from four geographic regions of Thailand was studied in order to determine an association between arabinose assimilation of B. pseudomallei and the uneven distribution of melioidosis found among these four areas. A total of 830 isolates of B. pseudomallei (412 patient isolates and 418 soil isolates) collected from the patients and soil in four regions of Thailand in 1997 were tested for an ability to grow on a minimal agar medium supplemented with L-arabinose. All patient isolates except one could not utilise arabinose (Ara-). For 418 soil isolates, 232 (55.5%) isolates were identified as Ara type. They comprised 180 (62.5%), 36 (46.8%), 6 (35.3%) and 10 (27.8%) isolates derived from northeastern, southern, northern and central regions respectively. The ratios of Ara- to Ara, were 1.7, 0.9. 0.5 and 0.4 among isolates collected from northeastern, southern, northern and central regions respectively. The prevalence of Ara- in soil isolates in northeast is significantly higher than those in other regions. This observation suggests that in addition to the presence of B. pseudomallei in soil which is one of the factors contributing to a burden of melioidosis in northeastern Thailand, the distribution of more virulent biotype (Ara-) soil isolates is a factor contributing to a high prevalence of melioidosis in northeastern Thailand as well.

Ribotype differences between clinical and environmental isolates of Burkholderia pseudomallei.

Burkholderia pseudomallei is isolated frequently from the soil in regions where the disease melioidosis occurs. However, recent surveys in Thailand have shown that the frequency of isolation of the organism from soil samples is not directly related to the incidence of melioidosis in an area. To determine whether strain populations of B. pseudomallei prevalent in soil are gentypically related to strains causing clinical disease, rRNA BamHI restriction fragment length polymorphisms (RFLP) of 139 soil environmental isolates and 228 human isolates were compared. Two groups of ribotype patterns were found. Group I comprised 37 different ribotype patterns which were characterised by five to eight hybridisation bands of 2.8- > 23 kb. All of these ribotypes were identified among the clinical isolates, and 18 of them were also found in 59 environmental isolates. Group II was represented by 12 ribotypes found only in environmental strains. These ribotype patterns comprised one to five bands in the size range 9- > 23 kb. All but one of the 73 isolates in this group grew on a minimal medium supplemented with L-arabinose. In contrast, only 3% of the 66 isolates from the environment with group I ribotype patterns could utilise this sugar as their sole energy source. These findings suggest that B. pseudomallei strains that utilise arabinose constitute a population that is genetically distinct from other environmental and clinical strains.

Isolation rates of Burkholderia pseudomallei among the four regions in Thailand.

This study aimed to compare the isolation rates of Burkholderia pseudomallei among community-based hospitals located in the central, north, northeast, and south of Thailand. A questionnaire inquiring about the number of isolation of B. pseudomallei from various clinical specimens during 1994-95 were mailed to 141 community-based hospitals. Of these, 125 hospitals (88.6%) responded to the questionnaire. Microbiological laboratory was not available in thirty hospitals. Data from 95 remaining hospitals with capability to do bacterial culture showed that B. pseudomallei was never isolated in 49 hospitals. Eleven, 9, 19 and 7 hospitals where B. pseudomallei has been isolated, are located in the central, north, northeast and south of Thailand respectively. From these 46 hospitals, a total of 1,131 strains of B. pseudomallei were isolated from 407,263 specimens in 1994 and 1,165 strains from 440,541 specimens in 1995. However, the isolation was most frequent in northeastern hospitals, which accounted for 890 and 964 strains in 1994 and 1995 respectively while only 94, 76, 71 and 83, 75, 43 strains were simultaneously isolated during the 2-year period in those located in central, north and south respectively. The isolation rates of B. speudomallei in 1994 and 1995 were 4.2 and 4.1 per 1,000 clinical specimens in northeastern hospitals as compared to 1.1, 1.8, 1.1 and 1.1, 1.2, 0.7 in those located in central, north and south respectively. Ubon Ratchathani, Nakhon Ratchasima, Buri Ram, Khon Kaen and Udon Thani were the five provinces which exhibited the highest isolation rates as follows; 244, 150, 147, 127, 100 and 218, 128, 114, 119, 58, in 1994 and 1995, respectively. It was concluded that B.pseudomallei was most commonly isolated in the northeast of Thailand. Under-recognition of B. pseudomallei may prevail not only in other parts of Thailand but in some areas of the northeast as well.

Undetectable anti-bacterial activity of Andrographis paniculata (Burma) wall. ex ness.

Andrographis paniculata (Burma) Wall. ex Ness (AP) is a herbal medicine and has been used for therapy of upper respiratory tract infection (URI) as well as acute diarrhea with reported efficacy of 75-100 per cent. To investigate whether anti-bacterial activity was responsible for the reported therapeutic success of AP, we carried out a number of studies. The first study was a direct assay of anti-bacterial activity of AP suspended in water. The tested pathogens included Salmonella, Shigella, E.coli, gr. A Streptococci and S.aureus. Anti-bacterial activity was not demonstrable even in a solution containing 25,000 mg per litre of crude powder. The second was designed to detect serum bactericidal activity after oral intake of stem and leaves of AP. Ten healthy volunteers were enrolled in the study. They received a single oral dose of AP (1, 2, 3 and 6 g) in a randomized, cross-over manner. The washout period was one week. Blood samples were taken at 0, 1, 2, 4, 8 and 24 hours after ingestion. Serum bactericidal activity was assayed by agar diffusion technique using Bacillus spores and five strains of each pathogen (Shigella, Salmonella typhi, S.aureus and gr. A Streptococci) incubated for 24 hours. Again serum bactericidal activity was not detected in any of the sera tested. In a third study, ninety-six rats were daily fed with high doses of AP ranging 0.12-24 g per kg body wt. for six months before sacrifice. Antibacterial activity was still undetectable when lung parenchyma and liver tissue was placed on culture media containing bacteria tested. In conclusion, anti-bacterial activity of AP is undetectable in our study.

A model for testing antiseptic efficacy: a preliminary study on Betadine and Germidine.

Efficacy of povidone iodine antiseptic, betadine and germidine, was tested against normal skin flora and four pathogenic bacteria namely S. aureus, S. epidermidis, E. coli and Pseudomonas aeruginosa by a new model. The study was performed on the volar surface of forearms of ten patients. First of all, the skin flora was cultured then 10(8) cu/ml of the tested organisms was applied by a cotton swab and left to dry for 1 minute before the culture was repeated. Betadine or germidine was applied over the area previously painted with the organism. The culture was taken 1 to 2 minutes thereafter. The results showed that this model was feasible and convenient. Betadine and germidine are efficacious against normal skin flora and pathogenic bacteria.

Patients colonized by antibiotic resistant bacteria--a potential source of infections in the medical wards.

Bacterial colonization was studied in 12 non-infected female patients admitted into one medical ward, Siriraj hospital, Bangkok, from March to June 1988. Swabs were taken on the first day of admission, then every other day until discharge, from six sites; i.e. anterior nares, vault of axilla, hands, anterior chest, abdomen and toe web. The times and total number of swabbing were 52 and 312 respectively. All patients were colonized with bacteria. S. epidermidis was found in all patients. S. aureus was found in 9 patients, 48 times (15.4%). Methicillin-resistant S. aureus (M.R.S.A.) was identified in 4 patients on 10 occasions (3.2%). Gram-negative bacilli were isolated in 11 of 12 patients, and the number of positive samples was 60 (19.2%). All bacteria were highly resistant to the commonly used antimicrobials. The study failed to show that colonization increased with the duration of hospitalization. It is concluded that the majority of patients who had been colonized with pathogenic bacteria were important sources of infections in the medical ward.