Prevalent emm types and superantigen gene patterns of group A Streptococcus in Thailand.

Group A Streptococcus (GAS) are globally distributed bacterial pathogens. We examined the emm genotypes, which are important indicators of virulence, of 349 clinical GAS isolates collected using two surveillance systems, i.e. Invasive Bacterial Infection Surveillance (IBIS) from 2010 to 2011 (234 isolates) and routine surveillance of clinically isolated bacteria from various hospitals during 1996-2011 (115 isolates) in Thailand. The major emm genotypes in IBIS samples were emm44 (12·0%), emm104 (6·8%), emm22 (5·6%), and emm81 (5·6%), whereas only one isolate (0·4%) had the emm1 genotype, which is significantly more common in invasive cases in the Western world. In samples collected during routine surveillance, emm238 (10·4%), emm44 (8·7%), and emm165 (7·0%) were dominant. The major superantigen gene profiles were similar between the groups, and 30·1% of isolates did not possess the phage-encoded superantigens (speA, speC, speH, speI, speK, speL, speM, ssa). Although most isolates exhibited limited gene profiles, emm44 isolates had highly variable gene profiles (15 patterns). We conclude that emm44 is the predominant GAS genotype in Thailand, and isolates varied in superantigen gene profiles.

Acinetobacter bacteraemia in Thailand: evidence for infections outside the hospital setting.

SUMMARY: Acinetobacter is a well-recognized nosocomial pathogen. Previous reports of community-associated Acinetobacter infections have lacked clear case definitions and assessment of healthcare-associated (HCA) risk factors. We identified Acinetobacter bacteraemia cases from blood cultures obtained <3 days after hospitalization in rural Thailand and performed medical record reviews to assess HCA risk factors in the previous year and compare clinical and microbiological characteristics between cases with and without HCA risk factors. Of 72 Acinetobacter cases, 32 (44%) had no HCA risk factors. Compared to HCA infections, non-HCA infections were more often caused by Acinetobacter species other than calcoaceticus-baumannii complex species and by antibiotic-susceptible organisms. Despite similar symptoms, the case-fatality proportion was lower in non-HCA than HCA cases (9% vs. 45%, P < 0·01). Clinicians should be aware of Acinetobacter as a potential cause of community-associated infections in Thailand; prospective studies are needed to improve understanding of associated risk factors and disease burden.

Pneumococcal antigen testing of blood culture broth to enhance the detection of Streptococcus pneumoniae bacteremia.

The purpose of this investigation was to enhance the detection of pneumococcal bacteremia cases using the Binax NOW® immunochromatographic test (ICT) on blood culture broth as part of surveillance in two rural Thailand provinces. Blood cultures were collected as clinically indicated from hospitalized patients. ICT was performed on broth from culture bottles flagged as positive by BactT/ALERT® (alarm-positive) but which failed to grow organisms on subculture. During the period May 2005-June 2007, ICT was positive on 43 (24%) of 182 alarm-positive blood cultures with no growth on subculture. Compared to pneumococcal bacteremia cases confirmed by culture, cases detected only by ICT had a longer median time from culture collection to incubation and a longer median time from alarm positivity to subculture, and were more likely to be from patients pretreated with antibiotics. In a subsequent surveillance period (July 2007-December 2009), ICT continued to detect additional pneumococcal cases, but in a lower proportion of samples (7 of 221, 3.2%). Recently, as part of a separate study, ICT applied to uninoculated blood culture broth produced weak-positive results, mandating caution if testing broth from patient blood cultures. The antigen testing of blood culture broth appears to enhance the detection of pneumococcal bacteremia, but a controlled evaluation is needed.

MRSA carriage in a tertiary governmental hospital in Thailand: emphasis on prevalence and molecular epidemiology.

We investigated prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) in a case-control study performed in a 900-bed tertiary governmental healthcare facility in Bangkok, Thailand. Multivariate unconditional logistic regression was used to identify risk profiles for MRSA carriage. Phage typing, pulsed-field gel electrophoresis (PFGE), polymorphisms of the coa and spa genes, hypervariable region (HVR) of SCCmec, multi-locus sequence typing (MLST), and identification of ST30/ST8 mosaic chromosome by heteroduplex-polymerase chain reaction (heteroduplex-PCR) were used to demonstrate a clonal relationship. Fifty-seven of 619 in-patients (9.2%) were positive for MRSA. Risk factors were being male, long admission, low modified McCabe score, history of MRSA infection, and use of broad spectrum cephalosporin. Molecular typing results indicated close relatedness among MRSA isolates. Successful epidemic subtypes were recovered from many different wards. However, all subtypes with different multi-locus sequence types were single locus variants (SLVs) of ST239. Heteroduplex-PCR gave two positive bands from ST8/ST30 mosaic chromosomal structures in all SLVs indicating all isolates were of the ST239 origin. The burden of MRSA nosocomial infections is high in the governmental tertiary hospital. The sole ST239 and its SLVs identified in this hospital is striking and calls for better policy for infection control and prevention.

Separation of 6-deoxy-heptan [correction of 6-deoxy-heptane] from a smooth-type lipopolysaccharide preparation of Burkholderia pseudomallei.

Smooth-type lipopolysaccharide (LPS) of Burkholderia pseudomallei has been reported to contain two kinds of O-antigenic polysaccharides, a 1,3-linked homopolymer of 6-deoxy-heptose and a polymer with a repeating unit of -->3)-glucose-(1-->3)-6-deoxy-talose-(1--> with O-acetyl or O-methyl modifications. A LPS preparation containing these two polysaccharides was separated by gel-permeation chromatography in this study. Chemical analysis of the separated fractions revealed the 6-deoxy-heptan [corrected] to be a polysaccharide without a lipid portion and the polymer of glucose and 6-deoxy-talose to be an O-antigenic polysaccharide of the LPS. This result was further supported by the assay of these polysaccharide molecules for macrophage activation activity. The 6-deoxy-heptan [corrected] showed no macrophage activation, indicating that this polysaccharide was not the LPS, but one of the capsular polysaccharides of B. pseudomallei.

Burkholderia uboniae sp. nov., L-arabinose-assimilating but different from Burkholderia thailandensis and Burkholderia vietnamiensis.

A polar multitrichous gram-negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA-DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B. thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9%. The strain was clearly differentiated from the type strain of B. thailandensis by physiological, bio-chemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition. Based on the results of 16S rDNA sequence analysis, DNA-DNA hybridization and phenotypic characterization, Burkholderia uboniae sp. nov. is herein proposed. The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand. Major respiratory quinone is ubiquinone-8(Q8). G+C content of DNA is 69.71%.

Nasopharyngeal carriage of penicillin-resistant Streptococcus pneumoniae among children with acute respiratory tract infections in Thailand: a molecular epidemiological survey.

The prevalence of penicillin-resistant Streptococcus pneumoniae in Thailand has dramatically increased over the last decade. During a national survey, which was conducted from 1992 to 1994, 37.2% of the pneumococci isolated from the nasopharynges of children with acute respiratory tract infections were penicillin resistant (MIC, >/=0.1 microg/ml). In order to investigate the prevalence and clonal relatedness of nasopharyngeal carriage of penicillin-resistant S. pneumoniae in Thailand, a molecular epidemiological survey was undertaken. To this end, 53 penicillin-resistant pneumococcal isolates from children who suffered from acute respiratory tract infections and who originated from five distinct regions of the country were characterized in detail. DNA fingerprint analysis demonstrated 13 clusters, i.e., genotypes shared by two or more strains, and 14 unique genotypes. The cluster size varied from 2 (nine clusters) to 11 strains (one cluster). Six of the 13 restriction fragment end labeling clusters consisted of two or more distinct serotypes, indicating frequent horizontal transfer of capsular genes. Geographical distribution of the genotypes among the five regions of Thailand demonstrated that only four genetic clusters were restricted to single areas of the country, whereas the other nine clusters represented isolates collected in two or more districts. These observations demonstrate that the majority of the genetic clusters are spread throughout the country. The most predominant genetic cluster, representing 21% of the isolates, was identical to the Spanish pandemic clone 23F. In addition, the second largest cluster matched the Spanish-French international clone 9V. These data indicate that the genetic clones 23F and 9V, which are widely spread throughout the world, are the most predominant multidrug-resistant pneumococcal clones in Thailand. Therefore, we conclude that these pandemic clones are primarily responsible for the increase in the prevalence of pneumococcal penicillin resistance in Thailand.

Characterization of three capsular polysacharides produced by Burkholderia pseudomallei.

Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei. When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced. CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories. When the bacterium was grown with the medium containing 5" glycerol. CP-2 was synthesized. CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1. Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides. CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously.

Incidence and clinical symptoms of Aeromonas-associated travellers' diarrhoea in Tokyo.

In a survey examining the causes of travellers' diarrhoea treated in Tokyo between July 1986 and December 1995, Aeromonas species were isolated from 1265 (5.5%) of 23,215 travellers returning from developing countries. Aeromonas species were the fourth most frequent enteropathogen isolated, following enterotoxigenic E. coli (8.5%), Salmonella spp. (7.6%) and Plesiomonas shigelloides (5.6%). Aeromonas species were found in 1191 (5.6%) of 21,257 patients with diarrhoea and in 74 (3.8%) of 1958 healthy individuals without diarrhoea. Mixed infection was observed in 512 (40.5%) cases. No significant difference in the prevalence of Aeromonas by year, season, age distributions, or sex was observed, but a slight difference was noted depending on the country where the travellers visited. Of the 1265 Aeromonas isolates, 893 strains (70.6%) were A. veronii biovar sobria, 330 (26.1%) were A. hydrophila, and 42 (3.3%) were A. caviae. The clinical symptoms of patients from whom Aeromonas species was isolated as the only potential enteric pathogen were almost similar, which were watery diarrhoea (about 60%), abdominal cramps (43%), fever (around 15%), and nausea or vomiting (13%). Although the severity of illness was milder than that of enterotoxigenic E. coli alone, these data suggest that Aeromonas species are important enteric pathogens in travellers' diarrhoea.

Molecular epidemiology of drug-resistant pneumococci: toward an international approach.

An international multicenter study was undertaken to investigate the epidemiological dynamics of penicillin-resistant pneumococci. We compared the molecular epidemiological characteristics of 205 penicillin-resistant isolates originating from The Netherlands, Thailand, United States, Spain, Greece, Poland, Cuba, Germany, Finland, United Kingdom, South Africa, Hungary, Portugal, Croatia, and the Czech Republic. Eighty-four distinct restriction fragment end labeling (RFEL) types were observed. Twenty-eight genetic types were shared by two or more strains. Five genetic clusters consisted of strains originating from different countries, illustrating dissemination of penicillin-resistant pneumococci among countries. The strains displaying the two predominant RFEL types corresponding with the pandemic clones 23F and 9V were found in 10 and 6 different countries, respectively. This clearly demonstrates the pandemic behavior of these two clones. Twelve out of the 28 genetic clusters contained two or more serotypes. This finding indicates frequent horizontal transfer of capsular genes. Within distinct RFEL types, identical penicillin binding protein (PBP) genotypes were often observed, suggesting a high frequency of horizontal transfer of penicillin resistance genes. The most predominant PBP type was found in 15 distinct RFEL types, comprised 44% of the entire collection, and was observed in 11 countries. The vast majority of the strains belonging to the pandemic clones 23F and 9V shared this predominant PBP type. We hypothesize that the clones 23F and 9V are responsible for the worldwide increase of penicillin-resistance, because they serve as a genetic reservoir for susceptible pneumococci to acquire penicillin resistance.

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.

Environmental surveillance of Legionella species in Thailand.

Legionellae was found in 57 per cent of 94 cooling towers and 21.8 per cent of 78 other environmental sources. These figures reveal that the existence of legionellae in cooling towers is more prevalent than that found in other environmental sources. Legionella pneumophila serogroup 1 was the most prevalent organism among the legionellae found in cooling towers and other environmental sources. The recovery of legionellae in each province was seasonally independent and was found throughout the year. This is the first report of an environmental survey representing the existence of legionellae in every region of Thailand.

Extraction and characterization of lipopolysaccharide from Pseudomonas pseudomallei.

The best yield of lipopolysaccharide (LPS) of Pseudomonas pseudomallei GIFU 12046 was obtained by extraction of defatted cells by phenol/chloroform/petroleum ether. The LPS showed a smooth character on SDS-polyacrylamide gel electrophoresis and contained D-glucose, L-glycero-D-manno-heptose, and D-glucosamine as the main sugar components, and 3-hydroxypalmitic acid as an amide-linked fatty acid. The growth conditions did not affect the electrophoresis profile and chemical composition of LPS. 2-Keto-3-deoxyoctonic acid was not detectable, and mild acid hydrolysis could not liberate free lipid A, suggesting that the linkage between inner core and lipid A was stable against acid hydrolysis, and the structure of this region is similar to that of P. cepacia, which has close taxonomic relationship with P. pseudomallei.

Species-differences in the pH-activity pattern of acid phosphatase in the family Enterobacteriaceae.

Nonspecific phosphatase activities were surveyed comparing major species of the family Enterobacteriaceae. The strains were subjected to a whole cell assay system with paranitrophenyl phosphate as substrate over a wide pH range and with a standardized number of bacterial cells. The overall results suggest that the general shape of the pH activity curve and the location of peaks (pH optimum) can be employed as a supplementary criterion to characterize species of Enterobacteriaceae.

Heat-stable and heat-labile components of nonspecific acid phosphatase detected in Pseudomonas pseudomallei.

In a whole cell assay system with p-nitrophenyl phosphate as substrate, strains of Pseudomonas pseudomallei showed a two-peak pattern in pH activity curve of acid phosphatase, suggesting the presence of two enzyme components different in pH optimum (4.2 and 5.2). The component of 5.2 pH optimum was detected in the outer membrane fraction and the activity was resistant to heating at 70 C for 30 min. The other component of 4.2 pH optimum was heat-labile. No substantial difference was observed in the enzymatic activity between R and S type colonies.

Growth and survival of Pseudomonas pseudomallei in acidic environments.

A study was made on the growth and survival of Pseudomonas pseudomallei in culture environments differing in nutrients, initial pH, and aeration, in comparison with Pseudomonas cepacia and Pseudomonas aeruginosa. The observations led us to a view that P. pseudomallei has the highest adaptability to acidic environments among the three species. Unlike the other species, it grew in heart infusion broth of initial pH 4.5 under aeration and survived keeping a high level (10(9) per ml) of viable counts for as long as 30 days. This sort of adaptation was found to be more evident in the media of poor nutrition and under limited aeration.

In vitro susceptibilities of Pseudomonas pseudomallei to 27 antimicrobial agents.

Clinical isolates of Pseudomonas pseudomallei isolated in Thailand from 1981 to 1989 were tested for their in vitro susceptibilities to 27 antimicrobial agents, including older and newer quinolones, broad-spectrum cephems, carbapenems, monobactams, penicillins, tetracyclines, chloramphenicol, rifamycin, sulfamethoxazole, trimethoprim, and fosfomycin. Tosufloxacin, meropenem, CS-533, and minocycline were active against P. pseudomallei at levels comparable to or even greater than those of antimicrobial agents tested in previous studies, such as ciprofloxacin, ceftazidime, imipenem, carumonam, and piperacillin. Drug-resistant P. pseudomallei was found in only 1% of the isolates. The drug-resistant P. pseudomallei isolates displayed a unique pattern of susceptibility to the above-listed drugs.

High activity of acid phosphatase of Pseudomonas pseudomallei as a possible attribute relating to its pathogenicity.

Phosphatase activities were compared quantitatively among selected species of pseudomonads. P. pseudomallei showed the highest activity of a bell-shaped pH pattern with a peak at around pH 5.0. P. cepacia had a similar pattern of milder intensity. In contrast, P. aeruginosa revealed an alkaline phosphatase activity with a pH optimum higher than 8.0, but the level of activity was much lower than those of the above two species. The enzymatic reactions of other species were slight or negligible at their optimum pH in the same test system. These data were discussed in reference to their growth behavior in different pH environments and also in connection with such recent information that the high activity of microbial acid phosphatase may be a favorable attribute to their intracellular parasitism.

Pseudomonas pseudomallei and melioidosis, with special reference to the status in Thailand.

Melioidosis is a long-known disease since 1912, but only quite recently we have obtained the knowledges about its actual clinical and epidemiological features. The disease is so unique in having a wide spectrum of disease course and clinical manifestation. The causative agent, P. pseudomallei, is free-living bacterium in the natural environments (soil and surface water) of tropical and subtropical areas. Just like legionnaires' disease, melioidosis is a good example of infectious disease in which pneumonia is produced by inhalation of contaminated soil dusts or water droplets. The infection becomes dormant for years, but with a chance of recrudescence under a variety of insults to the host resistance. The disease, may it be acute or chronic, will be symptomatically confused with malaria, typhoid fever, leptospirosis, septicemia caused by other gram-negative bacteria, tuberculosis and mycotic infections. Isolation of the causative agent from clinical specimens is the only reliable method for diagnosis. Because of the increasing clinical awareness and the development of diagnostic methods, the reported cases of melioidosis have numbered almost one thousand in Thailand during the past 20 years. This country has now the most ample clinical experiences on melioidosis. We have reviewed the history of melioidosis research from bacteriological, immunological, clinical and epidemiological viewpoints, especially including the recent reports in Thailand.