Aroonlug Lulitanond

Risk Factors for Melioidosis and Bacteremic Melioidosis

A case-control study was conducted in four hospitals in northeastern Thailand to identify risk factors for melioidosis and bacteremic melioidosis. Cases were patients with culture-proven melioidosis, and there were two types of controls (those with infections, i.e., with community-acquired septicemia caused by other bacteria, and those without infection, i.e., randomly selected patients admitted with noninfectious diseases to the same hospitals). Demographic data, clinical presentations, and suspected risk factors were analyzed. Diabetes mellitus, preexisting renal diseases, thalassemia, and occupational exposure, classified by the soil and water risk assessment, were confirmed to be significant risk factors for melioidosis and bacteremic melioidosis. Only diabetes mellitus was a significant factor associated with bacteremic melioidosis, as compared with nonbacteremia. A significant interaction was found between diabetes mellitus and occupational exposure. Thus, diabetic rice farmers would be the most appropriate population group for targeted control measures such as vaccination in the future.

Emergence of NDM-1- and IMP-14a-producing Enterobacteriaceae in Thailand

OBJECTIVES To detect carbapenemases in clinical isolates of Enterobacteriaceae collected from patients in a university hospital in Thailand between October 2010 and August 2011. METHODS A total of 4818 Enterobacteriaceae isolates were screened for the presence of carbapenemases by ertapenem and imipenem disc diffusion tests. All positive screening isolates were subjected to modified Hodge test, phenylboronic acid- and EDTA-carbapenem combined disc tests and two multiplex PCRs of bla(IMP), bla(VIM), bla(SPM), bla(SIM) and bla(GIM), and of bla(KPC), bla(NDM) and bla(OXA-48). Carbapenemase-producing isolates were typed by PFGE and then characterized by antimicrobial susceptibility tests. Conjugation was performed using a broth culture mating method. RESULTS Two isolates each of Escherichia coli, Klebsiella pneumoniae and Citrobacter freundii produced NDM-1, whereas two other isolates of K. pneumoniae produced IMP-14a. DNA fingerprints revealed that the metallo-β-lactamase (MBL)-producing isolates were of different strains except for clonal strains of C. freundii. In vitro transfer of carbapenem resistance was successful for the eight MBL-producing isolates. All MBL producers were susceptible to colistin and tigecycline. The six NDM-producing isolates were recovered from the urine of three patients, who had no history of travel outside Thailand. Interestingly, one patient had chronic urinary tract infections caused by a K. pneumoniae strain and two strains of E. coli producing NDM-1. CONCLUSIONS Surveillance of carbapenemases, particularly NDM-1, in Enterobacteriaceae is urgently needed to control and prevent the spread of these resistance determinants in our country.

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.

Discrimination of SHV β-Lactamase Genes by Restriction Site Insertion-PCR

ABSTRACT Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3′ end to modulate a restriction site. We have developed this technique to identify described mutations of the blaSHV genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV β-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly→Ala) (BsrI), and 240 (NruI) ofblaSHV genes. All amplimers of theblaSHV genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification ofblaSHV-6, blaSHV-8, and 12 novel blaSHV variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV β-lactamases and may be extended to the characterisation of other resistance determinants.

CMY-2, CMY-8b, and DHA-1 plasmid-mediated AmpC β-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae from a university hospital, Thailand

Between February 2005 and January 2006 in Srinagarind Hospital, Thailand, 44 from 1730 isolates (2.5%) of Escherichia coli and 8 from 982 isolates (0.8%) of Klebsiella pneumoniae were found to produce plasmid-mediated AmpC β-lactamases (pAmpCs) as detected by a cefoxitin-Hodge test followed by a multiplex polymerase chain reaction (PCR) technique. Fifteen of the 52 pAmpC-producing isolates also produced extended-spectrum β-lactamases. The ampC genes found in both organisms were bla(CMY-2) (46 isolates), bla(CMY-8b) (4 isolates), and bla(DHA-1) (2 isolates). These genes were present on plasmids. Twenty-five of the 46 CMY-2-producing isolates could transfer cefoxitin resistance to E. coli UB1637 by conjugation. More than 90% of the pAmpC-producing isolates were resistant to cefoxitin, but 80% to 90% of them were susceptible or intermediately susceptible to ceftazidime or cefotaxime. Enterobacterial repetitive intergenic consensus PCR analysis revealed that most isolates were of different strains, indicating the ease of transmission of these resistance determinants. This is the first report of CMY-2, CMY-8b, and DHA-1 β-lactamases in Thai isolates.

The First Vancomycin-Intermediate Staphylococcus aureus Strains Isolated from Patients in Thailand

ABSTRACT We screened 533 and 361 methicillin (meticillin)-resistant Staphylococcus aureus strains isolated in a university hospital in 2002 and 2003 and in 2006 and 2007, respectively, and identified 4 (0.8%) of the strains in the first group and 8 (2.2%) of the strains in second group as heterogeneous vancomycin-resistant S. aureus (heterogeneous VISA) strains and 3 (0.8%) of the strains in the second group as VISA strains. This is the first report of VISA strains isolated from patients in Thailand.

ST9 MRSA strains carrying a variant of type IX SCCmec identified in the Thai community.

BackgroundInfections caused by methicillin-resistant Staphylococcus aureus (MRSA) in Thailand occur most frequently in healthcare facilities. However, reports of community-associated MRSA are limited.MethodsWe characterized 14 MRSA isolates from outpatients (O-1 to O-14) by phenotypic and genotypic methods and compared them with 5 isolates from inpatients (I-1 to I-5). Thai MRSA isolates from a healthcare worker (N-1) and a pig (P-1) were also included as ST9 MRSA strains from other sources.ResultsAll MRSA isolates from the outpatients and inpatients were multidrug-resistant (resistant to ≥3 classes of antimicrobials). All of them except strains O-2 and I-3 carried type III SCCmec and belonged to agrI, coagulase IV, spa type t037 or t233, which related to ST239. The strain O-2 (JCSC6690) carried type IX SCCmec and belonged to agrII, coagulaseXIc, spa type t337 and ST9, whereas the strain I-3 carried a type III SCCmec and belonged to ST1429. Nucleotide sequence determination revealed that the type IX SCCmec element in strain O-2 was distinct from that in a Thai ST398 strain (JCSC6943) previously identified in 2011; nucleotide identities of ccrA and ccrB were 93 and 91%, respectively and several open reading frames (ORFs) at the joining regions were different. PCR experiments suggested that strain O-2 and N-1 carried similar SCCmec element, whereas that of strain P-1 was different, suggesting that distinct ST9-MRSA–IX clones might be spreading in this province.ConclusionsThe SCCmecIX-ST9 MRSA clones of distinct SCCmec subtypes might have emerged in the Thai community and might also have disseminated into the hospital.

Chronic Opisthorchis viverrini Infection Changes the Liver Microbiome and Promotes Helicobacter Growth

Adults of Opisthorchis viverrini reside in the biliary system, inducing inflammation of bile ducts and cholangitis, leading to hepatobiliary disease (HBD) including cholangiocarcinoma. O. viverrini infection also has major implications for the bacterial community in bile ducts and liver. To investigate this in chronic O. viverrini infection (≥ 8 months p.i.), bacterial genomic DNA from livers of hamsters and from worms was investigated using culture techniques, PCR for Helicobacter spp. and high-throughput next-generation sequencing targeting the V3-V4 hypervariable regions of prokaryotic 16S rRNA gene. Of a total of 855,046 DNA sequence reads, 417,953 were useable after filtering. Metagenomic analyses assigned these to 93 operational taxonomic units (OTUs) consisting of 80 OTUs of bacteria, including 6 phyla and 42 genera. In the chronic O. viverrini-infected group, bacterial community composition and diversity were significantly increased compared to controls. Sequences of Fusobacterium spp. were the most common (13.81%), followed by Streptococcus luteciae (10.76%), Escherichia coli (10.18%), and Bifidobacterium spp. (0.58%). In addition, Helicobacter pylori (0.17% of sequences) was also identified in the liver of chronic O. viverrini infections, but not in normal liver. The presence of H. pylori was confirmed by PCR and by use of an antibody against bacterial antigen, supporting the metagenomics data. The identities of bacteria cultured for enrichment suggested that chronic O. viverrini infection changes the liver microbiome and promotes Helicobacter spp. growth. There may be synergy between O. viverrini and the liver microbiome in enhancing immune response-mediated hepatobiliary diseases.

SCCmec IX in meticillin-resistant Staphylococcus aureus and meticillin-resistant coagulase-negative staphylococci from pigs and workers at pig farms in Khon Kaen, Thailand

Livestock-associated meticillin-resistant Staphylococcus aureus, clonal complex (CC) 398, has been reported in Europe, whereas CC9 MRSA has mostly been found in Asia. Therefore, we aimed to detect MRSA on pig farms in north-eastern Thailand. A total of 257 nasal swabs (159 samples from pigs and 98 from pig-farm workers) were collected from three pig farms in north-eastern Thailand from 2010 to 2011. MRSA isolates were confirmed for femA and mecA genes by PCR. The MICs of eight antimicrobials, namely vancomycin (VA), cefazolin (CZ), ofloxacin (OF), tetracycline (TET), erythromycin (ER), oxacillin (OX), cefoxitin (FOX) and gentamicin (GN), were tested by agar dilution method. The virulence genes for Panton-Valentine leukocidin toxin (lukSF-PV), toxic shock syndrome toxin-1 (tst) and α-haemolysin (hla) were detected by PCR. Strain typing was performed by staphylococcal cassette chromosome (SCC) mec, agr, spa and multilocus sequence typing. Four MRSA were isolated: three from workers and one from a pig. All the MRSA isolates were resistant to OX, GN, ER, TET and CZ, and they all carried hla only. Two MRSA from humans carried SCCmec II-sequence type (ST)764-agrII, whereas the two remaining MRSA (one each from a human and a pig) contained SCCmec IX-ST9-agrII. Interestingly, meticillin-resistant coagulase-negative Staphylococcus isolates carrying SCCmec IX were also obtained from five workers and three pigs. This study suggests that the SCCmec IX element is distributed among the Staphylococcus found in pigs and pig-farm workers, and pigs may be a reservoir for MRSA in the community.

Effect of acidic pH on the invasion efficiency and the type III secretion system of Burkholderia thailandensis

Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.