Chalit Komalamisra

Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study

The emergence of colistin-resistant Klebsiella pneumoniae (CRKP) is a major public health concern worldwide. In this study, the prevalence and molecular basis of colistin resistance in CRKP isolated from healthy individuals and patients in Lao PDR, Thailand, Nigeria and France were investigated. Stool samples were screened by culture for the presence of colistin-resistant Klebsiella spp. Whole-genome sequence analysis was used to decipher the molecular mechanism of colistin resistance in a blaNDM-1-positive in vitro-selected CRKP mutant. PCR amplification and sequencing of the mgrB genetic environment was performed for all CRKP isolates as well as control colistin-susceptible K. pneumoniae (CSKP) isolates recovered from the same stools. A total of 869 stool samples were screened for colistin-resistant Klebsiella spp., yielding 32 CRKP and 2 colistin-resistant Klebsiella oxytoca. Comparative whole-genome sequence analysis revealed that an in vitro-selected CRKP mutant had an insertion sequence in its mgrB gene, as well as missense mutations in other selected clones. Of the 34 colistin-resistant Klebsiella spp. isolates, 14 (41.2%; 13 CRKP and 1 K. oxytoca) from the four countries also had various defects in their mgrB genes, but no such defects were found in the CSKP controls (P<10(-4)). Few mutations were observed in pmrAB compared with mgrB among the CRKP isolates. The worldwide emergence of CRKP is a major public health concern. Detection and surveillance of such strains are warranted to prevent an uncontrollable pandemic. Inactivation of the PhoP/PhoQ regulator gene mgrB is associated with ≥40% of colistin resistance among the CRKP isolates observed in this study.

High prevalence of CTX-M β-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in Thailand

OBJECTIVES To determine the prevalence of CTX-M beta-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in a rural area of Thailand. METHODS Bacteria in stool specimens were screened for extended-spectrum beta-lactamase (ESBL) production on McConkey agar with cefotaxime and confirmed by the double-disc synergy test. Genetic detection and genotyping of CTX-M-type ESBL was performed by PCR with bacterial DNA extracted from isolates. RESULTS A markedly high number (82 of 141, 58.2%) of the specimens showed the presence of CTX-M beta-lactamase-producing Enterobacteriaceae, as confirmed by both phenotypic and genetic examinations. The majority of the CTX-M beta-lactamase-producing bacteria were Escherichia coli (85.1%). CONCLUSIONS The study revealed the wide dissemination of CTX-M beta-lactamase-producing Enterobacteriaceae in the healthy population.

Prevalence of and risk factors associated with faecal carriage of CTX-M β-lactamase-producing Enterobacteriaceae in rural Thai communities

OBJECTIVES To determine the prevalence of CTX-M β-lactamase-producing Enterobacteriaceae and to study the risk factors associated with faecal carriage in asymptomatic rural Thai people. METHODS In all, 417 stool samples were obtained from rural Thai people and screened for extended-spectrum β-lactamases (ESBLs) using MacConkey agar supplemented with 2 mg/L cefotaxime. Results were confirmed using cefotaxime and ceftazidime with and without clavulanic acid. The bla(CTX-M) genes were identified and genotyped using PCR with bacterial DNA samples. Multivariate analysis was performed to investigate risk factors associated with the faecal carriage of CTX-M producers. RESULTS The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae was 65.7%. The CTX-M-9 group (60.6%) was dominant, followed by the CTX-M-1 group (38.7%). Most of the bacteria were Escherichia coli (85.4%) and Klebsiella pneumoniae (4.7%). Of a total of 234 E. coli strains, 48.7% belonged to phylogenetic group A, 28.6% to group B1, 15.8% to group D and 6.8% to group B2. Most CTX-M producers were susceptible to carbapenems and amikacin, but resistant to tetracycline and gentamicin. In a multivariate logistic regression model, better education status (OR 2.245; 95% CI 1.297-3.884), history of hospitalization (OR 1.643; 95% CI 1.036-2.603) and the use of antibiotics within the last 3 months (OR 1.883; 95% CI 1.221-2.903) were independently associated with faecal carriage. CONCLUSIONS Faecal carriage of CTX-M-type ESBL-producing Enterobacteriaceae among asymptomatic individuals in rural Thailand remains alarmingly high, and previous antibiotic use and a history of hospitalization may contribute to its dissemination.

Discovery of Opisthorchis viverrini metacercariae in freshwater fish in southern Cambodia.

Small liver flukes, Clonorchis sinensis and Opisthorchis viverini, are fish-borne trematodes (FBTs) causing significant public health problems in Asia. While C. sinensis is distributing mainly in far east Asia, O. viverini is distributing in Indochina peninsula. Recently, however, the geographical distributions of those small liver flukes were proven to be far wider than expected. Nevertheless, little is known about the epidemiology of small liver flukes in Cambodia. The present study is, therefore, aimed at clarifying the status of small liver fluke infections in various species of freshwater fish in southern Cambodia. A total of 1479 freshwater fish, 1316 (89%) comprised of 20 different species of the cyprinoid family and 163 (11%) belonging to 8 families of non-cyprinoids, were collected during May 2007 and February 2008, and the presence of small liver fluke metacercariae was examined by the compression method. Small liver fluke metacercariae were found in 10 species of cyprinoids with the infection rate ranging 2.1-66.7% and the mean intensity of infection of 1.0-15.0 (range: 1-65). For the speciation, adult worms were obtained by experimental infection in hamsters. The small liver fluke found in this study were identified as Opisthorchis viverrini by the morphological features of adult worms, and this identification was confirmed by partial COI sequencing of the metacercariae.

Analysis of risk factors for a high prevalence of extended-spectrum {beta}-lactamase-producing Enterobacteriaceae in asymptomatic individuals in rural Thailand.

The prevalence of and risk factors associated with extended-spectrum β-lactamase (ESBL)-producing micro-organisms have not been well studied in healthy individuals. The aim of this study was to determine this in healthy individuals in Thailand. Stool samples and questionnaires obtained from 445 participants from three provinces in Thailand were analysed. The antimicrobial susceptibility of the isolates was assessed using phenotypic and genotypic methods. PCR analysis was performed to detect and group the bla(CTX-M) genes. The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae in the three provinces was as follows: 29.3 % in Nan (43/147), 29.9 % in Nakhon Si Thammarat (43/144) and 50.6 % in Kanchanaburi (78/154) (P<0.001). Of the 445 samples, 33 (7.4 %), 1 (0.2 %) and 127 (28.5 %) isolates belonged to the bla(CTX-M) gene groups I, III and IV, respectively. Escherichia coli was the predominant member of the Enterobacteriaceae producing CTX-M-type ESBLs (40/43, 39/43 and 70/78 isolates in Nan, Nakhon Si Thammarat and Kanchanaburi, respectively). No statistically significant association was observed between the presence of ESBL-producing bacteria and gender, age, education, food habits or antibiotic usage. However, the provinces that had the highest prevalence of ESBL-producing Enterobacteriaceae also had the highest prevalence of use and purchase of antibiotics without a prescription. Thus, this study revealed that faecal carriage of ESBL-producing Enterobacteriaceae is very high in asymptomatic individuals in Thailand, with some variations among the provinces. This high prevalence may be linked to antibiotic abuse.

Wide dissemination of extended-spectrum β-lactamase-producing Escherichia coli in community residents in the Indochinese peninsula

Recent studies have reported a widespread distribution of extended-spectrum β-lactamase (ESBL)-producing bacteria, not only in the nosocomial setting, but also in the community; some local communities in Southeast Asia have been reported to show a high prevalence of ESBL-producing bacteria. However, the details regarding the quantitative/qualitative state of ESBL-producing bacterial spread in Southeast Asia are currently unclear. Thus, the aim of this study was to assess the state of ESBL-producing bacterial spread in community residents from the Indochinese peninsula, as a representative region of Southeast Asia. In order to achieve this aim, local community residents in Laos and Vietnam were examined for fecal carriage of ESBL-producing Enterobacteriaceae, and the findings were compared with data from a previous study in Thailand which was conducted in the same manner as this study. Between 47.0%–70.2% of the Laotian and Vietnamese residents carried ESBL-producing CTX-M genotype Enterobacteriaceae. The most common sub-genotypes of CTX-M were CTX-M-1 (33.0%–47.5%) and CTX-M-9 (47.5%–64.1%), and these rates were similar among all three countries. Taken together, these results confirmed that ESBL-producing Enterobacteriaceae are widely disseminated in Indochinese countries, such as Thailand, Laos, and Vietnam.

Systematics of the subfamily Haplorchiinae (Trematoda: Heterphyidae), based on nuclear ribosomal DNA genes and ITS2 region.

Phylogenetic relationships of 6 species in the trematode subfamily Haplorchiinae were analyzed using small and large subunit of ribosomal DNA genes (18S rDNA and 28S rDNA) and internal transcribed spacer subunit II (ITS2) region as molecular markers. Maximum Likelihood and Bayesian inference analyses of combined rDNAs and ITS2 indicated a close relationship between the genera Haplorchis and Procerovum, while these two genera were distinct from Stellantchasmus falcatus. These phylogenetic relationships were consistent with the number of testes but not with the characters of the modification of the seminal vesicle or of the ventral sucker. Although three Haplorchis spp. were, together with Procerovum, in the same cluster, their mutual topology was incongruent between rDNA and ITS2 trees. Phylogenetic analyses using other molecular markers with more species are necessary to work out solid phylogenetic relationships among the species in this subfamily.

Serodiagnosis of human opisthorchiasis using cocktail and electroeluted Bithynia snail antigens.

Cocktail and electroeluted antigens from Bithynia goniomphalos, the snail intermediate host of Opisthorchis viverrini, were extracted and purified. The performance of these two antigens in the antibody detection of human opisthorchiasis was evaluated by indirect ELISA. Serum samples from people whose stool was either: (i). positive for Opisthorchis eggs (n=61); or (ii). positive for at least one of 19 other species of parasite (n=125); or (iii). clear of parasites (n=30) were tested. The sensitivity, specificity, positive predictive value and negative predictive value of ELISA using cocktail antigen were 88.5, 88, 78.2 and 94%, respectively; those of ELISA using eluted antigen (53 kDa) were 91.8, 98.4, 96.5 and 96.1%, respectively. Cross-reaction with the eluted antigen was seen in only one of four cases of hymenolepiasis and only one of 10 cases of strongyloidiasis. The kappa coefficients for ELISA in relation to stool examination were 0.84 (cocktail antigen) and 0.87 (eluted antigen). This study showed that Bithynia snail antigen could be used to replace worm antigen in the antibody detection of human O. viverrini infection.

Zoonotic potential of Enterocytozoon bieneusi among children in rural communities in Thailand

Enterocytozoon bieneusi is a common opportunistic intestinal pathogen worldwide. Genotype distribution of E. bieneusi differs by geography and host immunity. In order to investigate the prevalence, genotype characteristics, and host specificity of E. bieneusi in the community, we conducted a preliminary cross-sectional study among children in Western and Northern Thailand. Seventy-eight (78) and 102 stool samples were collected; the prevalence of E. bieneusi was 3.8% and 2.9% by nested PCR in Western and Northern Thailand, respectively. Three genotypes were identified: Genotype D predominated, followed by EbpC, and then novel genotype ETMK1. The first two genotypes have zoonotic potential. Analysis of the genetic proximity of the E. bieneusi ITS sequences from our study, compared with those published in genetic databases, showed that all positive samples were classified into Group 1, the largest group consisting of various host specificity. The present study demonstrates the possible zoonotic transmission of E. bieneusi in rural communities in Thailand. A large-scale investigation of both human and animal samples, as well as improvements in the available phylogenetic tools, will be required to elucidate transmission routes of E. bieneusi in this area.

Genetic differences in the rat lungworm, Angiostrongylus cantonensis (Nematoda: Angiostrongylidae), in Thailand.

This study surveyed the genetic differences among Angiostrongylus cantonensis (A. cantonensis) using the mitochondrial cytochrome b (cytb) gene. Partial cytb sequences were determined for 91 worms from eight locations in Thailand. Using morphological techniques, the nematodes were found to be A. cantonensis. Phylogenetic analysis found two main clades, which were subdivided into four subclades (clusters). Haplotype network analysis showed that 11 distinct cytb haplotypes were also present in four groups of A. cantonensis. There was no observable relationship between the genetic differentiation of gene flow and geographical distance. This low genetic variation and geographical distribution of A. cantonensis in each location indicates a founder effect, which may have resulted from multiple independent origins, and suggests that haplotypes migrated from endemic areas via human-related activities.