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There are few studies comparing proportion, frequency, mortality and mortality rate of antimicrobial-resistant (AMR) bacterial infections between tertiary-care hospitals (TCHs) and secondary-care hospitals (SCHs) in low and middle-income countries (LMICs) to inform infection control strategies. We evaluated bloodstream infections (BSIs) from 2012 to 2015 in 15 TCHs and 34 SCHs in Thailand. There were differences in the proportions (%) of BSI caused by AMR strains for some pathogens between TCHs and SCHs. Of 19,665 patients with AMR BSI, 6,746 (34.3%) died. Among patients with AMR BSI, there were no or minimal differences in mortality proportion for all AMR pathogens between TCHs and SCHs. However, the frequency and mortality rates of AMR BSI were considerably higher in TCHs for most pathogens. For example, the mortality rate of hospital-origin carbapenem-resistantAcinetobacter baumanniiBSI in TCHs was nearly three times higher than that in SCHs (10.2 vs. 3.6 per 100,000 patient-days at risk, mortality rate ratio 2.77; 95% confidence interval 1.71 to 4.48, p

Here, we report the whole-genome sequence of a methicillin-resistant Staphylococcus aureus strain harboring staphylococcal cassette chromosome mec (SCC mec ) type IX, isolated from a fatal sepsis case. Genomic analysis revealed that the isolate was sequence type 9 and spa type t3446, carrying multiple antimicrobial-resistant genes comprising mecA , blaZ , aac(6′)-aph(2″) , aadD , ant(6)-Ia , lsa (E), dfrG , tet (M), fexA , and lnu (B).

Here, we report the whole-genome sequence of a methicillin-resistant Staphylococcus aureus strain harboring staphylococcal cassette chromosome mec (SCCmec) type IX, isolated from a fatal bacteremic pneumonia case. Genomic analysis revealed that the isolate was sequence type 9 and spa type t3446, carrying multiple antimicrobial resistance genes comprising mecA, blaZ, aac(6′)-aph(2″), aadD, ant(6)-Ia, lsa(E), dfrG, tet(M), fexA, and lnu(B).

Here, we report the whole-genome sequence of multidrug-resistant Salmonella enterica serovar Cannstatt harboring mcr-1.1, isolated from a fatal sepsis case. Genomic analysis revealed that the isolate was sequence type 2390 carrying mcr-1.1, blaCTX-M-14, aac(3)IId, aac(6′)Iaa, floR, qnrS1, sul2, tetA, and tetM. Three Inc plasmids were observed, including the IncX4 plasmid containing mcr-1.1.

Many microbial species have been recognized as enteropathogens for humans. Here, we predicted the causative agents of acute diarrhea using data from multiplex quantitative PCR (qPCR) assays targeting 19 enteropathogens. For this, a case-control study was conducted at eight hospitals in Thailand. Stool samples and clinical data were collected from 370 hospitalized patients with acute diarrhea and 370 non-diarrheal controls. Multiple enteropathogens were detected in 75.7% and 13.0% of diarrheal stool samples using multiplex qPCR and bacterial culture methods, respectively. Asymptomatic carriers of enteropathogens were found among 87.8% and 45.7% of individuals by qPCR and culture methods, respectively. These results suggested the complexity of identifying causative agents of diarrhea. An analysis using the quantification cut-off values for clinical relevance drastically reduced pathogen-positive stool samples in control subjects from 87.8% to 0.5%, whereas 48.9% of the diarrheal stool samples were positive for any of the 11 pathogens. Among others, rotavirus, norovirus GII, Shigella/EIEC, and Campylobacter were strongly associated with acute diarrhea (P-value

Acute diarrheal diseases are causes of global public health concern, especially in developing countries. A variety of diarrhea-associated microbial species, including bacteria, viruses, and protozoa , have been recognized. Simplified methods for detecting a wide range of diarrheagenic enteric microbes can clarify the etiology and aid in the diagnosis of diarrheal diseases. Here, we report a quantitative real-time (q)PCR-based method for simultaneous detection of 24 targets from 19 microbes suspected of causing diarrhea in stool specimens. We first selected the 24 oligonucleotide primer sets and hydrolysis probes conjugated with the fluorescent reporter dyes FAM, NED, or ABY, along with an internal control, and the passive reference dye ROX to establish a single-plate panel assay. The 12-duplex qPCR panel showed high linearity, with R 2 values of 0.981–1.0 and limits of detection ranging from 1 to 103 fg for bacterial DNA (1–200 cells), 10–102 copies for viral DNA/RNA , and 10 fg for parasitic DNA (equivalent to approximately 1 parasite) per reaction. The accuracy and robustness of the assay was demonstrated in experiments using clinical stool specimens. This platform is low cost and easily customizable, and can be applied to various types of qPCR instruments and experimental designs for surveillance of acute diarrhea.

Vibrio cholerae inhabits aquatic environments worldwide and has over 200 recognized serogroups classified by O-polysaccharide specificity. Here, we report that V. cholerae selects either of two genetic traits during their evolution. Sequencing of the specific gene locus MS6_A0927 revealed that 339 of 341 strains of V. cholerae and closely related Vibrio species originating from 34 countries over a century carried either metY (M) (~1,269 bp) or luxR-hchA (LH) (~1,600 bp) genes, and consequently those vibrios were separated into two clusters, M (45.4%) and LH (54.6%). Only two strains contained both M and LH in the same locus. Moreover, extensive polymorphisms in those genes were detected in M and LH with 79 and 46 sequence variations, respectively. V. cholerae O1 strains isolated from cholera outbreaks worldwide, and some non-O1 strains evolving from O1 via exchange of genes encoding cell surface polysaccharides possessed LH alleles. Analysis of polymorphisms in the gene locus implicated a high degree of genetic diversity and identical subpopulations among the V. cholerae species.

Cholera, caused by Vibrio cholerae, remains a global threat to public health. In Myanmar, the availability of published information on the occurrence of the disease is scarce. We report here that cholera incidence in Mandalay generally exhibited a single annual peak, with an annual average of 312 patients with severe dehydration over the past 5 years (since 2011) and was closely associated with the rainy season. We analyzed cholera outbreaks, characterized 67 isolates of V. cholerae serogroup O1 in 2015 from patients from Mandalay, and compared them with 22 V. cholerae O1 isolates (12 from Mandalay and 10 from Yangon) in 2014. The isolates carried the classical cholera toxin B subunit (ctxB), the toxin-coregulated pilus A (tcpA) of Haitian type, and repeat sequence transcriptional regulator (rstR) of El Tor type. Two molecular typing methods, pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis (MLVA), differentiated the 89 isolates into seven pulsotypes and 15 MLVA profiles. Pulsotype Y15 and one MLVA profile (11, 7, 7, 16, 7) were predominantly found in the isolates from cholera outbreaks in Mandalay, 2015. Pulsotypes Y11, Y12, and Y15 with some MLVA profiles were detected in the isolates from two remote areas, Mandalay and Yangon, with temporal changes. These data suggested that cholera spread from the seaside to the inland area in Myanmar.

Objectives Escherichia coli isolates carrying the mcr-1 gene are rarely reported in diarrhoeal patients. Here we report the draft genome sequence of a colistin-resistant E. coli isolated from a hospitalised patient with acute diarrhoea in Thailand. Methods Whole genomic DNA of the colistin-resistant E. coli isolate (MSF11) was extracted and was sequenced using an Ion Torrent sequencer with 400-bp read chemistry. The draft genome sequence of MSF11 was analysed with regard to multilocus sequence type (ST), serotype, acquired antimicrobial resistance genes, plasmid replicon types and virulence genes using tools from the Center for Genomic Epidemiology. Results E. coli strain MSF11 was serotype OUT:H10 and ST226. Acquired antimicrobial resistance genes [blaCTX-M-15, qnrS1, catA2, mdf(A) and mcr-1.1] and virulence-related genes (astA and gad) were identified. The mcr-1 gene contained a single nucleotide polymorphism at position 27 (C → T) of the prototype, and the variant gene was associated with an IncX4-type plasmid. This plasmid-borne colistin resistance mediated by the mcr-1 variant has been observed among colistin-resistant strains from humans, animals and the environment previously reported in Thailand, although the STs and serotypes of the E. coli strains were different. Conclusions An mcr-1 variant was identified in an E. coli isolate harbouring the EAST1 (enteroaggregative E. coli heat-stable toxin 1) gene (astA) from a human diarrhoeal stool specimen. This study highlights the potential risk of dissemination of colistin-resistant E. coli in view of the prevalence of the variant gene on IncX4-type plasmids.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses.

The resilience of Burkholderia pseudomallei, the causative agent of melioidosis, was evaluated in control soil microcosms and in soil microcosms containing NaCl or FeSO4 at 30°C. Iron (Fe(II)) promoted the growth of B. pseudomallei during the 30-day observation, contrary to the presence of 1.5% and 3% NaCl. Scanning electron micrographs of B. pseudomallei in soil revealed their morphological alteration from rod to coccoid and the formation of microcolonies. The smallest B. pseudomallei cells were found in soil with 100 μM FeSO4 compared with in the control soil or soil with 0.6% NaCl (P < 0.05). The colony count on Ashdown's agar and bacterial viability assay using the LIVE/DEAD(®) BacLight(™) stain combined with flow cytometry showed that B. pseudomallei remained culturable and viable in the control soil microcosms for at least 120 days. In contrast, soil with 1.5% NaCl affected their culturability at day 90 and their viability at day 120. Our results suggested that a low salinity and iron may influence the survival of B. pseudomallei and its ability to change from a rod-like to coccoid form. The morphological changes of B. pseudomallei cells may be advantageous for their persistence in the environment and may increase the risk of their transmission to humans.

Melioidosis is highly prevalent in Northeast Thailand which is associated with high incidence of Burkholderia pseudomallei present in the soil of this region. B. pseudomallei when present in biofilm becomes resistant to numerous environmental factors and also to certain antibiotics. In this study, we examined the effects of several environmentally relevant factors (salinity, iron, manganese and temperature) on biofilm formation of four clinical ribotypes of B. pseudomallei commonly found in Northeast Thailand. The results showed that biofilm formation increased when B. pseudomallei were grown in modified Vogel and Bonner's medium containing 0.85-1.7 M NaCl or 100-500 microM iron (FeSO4). Low temperature (20 degrees C) also induced more biofilm formation than 30 degrees C or 37 degrees C. On the other hand, protease production and bacterial motility were adversely affected but not in the case of low temperature. Results from this study should be useful in the development of prevention measures or controlling B. pseudomallei biofilm formation in the environment.