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.

Mobile colistin-resistant genes (mcr) have become an increasing public health concern. Since the first report of mcr-1 in Thailand in 2016, perspective surveillance was conducted to explore the genomic characteristics of clinical carbapenem-resistant Enterobacterales (CRE) isolates harboring mcr in 2016–2019. Thirteen (0.28%) out of 4,516 CRE isolates were found to carry mcr genes, including 69.2% (9/13) of E. coli and 30.8% (4/13) of K. pneumoniae isolates. Individual mcr-1.1 was detected in eight E. coli (61.5%) isolates, whereas the co-occurrence of mcr-1.1 and mcr-3.5 was seen in only one E. coli isolate (7.7%). No CRE were detected carrying mcr-2, mcr-4, or mcr-5 through to mcr-9. Analysis of plasmid replicon types carrying mcr revealed that IncX4 was the most common (61.5%; 8/13), followed by IncI2 (15.4%; 2/13). The minimum inhibitory concentration values for colistin were in the range of 4–16 μg/ml for all CRE isolates harboring mcr, suggesting they have 100% colistin resistance. Clermont phylotyping of nine mcr-harboring carbapenem-resistant E. coli isolates demonstrated phylogroup C was predominant in ST410. In contrast, ST336 belonged to CC17, and the KL type 25 was predominant in carbapenem-resistant K. pneumoniae isolates. This report provides a comprehensive insight into the prevalence of mcr-carrying CRE from patients in Thailand. The information highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harboring CRE and the need for rational drug use in all sectors.

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.

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 cutoff values for clinical relevance drastically reduced pathogenpositive 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 < 0.001). Characteristic clinical symptoms, epidemic periods, and age-related susceptibility to infection were observed for some enteropathogens. Investigations based on qPCR approaches covering a broad array of enteropathogens might thus improve our understanding of diarrheal disease etiology and epidemiological trends. Diarrheal diseases are one of the major causes of mortality and morbidity worldwide, especially during the first 5 years of life for individuals subjected to malnutrition 1-3. Diarrhea can be defined by increased stool frequency, liquidity, or volume 4. A wide range of enteropathogens including bacteria, viruses, and protozoa have been