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Objectives Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant Enterobacterales (CRE). Plasmid-mediated colistin resistance, in particular the mcr-1 gene, has been identified and WGS is the go-to method in identifying plasmids carrying mcr-1 genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying mcr-1. Methods ODM is a single-molecule technique, which we have demonstrated can be used for identifying plasmids harbouring antibiotic resistance genes. We here applied the technique to plasmids isolated from 12 clinical Enterobacterales isolates from patients at a major hospital in Thailand and verified our results using Nanopore long-read sequencing. Results We successfully identified plasmids encoding the mcr-1 gene and, for the first time, demonstrated the ability of ODM to identify resistance gene sites in small (∼30 kb) plasmids. We further identified blaCTX-M genes in different plasmids than the ones encoding mcr-1 in three of the isolates studied. Finally, we propose a cut-and-stretch assay, based on similar principles, but performed using surface-functionalized cover slips for DNA immobilization and an inexpensive microscope with basic functionalities, to identify the mcr-1 gene in a plasmid sample. Conclusions Both ODM and the cut-and-stretch assay developed could be very useful in identifying plasmids encoding antibiotic resistance in hospitals and healthcare facilities. The cut-and-stretch assay is particularly useful in low- and middle-income countries, where existing techniques are limited.

Background Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) have continually grown as a global public health threat, with significant mortality rates observed across the world. We examined the clinical data from patients with CPE infections and their outcomes, concentrating on Klebsiella pneumoniae isolates. We analysed the clinical information, performed antimicrobial susceptibility testing, and conducted molecular epidemiological and genomic analyses on the isolates to identify patterns in the data. Methods The clinical characteristics of 33 hospitalised patients with confirmed CPE, including patient-related factors associated with the development of CPE infections, were examined. Patients were divided according to whether they were “colonised” or “infected” with CPE and by the timing and frequency of their rectal swab collections, from which 45 swabs were randomly selected for analysis. CPE isolates were purified, and antimicrobial susceptibility tests performed. Whole genome sequences of these isolates were determined and analysed to compute bacterial multilocus sequence types and plasmid replicon types, infer phylogenetic relationships, and identify antimicrobial resistance and virulence genes. Results Altogether, 88.9% (40/45) of the CPE isolates were K. pneumoniae. The most abundant carbapenemase gene family in the K. pneumoniae isolates (33/39) was blaOXA-232, with blaNDM-1 additionally identified in 19 of them. All CPE isolates carrying either blaOXA-232 or blaNDM-1 were resistant to meropenem, but only 40 from 45 were susceptible to colistin. Among the CPE-infected patients (n = 18) and CPE-colonised patients who developed CPE infections during the study (n = 3), all but one received standard colistin-based combination therapy. Phylogenetic analysis revealed the polyclonal spread of carbapenemase-producing K. pneumoniae (CPKP) within the patient population, with the following two major subclades identified: ST16 (n = 15) and ST231 (n = 14). CPKP-ST231 had the highest virulence score of 4 and was associated with primary bacteraemia. The siderophores yersiniabactin and aerobactin, considered to be important virulence factors, were only identified in the CPKP-ST231 genomes. Conclusions This study has revealed the genomic features of colonising CPE isolates, focusing on antimicrobial resistance and virulence determinants. This type of multi-layered analysis can be further exploited in Thailand and elsewhere to modify the regimes used for empirical antibiotic treatment and improve the management strategies for CPE infections in hospitalised patients.

Additional file 1: Table S1. Detailed clinical information of 33 carbapenemase-producing Enterobacteriaceae (CPE) patients described in the study. Table S2. Antimicrobial resistance determinants for 45 carbapenemase-producing Enterobacteriaceae isolates identified from whole genome sequencing analysis.

Background: Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) have continually grown as a global public health threat, with significant mortality rates observed across the world. We examined the clinical data from patients with CPE infections and their outcomes, concentrating on Klebsiella pneumoniae isolates. We analysed the clinical information, performed antimicrobial susceptibility testing, and conducted molecular epidemiological and genomic analyses on the isolates to identify patterns in the data.Methods: The clinical characteristics of 33 hospitalised patients with confirmed CPE, including patient-related factors associated with the development of CPE infections, were examined. Patients were divided according to whether they were “colonised” or “infected” with CPE and by the timing and frequency of their rectal swab collections, from which 45 swabs were randomly selected for analysis. CPE isolates were purified, and antimicrobial susceptibility tests performed. Whole genome sequences of these isolates were determined and analysed to compute bacterial multilocus sequence types and plasmid replicon types, infer phylogenetic relationships, and identify antimicrobial resistance and virulence genes.Results: Altogether, 88.9% (40/45) of the CPE isolates were K. pneumoniae. The most abundant carbapenemase gene family in the K. pneumoniae isolates (33/39) was blaOXA-232, with blaNDM-1 additionally identified in 19 of them. All CPE isolates carrying either blaOXA-232 or blaNDM-1 were resistant to meropenem, but only 40 from 45 were susceptible to colistin. Among the CPE-infected patients (n=18) and CPE-colonised patients who developed CPE infections during the study (n=3), all but one received standard colistin-based combination therapy. Phylogenetic analysis revealed the polyclonal spread of carbapenemase-producing K. pneumoniae (CPKP) within the patient population, with the following two major subclades identified: ST16 (n=15) and ST231 (n=14). CPKP-ST231 had the highest virulence score of 4 and was associated with primary bacteraemia. The siderophores yersiniabactin and aerobactin, considered to be important virulence factors, were only identified in the CPKP-ST231 genomes.Conclusions: This study has revealed the genomic features of disease-causing or colonising CPE isolates, focusing on antimicrobial resistance and virulence determinants. This type of multi-layered analysis can be further exploited in Thailand and elsewhere to modify the regimes used for empirical antibiotic treatment and improve the management strategies for CPE infections in hospitalised patients.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) in patients admitted to hospitals pose a great challenge to treatment. The genes causing resistance to carbapenems are mostly found in plasmids, mobile genetic elements that can spread easily to other bacterial strains, thus exacerbating the problem. Here, we studied 27 CR-KP isolates collected from different types of samples from 16 patients admitted to the medical ward at Siriraj Hospital in Bangkok, Thailand, using next generation sequencing (NGS) and optical DNA mapping (ODM). The majority of the isolates belonged to sequence type (ST) 16 and are described in detail herein. Using ODM, we identified the plasmid carrying the blaNDM-1 gene in the ST16 isolates and the plasmids were very similar, highlighting the possibility of using ODM of plasmids as a surrogate marker of nosocomial spread of bacteria. We also demonstrated that ODM could identify that the blaCTX-M-15 and blaOXA-232 genes in the ST16 isolates were encoded on separate plasmids from the blaNDM-1 gene and from each other. The other three isolates belonged to ST147 and each of them had distinct plasmids encoding blaNDM-1.

Background Increasing awareness of and understanding about antimicrobial resistance (AMR), promoting changes in behavior, and monitoring of AMR in the community are challenging, as AMR is associated with many contributing factors that are difficult to assess individually. This study aimed to determine the effectiveness of a community-based AMR campaign for improving awareness, understanding, and behavior relating to antibiotic use and AMR in Thailand and to assess if fecal carriage of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae could be a proxy composite indicator of AMR in the community. Methods This study was conducted in 4 communities that are home to approximately 400 000 people. A self-administered questionnaire on awareness, understanding, and behavior relating to antibiotic use and AMR was responded to by 20 521 and 19 634 adults before and immediately after the AMR campaign, respectively, at the household level. Fecal samples were collected from 534 adults before the AMR campaign and from 709 adults at 18 months after the AMR campaign to determine presence of ESBL-producing Enterobacteriaceae. Results Awareness, understanding, and behavior relating to antibiotic use and AMR, as assessed by a self-administered questionnaire, were significantly improved after the AMR campaign. The fecal carriage rate of ESBL-producing Enterobacteriaceae decreased from 66.5% before to 44.6% after the AMR campaign (P < .01). Conclusions Our AMR campaign was effective for improving awareness, understanding, and behavior relating to antibiotic use and AMR among people in the community at the household level, and the prevalence of fecal carriage of ESBL-producing Enterobacteriaceae seemed to be decreased after the AMR campaign. Fecal carriage rate of ESBL-producing Enterobacteriaceae may be a proxy composite indicator of AMR in the community., Fecal carriage of ESBL-producing Enterobacteriaceae in adults in community decreased from 66.5% to 44.6% after the implementation of a household-level antimicrobial resistance campaign in community. Fecal carriage rate of ESBL-producing Enterobacteriaceae may be a proxy composite indicator of antimicrobial resistance in community.

Introduction Iron chelator has previously demonstrated fungicidal effects. This study aimed to investigate the antifungal activity of the iron chelators deferoxamine (DFO) and deferasirox (DSX) against Cryptococcus. Materials and methods Cryptococcus neoformans and Cryptococcus gattii were used to determine the minimal inhibitory concentrations (MICs) of DFO and DSX, and the fractional inhibitory concentration index (FICI) of DFO and DSX when combined with amphotericin B (AMB). Expression of cryptococcal CFT1, CFT2, and CIR1 genes was determined using real-time polymerase chain reaction (PCR). Results Neither DFO nor DSX alone showed antifungal activity against Cryptococcus strains. When combined with AMB, the MICs of DFO and DSX decreased from > 200 μg/mL to 6.25 or 12.5 μg/mL. The MIC of AMB decreased one-fold dilution in most strains when combined with iron chelators. The FICI of DFO + AMB and DSX + AMB was 0.5 and 1, respectively. C. neoformans showed significant growth retardation when incubated with a combination of sub-MIC concentrations of AMB and DFO; whereas, C. gattii demonstrated lesser growth retardation in DFO + AMB. No cryptococcal growth retardation was observed when DSX was combined with AMB. When C. neoformans was grown in DFO, the CFT1, CFT2, and CIR1 proteins were expressed 1.7, 2.0, and 0.9 times, respectively. When C. neoformans was grown in DSX, the CFT1, CFT2, and CIR1 genes were expressed 0.5, 0.6, and 0.3 times, respectively. Conclusion Synergistic antifungal activity of combination DFO and AMB was observed in Cryptococcus. Relatively increased CFT1 and CFT2 expression may be associated with the effect of DFO that inhibits the growth of fungi.

Background Colonization with multidrug-resistant (MDR) bacteria is a major risk factor for developing subsequent MDR infections. Methods We performed a prospective surveillance study in hospitalized patients at Siriraj Hospital. Nasal cavity, throat, inguinal area and rectal swabs were obtained within the first 48-h after admission, on day-5 after hospitalization and then every 7 days until discharge. Target bacteria included extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL), carbapenem-resistant-P.aeruginosa (CR-PA), carbapenem-resistant-A.baumannii (CR-AB) and methicillin-resistant S.aureus (MRSA). Results From January 2013–December 2014, 487 patients were enrolled. The baseline prevalence of colonization by ESBL, CR-PA, CR-AB and MRSA at any site was 52.2%, 6.8%, 4.7% and 7.2%, respectively. After 3-week of hospitalization, the prevalence of colonization by ESBL, CR-PA, CR-AB and MRSA increased to 71.7%, 47.2%, 18.9% and 18.9%, respectively. Multivariable analysis revealed that diabetes mellitus and recent cephalosporin exposure were the independent risk factors for baseline colonization by ESBL. The independent risk factors for CR-AB and/or CR-PA colonization were cerebrovascular diseases, previous hospitalization, transfer from another hospital/a LTCF and previous nasogastric tube use, whereas those for MRSA colonization were previous fluoroquinolone exposure and previous nasogastric tube use. Conclusions The baseline prevalence of colonization by ESBL was relatively high, whereas the baseline prevalence of colonization by CR-PA, CR-AB and MRSA was comparable to previous studies. There was an increasing trend in MDR bacteria colonization after hospitalization.

The aim of this study was to determine the prevalence of antibiotic-resistant bacteria, especially extended-spectrum beta-lactamase (ESBL) producing Escherichia coli, in samples from healthy adults, foods, food animals, and the environment in selected areas of Thailand.Samples were collected from stool specimens from adult food factory and food animal farm workers, fresh and cooked foods sold at markets, rectal swabs of healthy pigs and chickens, fresh pork meat from slaughterhouses, water samples from canals as well as fish and shrimp farm ponds, and stagnant water sources on pig farms. Antibiotic susceptibility was determined using the disk diffusion or agar dilution methods. Extended-spectrum beta-lactamase production was assayed using a double disk diffusion method.Among 544 healthy adult food factory workers, 75·5% were positive for ESBL producing E. coli, while 77·3% of E. coli isolated from 30 healthy animal farm workers were positive. Amongst healthy food animals, ESBL producing status among E. coli isolates were more commonly detected in pigs (76·7%) than broilers (40%). Extended-spectrum beta-lactamase producing E. coli seemed to be more prevalent in fresh meat samples than in fresh vegetables, in fresh foods than in cooked foods, and in water samples collected from the animal farms than those from canals and fish and shrimp ponds.Extended-spectrum beta-lactamase producing E. coli isolates are prevalent amongst healthy individuals, foods along the food production chain from farms to consumers, and in the environment in selected areas in Thailand.

To generate epidemiological information regarding antibiotic use and antimicrobial resistance (AMR) in targeted communities for use by the Thailand AMR Containment and Prevention Program.A survey of antibiotics sold by 215 grocery stores and retail shops located in the target communities was done by the local people who were instructed to purchase specified antibiotics and to present to such stores and shops with symptoms of sore throat, backache, common cold, acute diarrhea, inflamed uterus, and dysuria. The purchased drugs were then identified and recorded. Contamination of extended-spectrum β-lactamase (ESBL)-producing E. coli was identified in 174 samples of foods and open water sources collected from the target communities. Carriage of ESBL-producing E. coli in gastrointestinal tracts of 534 adults living in the target communities was performed by stool sample culture. One thousand three hundred one patients with upper respiratory infection (URI) and 235 patients with acute diarrhea who attended the tambon health promoting hospitals located in the target communities were monitored for their clinical outcomes of treatments. The patients with URI and acute diarrhea with no indication of antibiotic received symptomatic treatments as appropriate and they were followed via telephone contact every few days until all symptoms related to URI and acute diarrhea disappeared. The data were analyzed by descriptive statistics.Antibiotics were sold to the local people who were presenting with common ailments at many grocery stores and retail shops in their respective communities. In almost all cases, antibiotics were inappropriately given. Overall prevalence of ESBL-producing E. coli contamination in foods and open water sources was 26.4%. ESBL-producing E. coli was isolated from fresh meat and open water sources in many samples. Overall prevalence of ESBL-producing E. coli carriage in gastrointestinal tracts of the adults cultured was 66.5%. All patients with URI and acute diarrhea who had no indication of antibiotics and did not receive antibiotics had either cure or favorable response within seven days of start of symptomatic treatment.Antibiotics are widely available and are inappropriately sold and given by grocery stores and retail shops located within local communities in Thailand. Antibiotic-resistant bacteria commonly and freely circulate within the community. Patients with URI and acute diarrhea with no indication for antibiotic therapy can be treated without antibiotics. Findings and observations from this study will be used as part of a social marketing campaign on prevention and containment of AMR to educate people living within communities in Thailand.

Skin and soft tissue fungal infections with Apophysomyces elegans, Fusarium solani, Cladophialophora bantiana have been reported in survivors from 2004 Indian ocean Tsunami. We report the first case of primary cutaneous cryptococcosis caused by Cryptococcus gattii VGII in a Tsunami survivor from Thailand.