Molecular characterization of multidrug-resistant ESKAPEE pathogens from clinical samples in Chonburi, Thailand (2017-2018).

BACKGROUND: ESKAPEE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli are multi-drug resistant (MDR) bacteria that present increasing treatment challenges for healthcare institutions and public health worldwide. METHODS: 431 MDR ESKAPEE pathogens were collected from Queen Sirikit Naval Hospital, Chonburi, Thailand between 2017 and 2018. Species identification and antimicrobial resistance (AMR) phenotype were determined following CLSI and EUCAST guidelines on the BD Phoenix System. Molecular identification of antibiotic resistant genes was performed by polymerase chain reaction (PCR), real-time PCR assays, and whole genome sequencing (WGS). RESULTS: Of the 431 MDR isolates collected, 1.2% were E. faecium, 5.8% were S. aureus, 23.7% were K. pneumoniae, 22.5% were A. baumannii, 4.6% were P. aeruginosa, 0.9% were Enterobacter spp., and 41.3% were E. coli. Of the 401 Gram-negative MDR isolates, 51% were carbapenem resistant, 45% were ESBL producers only, 2% were colistin resistance and ESBLs producers (2%), and 2% were non-ESBLs producers. The most prevalent carbapenemase genes were blaOXA-23 (23%), which was only identified in A. baumannii, followed by blaNDM (17%), and blaOXA-48-like (13%). Beta-lactamase genes detected included blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, blaCMY, blaPER and blaVEB. Seven E. coli and K. pneumoniae isolates showed resistance to colistin and carried mcr-1 or mcr-3, with 2 E. coli strains carrying both genes. Among 30 Gram-positive MDR ESKAPEE, all VRE isolates carried the vanA gene (100%) and 84% S. aureus isolates carried the mecA gene. CONCLUSIONS: This report highlights the prevalence of AMR among clinical ESKAPEE pathogens in eastern Thailand. E. coli was the most common MDR pathogen collected, followed by K. pneumoniae, and A. baumannii. Carbapenem-resistant Enterobacteriaceae (CRE) and extended spectrum beta-lactamases (ESBLs) producers were the most common resistance profiles. The co-occurrence of mcr-1 and mcr-3 in 2 E. coli strains, which did not affect the level of colistin resistance, is also reported. The participation of global stakeholders and surveillance of MDR remain essential for the control and management of MDR ESKAPEE pathogens.

Travelers' Diarrhea in Thailand: A Quantitative Analysis Using TaqMan® Array Card.

Background: Travelers' diarrhea (TD) is a common illness experienced by travelers from developed countries who visit developing countries. Recent questionnaire-based surveillance studies showed that approximately 6%-16% of travelers experienced TD while visiting Thailand; however, a majority of TD information was limited mainly to US military populations. Methods: A TD surveillance study was conducted at Bumrungrad International Hospital in 2012-2014 in Bangkok, Thailand. Enteropathogens were identified using conventional methods and the TaqMan® array card (TAC), which uses real-time polymerase chain reaction for the simultaneous detection of multiple pathogens. Analyses to determine pathogen-disease and symptoms association were performed to elucidate the clinical relevance of each enteropathogen. Results: TAC identified more pathogens per sample than conventional methods. Campylobacter spp. were the most prevalent, followed by the diarrheagenic Escherichia coli and norovirus GII. These agents had significant pathogen-disease associations as well as high attributable fractions among diarrheal cases. A wide range of pathogen loads for Campylobacter spp. was associated with TD, while heat-labile toxin enterotoxigenic Escherichia coli was associated with an increased pathogen load. Most cases were associated with inflammatory diarrhea, while Campylobacter spp. and Shigella spp. were associated with dysentery. Conclusions: A pan-molecular diagnostic method such as TAC produces quantifiable and comparable results of all tested pathogens, thereby reducing the variability associated with multiple conventional methods. This allows better determination of the clinical relevance of each diarrhea etiologic agent, as well as their geographical relevance in Thailand.

Multidrug-Resistant Shigella Infections in Patients with Diarrhea, Cambodia, 2014-2015.

We observed multidrug resistance in 10 (91%) of 11 Shigella isolates from a diarrheal surveillance study in Cambodia. One isolate was resistant to fluoroquinolones and cephalosporins and showed decreased susceptibility to azithromycin. We found mutations in gyrA, parC, ß-lactamase, and mphA genes. Multidrug resistance increases concern about shigellosis treatment options.

Bacterial pathogens isolated from raw meat and poultry compared with pathogens isolated from children in the same area of rural Thailand.

To better understand the epidemiology of bacterial food borne pathogens in children, in relation to pathogens in meats from a market in rural Thailand, we collected 73 cultures samples from raw chicken, pork and fish at a local market where diarrheal disease surveillance was conducted. Standard methods were employed to isolate, identify and serotype enteric pathogens from children and food samples. Antibiotic susceptibility testing was performed. Ninety-seven percent of food samples were contaminated with at least one enteric pathogen. The pathogens most commonly isolated from food were Salmonella spp (84%), Arcobacter butzleri (74%) and Campylobacter spp (51%). The most common serovars of Salmonella obtained from humans with diarrhea were S. Risen, S. Stanley and S. Anatum. Most common serovars of Salmonella isolated from food were S. Anatum, S. Stanley, and S. Corvallis. Fifty-one percent and 25% of children infected with Salmonella and Campylobacter, respectively, infected with the same serotypes isolated from food samples, suggesting these pathogens are widespread in food and humans. Pulsed-field gel analysis of Salmonella spp revealed 65 pulsotypes, but no point-sources of salmonellosis were identified. Joint epidemiologic/laboratory studies are useful to describe the epidemiology of enteric pathogens in rural populations.

Description of novel capsule biosynthesis loci of Campylobacter jejuni clinical isolates from South and South-East Asia.

Campylobacter jejuni is a major cause of bacterial diarrhea worldwide and associated with numerous sequela, including Guillain-Barré Syndrome, inflammatory bowel disease, reactive arthritis, and irritable bowel syndrome. C. jejuni is unusual for an intestinal pathogen in its ability to coat its surface with a polysaccharide capsule (CPS). The genes responsible for the biosynthesis of the phase variable CPS is located in the hypervariable region of C. jejuni genome which has been used to develop multiplex PCR to classify CPS types based on the Penner serotypes. However, there still are non-typable CPS C. jejuni by the current multiplex PCR scheme. The application of the next generation sequencing and whole genome analysis software were used for the identification of novel capsule biosynthesis of C. jejuni isolates. Unique PCR primers were designed to identify these new capsule biosynthesis loci. The designed primers sets were combined in a new multiplex mix called epsilon. The unique sequences provide an additional information of the biosynthesis loci responsible for some of the common CPS sugars/residues such as heptose, deoxtyheptose and MeOPN among C. jejuni in this new group of CPS multiplex assay. This new primer complements the current C. jejuni multiplex capsule typing system and will help in identifying previously untypeable capsule locus of C. jejuni isolates.

Etiology of Acute Diarrheal Disease and Antimicrobial Susceptibility Pattern in Children Younger Than 5 Years Old in Nepal.

Diarrhea is a common cause of morbidity and mortality among children younger than 5 years in developing countries. Children from 3 to 60 months of age were recruited from two hospitals in Nepal- Bharatpur Hospital, Bharatpur, and Kanti Children's Hospital, Kathmandu-in 2006 to 2009. Stool specimens collected from 1,200 children with acute diarrhea (cases) and 1,200 children without diarrhea (control subjects) were examined for a broad range of enteropathogens by standard microbiology, including microscopy, enzyme immunoassay for viral pathogens (adenovirus, astrovirus, and rotavirus) and protozoa (Giardia, Cryptosporidium, and Entamoeba histolytica), as well as by using reverse transcription real-time polymerase for norovirus. Antimicrobial susceptibility testing was performed using the disk diffusion method. Overall, rotavirus (22% versus 2%), norovirus (13% versus 7%), adenovirus (3% versus 0%), Shigella (6% versus 1%), enterotoxigenic Escherichia coli (8% versus 4%), Vibrio (7% versus 0%), and Aeromonas (9% versus 3%) were identified significantly more frequently in cases than control subjects. Campylobacter, Plesiomonas, Salmonella, and diarrheagenic E. coli (enteropathogenic, enteroinvasive, enteroaggregative) were identified in similar proportions in diarrheal and non-diarrheal stools. Campylobacter was resistant to second-generation quinolone drugs (ciprofloxacin and norfloxacin), whereas Vibrio and Shigella were resistant to nalidixic acid and trimethoprim/sulfamethoxazole. This study documents the important role of rotavirus and norovirus in acute diarrhea in children younger than 5 years, followed by the bacteria Shigella, enterotoxigenic E. coli, Vibrio cholera, and Aeromonas. Data on the prevalence and epidemiology of enteropathogens identify potential pathogens for public health interventions, whereas pathogen antibiotic resistance pattern data may provide guidance on choice of therapy in clinical settings.

Virulence genes and antimicrobial susceptibilities of hemolytic and nonhemolytic Escherichia coli isolated from post-weaning piglets in central Thailand.

The purpose of this study was to compare the existence of virulence genes in hemolytic Escherichia coli (HEC) and nonhemolytic E. coli (NHEC) isolated from weaner pigs in Thailand, and to determine their susceptibility to 10 antimicrobial agents. A total of 304 E. coli isolates were obtained from 90 piglets with diarrhea and 110 healthy piglets. Of these, 74 HEC isolates were obtained from 70 pigs with diarrhea, and 4 were obtained from 4 healthy pigs, while 190 and 40 NHEC were recovered from 110 healthy and 20 pigs with diarrhea, respectively. A ten digoxigenin (DIG)-labeled probe system was utilized for detecting genes encoding virulence-associated toxins and proteins in these isolates, and the minimal inhibitory concentration values against 10 antimicrobials were determined by means of the agar dilution technique. In total, 70.3% of the HEC isolates contained an exotoxin gene, lth, estp or stx2e, whereas 2.6% of the NHEC isolates hybridized with a gene probe for estp or stx2e. Over 90% of the isolates were resistant to most agents other than colistin and halquinol. The MIC(90) values of the HEC isolates for halquinol and colistin were 4 and 8 times greater than those of the NHEC isolates, respectively. The results represent the first characterization of resistant pathogenic E. coli distributed in the Thai pig industry. Amongst the HEC isolates, there appeared to be an association between the presence of some exotoxin genes, including lth, estp and stx2e, and reduced antimicrobial susceptibility.

Emergence and properties of fluoroquinolone resistant Salmonella enterica serovar Typhi strains isolated from Nepal in 2002 and 2003.

A total of 171 Salmonella enterica serovar Typhi strains isolated from Nepal, mostly from patients with typhoid fever in 2002-2003, were tested for antimicrobial susceptibility by disk diffusion assay. Selected S. enterica serovar Typhi isolates were tested for MICs by E-test for ceftriaxone, ciprofloxacin and ofloxacin. Mutations of DNA gyrase gyrA and gyrB and topoisomerase IV parC and parE were identified by sequencing of PCR amplicons. By disk diffusion assay, 75/171 S. enterica serovar Typhi isolates were resistant to nalidixic acid, ampicillin, choramphenicol, streptomycin, tetracycline, sulfisoxazole, and trimethroprim/sulfamethoxazoles. Multiple drug resistance to the 7 antimicrobials was most predominant among S. enterica serovar Typhi isolates in this study. Resistance to nalidixic acid was detected in 76/111 and 56/60 of total isolates collected in 2002 and 2003, respectively. Nalidixic acid-resistant isolates in 2002 and 2003 showed MIC range for ciprofloxacin of 0.125-0.250 mg/l. Nalidixic acid-resistant isolates contained point mutations in gyrA and parC but not gyrB and parE. The gyrA mutation of nalidixic acid-resistant isolates obtained in 2002 and 2003 had amino acid substitution at position 83 of Serine-->Tyrosine and Serine-->Phenylalanine, respectively. Two different mutations of gyrA were detected among nalidixic acid-resistant isolates. Thus it is necessary to monitor mutation in DNA topoisomerase associated with increases in quinolones resistance.

Distribution of genes related to Type 6 secretion system and lipooligosaccharide that induced ganglioside mimicry among Campylobacter jejuni isolated from human diarrhea in Thailand.

BACKGROUND: Campylobacter jejuni (C. jejuni) is one of the most common bacteria responsible for human gastroenteritis worldwide. The mode of human transmission is foodborne infections due to consumption of contaminated food, especially poultry. Type 6 secretion systems (T6SS) were described recently as Campylobacter virulence mechanisms. Furthermore, infection sequelae associated with neurological disorders like Guillain-Barré (GBS) and Miller Fisher (MF) syndromes can become serious health problems in some patients after Campylobacter gastroenteritis. Our objective was to determine the distribution of these virulence genes among C. jejuni isolated from stool of human diarrhea. METHODS: A total of 524 C. jejuni strains from travelers and pediatric cases of acute diarrhea in Thailand were selected for this study. All isolates belonged to one of 20 known capsule types and all were assayed by PCR for T6SS, a hemolysin co-regulated protein (hcp) gene, and GBS-associated genes (cgtA, cgtB, cstII HS19 and cstII HS2 ) which are involved in sialic acid production in the lipooligosaccharide (LOS) cores of C. jejuni. The distribution of these genes are summarized and discussed. RESULTS: Of all isolates with these 20 capsule types identified, 328 (62.6%) were positive for hcp, ranging from 29.2 to 100% among 10 capsule types. The GBS-associated LOS genes were detected among 14 capsule type isolates with 24.4% and 23.3% of C. jejuni isolates possessed either cstII HS19 or all three genes (cgtA, cgtB and cstII HS19 ), which were classified as LOS classes A and B whereas 9.2% of C. jejuni isolates possessing cstII HS2 were classified as LOS class C. The C. jejuni isolates of LOS A, B, and C together accounted for 56.9% of the isolates among 14 different capsule types while 31.1% of all C. jejuni isolates did not possess any GBS-associated genes. No significant difference was detected from C. jejuni isolates possessing GBS-associated LOS genes among travelers and children, but changes between those with hcp were significant (p < 0.05). CONCLUSIONS: Our results suggested a high diversity of hcp and GBS-associated LOS genes among capsule types of C. jejuni isolated from Thailand.

Enteric etiological surveillance in acute diarrhea stool of United States Military Personnel on deployment in Thailand, 2013-2017.

BACKGROUND: Diarrhea remains a major public health problem for both civilian and military populations. This study describes the prevalence of acute diarrheal illness etiological agents, their antibiotic resistance distribution patterns, the resulting impact upon military force health protection, and potential prevention and treatment strategies. RESULTS: Forty-eight acute diarrhea stool samples from US military personnel deployed to Thailand from 2013-2017 were screened for enteric pathogens using ELISA, the TaqMan Array Card (TAC), and conventional microbiological methods. These isolates were also evaluated using antimicrobial susceptibility testing (AST) against ampicillin (AMP), azithromycin (AZM), ceftriaxone (CRO), ciprofloxacin (CIP), nalidixic acid (NA), erythromycin (ERY), and trimethoprim-sulfamethoxazole (SXT) using commercial methodology. Susceptibility results were interpreted following the CLSI and NARM guidelines. Questionnaire data obtained from 47/48 volunteers indicated that 89.4% (42/47) reported eating local food and the most common clinical symptoms were nausea and abdominal pain (51%; 24/47). Multiple bacterial species were identified from the 48 stool samples with diarrhea etiological agents being detected in 79% (38/48) of the samples distributed as follows: 43.8% (21/48) Campylobacter jejuni and Campylobacter species, 42% (20/48) diarrheagenic Escherichia coli, and 23% (11/48) Salmonella. Co-infections were detected in 46% (22/48) of the samples. All C. jejuni isolates were resistant to CIP and NA. One C. jejuni isolate exhibited resistance to both AZM and ERY. Lastly, an association between exposure to poultry and subsequent detection of the diarrhea-associated pathogens E. coli and P. shigelloides was significant (p < 0.05). CONCLUSION: The detection of Campylobacter isolates with CIP, AZM and ERY resistance has critical force health protection and public health implications, as these data should guide effective Campylobacteriosis treatment options for deployed military members and travelers to Southeast Asia. Additional research efforts are recommended to determine the association of pathogen co-infections and/or other contributing factors towards diarrheal disease in military and traveler populations. Ongoing surveillance and AST profiling of potential disease-causing bacteria is required for effective disease prevention efforts and treatment strategies.

Determination of azithromycin heteroresistant Campylobacter jejuni in traveler's diarrhea.

Campylobacter is the most common cause of traveler's diarrhea (TD) and human bacterial gastroenteritis. A heteroresistant Campylobacter jejuni (C. jejuni) isolate, identified by microbiological methods and characterized with molecular techniques, was obtained from a traveler in Nepal suffering TD. The presence of atypical colonies within the clear zone of inhibition was the first evidence of an atypical phenotype, leading to additional characterization of this heteroresistant strain. Antimicrobial susceptibility testing (AST) and population analysis profiling (PAP) demonstrated heteroresistance to azithromycin (AZM), a first-line antibiotic treatment for Campylobacter infections. Molecular analysis indicated a point mutation occurred on the 23S rRNA gene at the A2075G transitions, and the number of mutated gene copies was proportional to AZM resistance. Heteroresistant C. jejuni subpopulations from acute TD are likely underestimated, which may lead to treatment failures, as was the case for this patient. The presence of a heteroresistant strain in a high antibiotic environment may select for additional drug resistance and enable distribution into hospital and local communities.

Epidemiology and etiology of Traveler's diarrhea in Bangkok, Thailand, a case-control study.

BACKGROUND: Traveler's diarrhea (TD) is a common health problem among visitors from developed to developing countries. Although global and regional estimates of pathogen distribution are available, the etiology of diarrhea among non-military travelers to Thailand is largely unknown. METHODS: A prospective TD case-control study was conducted among adult travelers from developed countries at a prominent hospital in Bangkok, Thailand during 2001-2003. Stool samples were collected from acute TD cases and non-diarrheal controls and analyzed for bacterial, viral, and protozoan pathogens by microbiology, ELISA or PCR methods. Calculation of adjusted odd ratios for risk factors was performed by logistic regression using STATA statistical software. RESULTS: Stool samples were collected and analyzed from 389 TD cases and 400 non-diarrhea controls. At least one pathogen was detected in 227 (58%) cases and 124 (31%) controls. Plesiomonas (14%), Vibrio (14%), Campylobacter (14%), and norovirus (12%) were the most frequently isolated pathogens among cases and significantly associated with diarrhea at p = 0.006, p < 0.001, p < 0.001, p < 0.001, respectively. Shigella (3%) and ETEC (8%), detected in lower prevalence, also showed significant association with TD at p < 0.001 and p = 0.002, respectively. Travelers from East Asian countries had an increased risk of Vibrio infection (Crude odds ratio: 3.1, p-value = 0.001); travelers from the United States, Canada, and Europe had an increased risk of Campylobacter infection (Crude odds ratio: 2.6, p-value = 0.001); and travelers from Australia and New Zealand had an increased risk of Salmonella infection (Crude odds ratio: 3.2, p-value = 0.009). CONCLUSIONS: Etiology of TD in Thailand is mainly of bacterial origin. Plesiomonas, Vibrio, and norovirus are underappreciated diarrheagenic pathogens. In our study, the origin of the traveler plays an important role in diarrhea etiology. Understanding variations in TD severity and etiology among travelers from different regions warrants further study.

Characterization of two Campylobacter jejuni strains for use in volunteer experimental-infection studies.

The development of vaccines against Campylobacter jejuni would be facilitated by the ability to perform phase II challenge studies. However, molecular mimicry of the lipooligosaccharide (LOS) of most C. jejuni strains with human gangliosides presents safety concerns about the development of Guillain-Barré syndrome. Clinical isolates of C. jejuni that appeared to lack genes for the synthesis of ganglioside mimics were identified by DNA probe analyses. Two clinical isolates from Southeast Asia (strains BH-01-0142 and CG8421) were determined to express the LOS type containing N-acetyl quinovosamine. No ganglioside structures were observed to be present in the LOSs of these strains, and pyrosequence analyses of the genomes of both strains confirmed the absence of genes involved in ganglioside mimicry. The capsule polysaccharide (CPS) of BH-01-0142 was determined to be composed of galactose (Gal), 6-deoxy-ido-heptose, and, in smaller amounts, D-glycero-D-ido-heptose, and the CPS of CG8421 was observed to contain Gal, 6-deoxy-altro-heptose, N-acetyl-glucosamine, and minor amounts of 6-deoxy-3-O-Me-altro-heptose. Both CPSs were shown to carry O-methyl-phosphoramidate. The two genomes contained strain-specific zones, some of which could be traced to a plasmid origin, and both contained a large chromosomal insertion related to the CJEI3 element of C. jejuni RM1221. The genomes of both strains shared a high degree of similarity to each other and, with the exception of the capsule locus of CG8421, to the type strain of the HS3 serotype, TGH9011.

Diarrheagenic Escherichia coli and Shigella strains isolated from children in a hospital case-control study in Hanoi, Vietnam.

This case-control study detected and characterized Shigella and diarrheagenic Escherichia coli (DEC) types among Vietnamese children less than 5 years old. In 249 children with diarrhea and 124 controls, Shigella spp. was an important cause of diarrhea (P < 0.05). We used multiplex PCR and DNA probes to detect enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), enteropathogenic E. coli (EPEC), attaching and effacing E. coli (A/EEC), verocytotoxin-producing E. coli (VTEC), and enterotoxigenic E. coli (ETEC). The prevalences of DEC in the diarrhea and control groups were 25.7 and 10.5%, respectively. In 62 children with diarrhea, 64 DEC strains included 22 EAggEC (8.8%), 2 EIEC (0.8%), 23 A/EEC (9.2%), 7 EPEC (2.8%), and 10 ETEC strains (4.0%). Among controls, 13 DEC strains included 5 EAggEC strains (4.0%), 7 A/EEC strains (5.6%), and 1 EPEC strain. The characterization of DEC by serotypes, antimicrobial susceptibility patterns, virulence genes, and pulsed-field gel electrophoresis showed the occurrence of many different and highly heterogenic DEC subtypes, but common serotypes were found among ETEC, EIEC and EPEC, respectively. Serotyping was used to distinguish between A/EEC and EPEC. However, A/EEC, EPEC, and EAggEC were isolated at high frequency from both cases and controls. Further in-depth studies are needed to better understand important virulence factors of DEC, especially A/EEC, EPEC, and EAggEC.

Genomic Characterization of Nonclonal mcr-1-Positive Multidrug-Resistant Klebsiella pneumoniae from Clinical Samples in Thailand.

Multidrug-resistant Klebsiella pneumoniae strains are one of the most prevalent causes of nosocomial infections and pose an increasingly dangerous public health threat. The lack of remaining treatment options has resulted in the utilization of older drug classes, including colistin. As a drug of last resort, the discovery of plasmid-mediated colistin resistance by mcr-1 denotes the potential development of pandrug-resistant bacterial pathogens. To address the emergence of the mcr-1 gene, 118 gram-negative Enterobacteriaceae isolated from clinical samples collected at Queen Sirikit Naval Hospital in Chonburi, Thailand were screened for colistin resistance using automated antimicrobial susceptibility testing and conventional PCR screening. Two K. pneumoniae strains, QS17-0029 and QS17-0161, were positive for mcr-1, and both isolates were sequenced to closure using short- and long-read whole-genome sequencing. QS17-0029 carried 16 antibiotic resistance genes in addition to mcr-1, including 2 carbapenemases, blaNDM-1 and blaOXA-232. QS17-0161 carried 13 antibiotic resistance genes in addition to mcr-1, including the extended-spectrum ß-lactamase blaCTX-M-55. Both isolates carried multiple plasmids, but mcr-1 was located alone on highly similar 33.9 Kb IncX4 plasmids in both isolates. The IncX4 plasmid shared considerable homology to other mcr-1-containing IncX4 plasmids. This is the first report of a clinical K. pneumoniae strain from Thailand carrying mcr-1 as well as the first strain to simultaneously carry mcr-1 and multiple carbapenemase genes (QS17-0029). The identification and characterization of these isolates serves to highlight the urgent need for continued surveillance and intervention in Southeast Asia, where extensively drug-resistant pathogens are being increasingly identified in hospital-associated infections.

Extended-spectrum ß-lactamase prevalence and virulence factor characterization of enterotoxigenic Escherichia coli responsible for acute diarrhea in Nepal from 2001 to 2016.

Background: Multidrug-resistant (MDR) Gram-negative bacterial species are an increasingly dangerous public health threat, and are now endemic in many areas of South Asia. However, there are a lack of comprehensive data from many countries in this region determining historic and current MDR prevalence. Enterotoxigenic Escherichia coli (ETEC) is a leading cause of both acute infant diarrhea and traveler's diarrhea in Nepal. The MDR prevalence and associated resistance mechanisms of ETEC isolates responsible for enteric infections in Nepal are largely unknown. Methods: A total of 265 ETEC isolates were obtained from acute diarrheal samples (263/265) or patient control samples (2/265) at traveler's clinics or regional hospitals in Nepal from 2001 to 2016. Isolates were screened for antibiotic resistance, to include extended spectrum beta-lactamase (ESBL) production, via the Microscan Automated Microbiology System. ETEC virulence factors, specifically enterotoxins and colonization factors (CFs), were detected using multiplex PCR, and prevalence in the total isolate population was compared to ESBL-positive isolates. ESBL-positive isolates were assessed using multiplex PCR for genetic markers potentially responsible for observed resistance. Results: A total of 118/265 (44.5%) ETEC isolates demonstrated resistance to ≥2 antibiotics. ESBL-positive phenotypes were detected in 40/265 isolates, with isolates from 2008, 2013, 2014, and 2016 demonstrating ESBL prevalence rates of 1.5, 34.5, 31.2, and 35.0% respectively. No difference was observed in overall enterotoxin characterization between the total ETEC and ESBL-positive populations. The CFs CS2 (13.6%), CS3 (25.3%), CS6 (30.2%), and CS21 (62.6%) were the most prevalent in the total ETEC population. The ESBL-positive ETEC isolates exhibited a higher association trend with the CFs CS2 (37.5%), CS3 (35%), CS6 (42.5%), and CS21 (67.5%). The primary ESBL gene identified was blaCTX-M-15 (80%), followed by blaSHV-12 (20%) and blaCTX-M-14 (2.5%). The beta-lactamase genes blaTEM-1 (40%) and blaCMY-2 (2.5%) were also identified. It was determined that 42.5% of the ESBL-positive isolates carried multiple resistance genes. Conclusion: Over 30% of ETEC isolates collected post-2013 and evaluated in this study demonstrated ESBL resistance. Persistent surveillance and characterization of enteric ETEC isolates are vital for tracking the community presence of MDR bacterial species in order to recommend effective treatment strategies and help mitigate the spread of resistant pathogens.

Antimicrobial resistant Helicobacter fennelliae isolated from non-diarrheal child stool sample in Battambang, Cambodia.

Helicobacter fennelliae (H. fennelliae) is associated with human gastroenteritis; however, H. fennelliae was isolated and confirmed by phenotypic and genotypic identification from a non-diarrheal child stool sample in Cambodia. Antimicrobial susceptibility testing demonstrated that this isolate had a high minimal inhibitory concentration against macrolides and quinolones, which are first-line antibiotic treatment choices for Campylobacter infections. Consequently, macrolides and quinolones were likewise expected to be ineffective against Campylobacter-like organisms such as H. fennelliae. This isolate warranted further genetic characterization to better understand associated antibiotic resistance mechanisms. Resistant pathogens from asymptomatic diarrheal cases are likely underestimated, and as such colonized individuals may spread resistant organisms to local community members and the environment.

Antibiotic resistance, molecular characterizations, and clinical manifestations of Campylobacteriosis at a military medical center in Hawaii from 2012-2016: a retrospective analysis.

Hawaii has one of the highest incidences of Campylobacteriosis in the United States, but there remains little published data on circulating strains or antimicrobial resistance. We characterized 110 clinical Campylobacter isolates (106 C. jejuni, 4 C. coli) processed at Tripler Army Medical Center in Honolulu, HI from 2012-2016. Twenty-five percent of C. jejuni isolates exhibited fluoroquinolone (FQ) resistance, compared with 16% for tetracycline (TET), and 0% for macrolides. Two of the four C. coli isolates were resistant to FQ, TET, and macrolides. C. jejuni isolates further underwent multilocus sequence typing, pulsed-field gel electrophoresis, and molecular capsular typing. Nineteen capsule types were observed, with two capsule types (HS2 and HS9) being associated with FQ resistance (p < 0.001 and p = 0.006, respectively). HS2 FQ-resistant isolates associated with clonal complex 21, possibly indicating clonal spread in FQ resistance. Macrolides should be considered for treatment of suspect cases due to lack of observed resistance.

Rotavirus disease in young children from Hanoi, Vietnam.

BACKGROUND: Pathogen prevalences and antimicrobial susceptibilities are essential for the rational development of preventive strategies for diarrheal diseases, but little recent information from Vietnam is available. We prospectively studied the prevalence of enteric pathogens in children less than 5 years of age with acute diarrhea and in nondiarrhea controls in a city hospital in Hanoi, Vietnam for 1 year. METHODS: Enteric bacteria and viruses were detected by standard culture methods, and enzyme immunoassay in 291 cases and 291 controls. RESULTS: Detection rates of viral pathogens among cases and controls were 31% and 3% for rotavirus, 12% and 1% for astrovirus and 4% and 1% for adenovirus. For bacterial pathogens, Aeromonas, Shigella, Salmonella, Campylobacter and enterotoxigenic E. coli were isolated from cases and controls in 15% and 8%, 9% and 1%, 7% and 1%, 4% and 0%, and 3% and 0%, respectively. The isolation of bacterial and viral pathogens except for adenovirus was significantly lower in controls than cases. Fluoroquinolones were effective against most bacterial enteropathogens, but resistance was observed in 27% of Campylobacter isolates. CONCLUSIONS: Viral etiologic agents especially rotavirus were the most important cause of acute diarrhea in children less than 5 years of age in Hanoi. The burden of rotavirus in young children in Hanoi warrants consideration of using the recently released rotavirus vaccine.

Intestinal parasitic infections among pre-school children in Sangkhlaburi, Thailand.

This study was conducted to investigate the presence of intestinal parasites among pre-school children (aged 3 months to 5 years) in Sangkhlaburi, a rural district in the west of Thailand along the Thai-Myanmar border. Stool specimens were collected from October 2001 through October 2002. A total of 472 pre-school children, 233 males and 239 females, 236 children with diarrhea and 236 asymptomatic children were recruited for the study. Each specimen was processed and examined by direct wet smear, modified acid fast stain, formalin-ethylacetate sedimentation concentration technique, and trichrome stain. In detecting Giardia lamblia and Cryptosporidium species ProSpecT Microplate assays (Alexon-Trend, Lenexa, KS) were performed. There were 107 individuals (22.7%), 41 diarrheal and 66 asymptomatic children, infected with intestinal parasites. The most frequent parasites identified in cases and controls were G. lamblia and Cryptosporidium spp. Eighteen specimens (3.8%) showed mixed parasite infections. Highest proportion of intestinal parasites occurred during the rainy season (June-October).