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.

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.

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.

In-vitro Studies of Anti-EGFR Tyrosine Kinase Activity of Thai nutraceutical Plants.

Functional foods have emerged as a new approach to improve human health in term of nutraceutical to prevent people from illness rather than cure patients through medical treatment. In Asian society, particularly in Thailand, the utilizations of functional ingredients have been integrated in every parts of ordinary life. In this study, the tyrosine kinase activity of epidermal growth factor receptor (EGFR) inhibiting properties of 23 Thai's herbs-ethanol extracts have been examined. The crude extracts of only four species that inhibit the activity of EGFR-tyrosine kinase, Azadirachta indica (neem, Sa-dao), Brucea javanica (L.) Merr. (Rajadad), Hibiscus sabdariffa L. (Roselle, Krachiap daeng), and Saccharum chinensis Roxb. (Red sugar cane). Moreover, only ethanol extractions from A. indica and B. javanica were also showed antitumor effect to non-small cell lung cancer, A549 cells.

First report of the mcr-1 colistin resistance gene identified in two Escherichia coli isolates from clinical samples, Philippines, 2018.

OBJECTIVES: The first report of a plasmid-borne colistin resistance gene (mcr-1) detected in an Escherichia coli isolate from China heralded the emergence of pandrug-resistant bacteria. Since then, the mcr-1 gene has been detected worldwide, but to date it has not been reported in the Philippines. METHODS: In this study, 123 antimicrobial-resistant isolates collected from January-June 2018 from patients admitted to a tertiary hospital in Manila, Philippines, were characterised. Biochemical identification and antimicrobial susceptibility testing were performed using a BD Phoenix™ M50 system with NMIC/ID-95 panel. Conventional PCR was performed to detect the genes mcr-1 to mcr-5, and short- and long-read whole-genome sequencing was performed. RESULTS: Two mcr-1-positive E. coli clinical isolates from separate patients harboured mcr-1 on an IncI2 plasmid. One isolate was shown to carry 12 antimicrobial resistance genes (ARGs) in addition to mcr-1, including the extended-spectrum ß-lactamase blaCTX-M-55, whilst the other E. coli isolate carried 6 ARGs in addition to mcr-1. CONCLUSION: Both patients had no prior colistin treatment recorded in their medical history and no travel history outside of the country within the past 6 months from the date of hospital admission, indicating local transmission and acquisition of the colistin-resistant strain from either community or hospital settings within the Philippines. This report should serve as a signal to local public-health officials of the need to intensify surveillance efforts and to increase vigilance and implementation of antimicrobial stewardship programmes to contain and slow the spread of antimicrobial resistance.

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.

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.

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.

Incidence of Campylobacter concisus and C. ureolyticus in traveler's diarrhea cases and asymptomatic controls in Nepal and Thailand.

BACKGROUND: Campylobacter concisus and C. ureolyticus have emerged in recent years as being associated with acute and prolonged gastroenteritis and implicated in the development of inflammatory bowel diseases. However, there are limited data on the prevalence of these microorganisms in Southeast Asia. In this study, 214 pathogen-negative stool samples after laboratory examination for common enteric pathogens to include C. jejuni and C. coli by culture from two case-control traveler's diarrhea (TD) studies conducted in Thailand (cases = 26; controls = 30) and Nepal (cases = 83; controls = 75) respectively were assayed by PCR for the detection of Campylobacter 16S rRNA and two specific heat shock protein genes specific for C. concisus (cpn60) and C. ureolyticus (Hsp60) respectively. RESULTS: Campylobacter 16S rRNA was detected in 28.5% (61/214) of the pathogen-negative TD stool samples (CIWEC Travel Medicine Clinic, Kathmandu, Nepal: cases = 36, control = 14; Bamrungrad International Hospital, Bangkok, Thailand: cases = 9, controls = 2). C. consisus was identified significantly more often in TD cases in Nepal (28.9%; 24/83) as compared to controls (4%; 3/75) (OR = 9.76; 95% CI 2.80-34.02; P = 0.0003) while C. consisus was detected in only two cases (2/26; 7.7%) and none of the controls stool samples from Thailand. C. ureolyticus was detected in four cases (4.8%; 4/83) and four controls (5.3%; 4/75) and in one case (3.8%; 1/26) and one control (3.1%; 1/30) from Nepal and Thailand respectively. C. jejuni and C. coli were isolated in 18.3 and 3.4% of the cases and in 4.0 and 1.4% of the controls in stool samples from both Thailand and Nepal respectively while C. concisus nor C. ureolyticus were not tested for in these samples. CONCLUSION: These findings suggest that C. concisus potentially is a pathogen associated with TD in Nepal. To our knowledge, this is the first report of C. concisus and C. ureolyticus detected from traveler's diarrhea cases from travelers to Nepal and Thailand.

Introduction and establishment of fluoroquinolone-resistant Shigella sonnei into Bhutan.

Shigella sonnei is a major contributor to the global burden of diarrhoeal disease, generally associated with dysenteric diarrhoea in developed countries but also emerging in developing countries. The reason for the recent success of S. sonnei is unknown, but is likely catalysed by its ability to acquire resistance against multiple antimicrobials. Between 2011 and 2013, S. sonnei exhibiting resistance to fluoroquinolones, the first-line treatment recommended for shigellosis, emerged in Bhutan. Aiming to reconstruct the introduction and establishment of fluoroquinolone-resistant S. sonnei populations in Bhutan, we performed whole-genome sequencing on 71 S. sonnei samples isolated in Bhutan between 2011 and 2013.We found that these strains represented an expansion of a clade within the previously described lineage III, found specifically in Central Asia. Temporal phylogenetic reconstruction demonstrated that all of the sequenced Bhutanese S. sonnei diverged from a single ancestor that was introduced into Bhutan around 2006. Our data additionally predicted that fluoroquinolone resistance, conferred by mutations in gyrA and parC, arose prior to the introduction of the founder strain into Bhutan. Once established in Bhutan, these S. sonnei had access to a broad gene pool, as indicated by the acquisition of extended-spectrum ß-lactamase-encoding plasmids and genes encoding type IV pili. The data presented here outline a model for the introduction and maintenance of fluoroquinolone-resistant S. sonnei in a new setting. Given the current circulation of fluoroquinolone-resistant S. sonnei in Asia, we speculate that this pattern of introduction is being recapitulated across the region and beyond.

Molecular characterization and PCR-based replicon typing of multidrug resistant Shigella sonnei isolates from an outbreak in Thimphu, Bhutan.

BACKGROUND: Shigella species are an important cause of diarrhea in developing countries. These bacteria normally acquire their antibiotic resistance via several different mobile genetic elements including plasmids, transposons, and integrons involving gene cassettes. During a diarrhea surveillance study in Thimphu, Bhutan in June and July, 2011, Shigella sonnei were isolated more frequently than expected. This study describes the antibiotic resistance of these S. sonnei isolates. METHODS: A total of 29 S. sonnei isolates from Thimphu, Bhutan was characterized for antimicrobial susceptibility by disc diffusion assay and minimum inhibitory concentration (MIC) assay. All isolates were tested by pulsed-field gel electrophoresis (PFGE) with restriction enzyme XbaI and were tested for plasmid. The plasmid patterns and the PFGE patterns were analyzed by Bionumerics software. DNA sequencing was performed on amplified products for gyraseA gene and class 1 and class 2 integrons. S. sonnei isolates were classified for incompatibility of plasmids by PCR-based replicon typing (PBRT). RESULTS: These S. sonnei were resistant to multiple drugs like ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, streptomycin, and tetracycline but susceptible to azithromycin. All isolates had class 2 integrons dfrA1, sat1 and aadA1 genes. Two point mutations in Gyrase A subunit at position Ser83Leu and Asp87Gly were detected in these quinolone resistant isolates. The plasmid and PFGE patterns of S. sonnei isolates suggested a clonal relationship of the isolates. All isolates carried common ColE plasmid. ColE plasmid co-resided with B/O plasmid (nine isolates) or I1 plasmid (one isolate). CONCLUSIONS: The characteristics of 29 S. sonnei isolates from Thimphu, Bhutan in June and July, 2011 are identical in PFGE, plasmid and resistance pattern. This study suggests that these recent S. sonnei isolates are clonally related and multidrug-resistant.