Presence and characterization of blaNDM-1-positive carbapenemase-producing Klebsiella pneumoniae from outpatients in Thailand.

BACKGROUND: Presently, community-associated carbapenemase-producing Enterobacterales (CPE) remains largely unknown and require public attention. This study aimed to investigate the presence of CPE from outpatients in Thailand. METHODS: Non-duplicate stool (n = 886) and urine (n = 289) samples were collected from outpatients with diarrhea and urinary tract infection, respectively. Demographic data and characteristics of patients were collected. Isolation of CPE was performed by plating enrichment culture on agar supplemented with meropenem. Carbapenemase genes were screened by PCR and sequencing. CPE isolates were phenotypically and genotypically characterized. RESULTS: Fifteen samples (1.3%, 14 stool and 1 urine) yielded blaNDM-1-positive carbapenemase-producing Klebsiella pneumoniae (CPKP). Additional resistance to colistin and tigecycline was observed in 53.3% and 46.7% of isolates, respectively. Age >60 years was identified as a risk factor for patients with CPKP (P < 0.001, adjusted odds ratio = 11.500, 95% confidence interval = 3.223-41.034). Pulsed field gel electrophoresis revealed genetic diversity of CPKP isolates; however, clonal spread has been observed. ST70 (n = 4) was common, followed by ST147 (n = 3). blaNDM-1 from all isolates were transferable and mainly resided on IncA/C plasmid (80%). All blaNDM-1 plasmids remained stable in bacterial host for at least 10 days in antibiotic-free environments, regardless of replicon types. CONCLUSION: This study demonstrates that the prevalence of CPE among outpatients in Thailand remains low and the spread of blaNDM-1-positive CPKP may be driven by IncA/C plasmid. Our results emphasize the need for a large-scale surveillance study to limit further spread of CPE in community.

Research Note: Longitudinal fecal shedding patterns and characterization of Salmonella enterica and mcr-positive Escherichia coli in meat-type ducks raised in an open-house system.

This longitudinal study aimed to determine the fecal shedding pattern and characterize Salmonella enterica and mcr-positive Escherichia coli from meat-type ducks raised in an open-house system in Thailand. Fecal samples (n = 1,475) were collected from ducks over a 6-month period. Overall, the detection rate of S. enterica was 5.4% and the highest fecal shedding rate was noted in 4-day-old ducklings (28.8%). Then, S. enterica shedding decreased to 10, 8, 4.7, and 0.7% when ducks reached the ages of 10 d, 17 d, 3 wk, and 4 wk, respectively. Seventy-nine isolates were recovered and Salmonella Amsterdam was the predominant serovar (79.7%). With respect to colistin-resistant E. coli, mcr-positive E. coli (colistin MICs = 8-16 µg/mL) was noted in ducks at the ages of 16 wk (6.0%) and 24 wk (18.7%). mcr-1 was the most common (75.7%), followed by mcr-3 (13.5%), and mcr-1 and mcr-3 co-carriage (10.8%). Most S. enterica isolates were susceptible to antibiotics and multidrug resistant (MDR) was found in only a single isolate. However, as many as 89.2% of mcr-positive E. coli were defined as MDR. Almost all S. enterica isolates (97.5-100%) carried several virulence genes involving in invasion, intracellular survival, and iron metabolism. Pulsed Field Gel Electrophoresis revealed that several mcr-positive E. coli isolates were clonally unrelated. Conjugative transfer of mcr-1, mcr-3 as well as co-transfer of mcr-1 and mcr-3 was observed with the frequencies ranging from 10-8 to 10-3. All mcr-1 resided on IncI2, while mcr-3 was associated with IncF and IncX4 plasmids. Our study provides the evidence of fecal shedding pattern of S. enterica and mcr-positive E. coli from meat-type ducks, highlighting the importance of duck farming in the dissemination of pathogenic bacteria that are potentially hazardous to human.

Risk Factors for Community-Acquired Urinary Tract Infections Caused by Multidrug-Resistant Enterobacterales in Thailand.

The dissemination of multidrug-resistant Enterobacterales (MDRE) in community settings is becoming a great concern. This study aimed to assess the incidence and risk factors associated with community-acquired urinary tract infections (CA-UTIs) caused by MDRE. A prospective case−control study was undertaken among patients with UTIs visiting an outpatient department in Phitsanulok Province, Thailand. Urine samples were collected and screened to include only patients with Enterobacterales infections. Risk factors were analyzed by multivariate logistic regression analysis. Of the 284 patients with CA-UTIs, 25.7% (n = 73) and 74.3% (n = 211) were positive for MDRE (case) and non-MDRE (control), respectively. Being a farmer was identified as an independent risk factor for MDRE-associated CA-UTIs (adjusted odds ratio = 3.101; 95% confidence interval = 1.272−7.564; p = 0.013). A total of 309 Enterobacterales isolates were recovered, and Escherichia coli was the most frequently detected (86.4%). The highest resistance rate was observed for ampicillin (67.0%), followed by ciprofloxacin (34.0%) and cotrimoxazole (32.7%), while resistance to third-generation cephalosporins (cefotaxime, ceftriaxone) and levofloxacin remained <20%. Resistance to ampicillin−gentamicin−cotrimoxazole was the most common pattern among MDRE isolates. Interestingly, we detected a colistin-resistant Enterobacter cloacae harboring mcr-9 (colistin MIC = 16 µg/mL). mcr-9 was transferable at high frequency (4.5 × 10−4) and resided on IncF plasmid. This study demonstrates that being a farmer is a risk factor for MDRE-associated CA-UTIs. Interestingly, this is the first report to identify mcr-9-positive E. cloacae from a Thai patient in the community.

Prevalence of blaCTX-M and Emergence of blaCTX-M-5-Carrying Escherichia coli in Chrysomya megacephala (Diptera: Calliphoridae), Northern Thailand.

This study was undertaken to assess the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) among blow fly (Chrysomya megacephala) populations in Northern Thailand. Of 600 blow flies collected from rural (n = 400) and urban (n = 200) areas, 334 blow flies carried ESBL-EC (55.7%). Prevalence of ESBL-EC in blow flies captured from rural areas was significantly higher than that from urban region (72.5% vs. 22.0%, p < 0.001). Susceptibility tests revealed that 68.6% of ESBL-EC possessed multidrug-resistant phenotypes. Coresistance to gentamicin (85%) was common, while resistance to ciprofloxacin was relatively low (18.0%). Of the 334 isolates, 253 isolates (75.7%) harbored blaCTX-M, in which blaCTX-M group 1 was predominant (56.5%), followed by blaCTX-M group 9 (39.1%). Interestingly, a single isolate was found to carry blaCTX-M-5, which resided on the IncA/C conjugative plasmid. This is the first report of blaCTX-M-5 from Thailand and its first identification in blow fly. Pulsed field gel electrophoresis (PFGE) demonstrated high genetic diversity among ESBL-EC isolates. Nevertheless, identical and closely related PFGE profiles were detected among isolates within the same regions and the regions which are several kilometers apart, suggesting that clonal transmission has occurred. Moreover, epidemiologically related isolates were observed between ESBL-EC from blow flies and human intestinal tract. This study provides evidences that blow flies, C. megacephala, are important reservoirs for ESBL-EC and could potentially act as vectors for the spread of ESBL-EC in a Thai community.

Acquisition of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in intensive care units in Thailand.

This study aimed to assess the prevalence and associated risk factors for extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EK) acquisition among patients staying in medical and surgical Intensive Care Units (ICU) in Northern Thailand. Rectal swabs were collected from 206 ICU patients upon admission and discharge. Overall, the ESBL-EK acquisition rate among patients during ICU stay was 29.6%. Acquisition rate was significantly higher for K. pneumoniae (20.9%) than that of E. coli (12.1%) (p = 0.024). Multivariate logistic regression analysis identified the use of third generation cephalosporin (p = 0.008) as a risk factor for ESBL-EK acquisition. Sixty-eight ESBL-EK isolates (25 E. coli and 43 K. pneumoniae) were recovered. The majority of ESBL-EK isolates (≥88%) were resistant to ceftazidime, cefepime and aztreonam. Fifty-two acquired ESBL-EK isolates (76.5%) were positive for blaCTX-M and 4 K. pneumoniae isolates simultaneously carried blaNDM-1. Our results reveal that ICU patients could acquire ESBL-EK during hospitalization and the use of third generation cephalosporin should be strictly controlled to prevent the acquisition of ESBL-EK among ICU patients.

First Detection and Genomic Insight into mcr-1 Encoding Plasmid-Mediated Colistin-Resistance Gene in Escherichia coli ST101 Isolated from the Migratory Bird Species Hirundo rustica in Thailand.

Background: This study aimed to investigate the occurrence of mcr-1 encoding plasmid-mediated colistin-resistance gene in Escherichia coli isolated from migratory birds in Thailand. Materials and Methods: A total of 178 cloacal swabs from migratory birds was sampled and isolated from 2016 to 2017 in Nan, Trang, and Bangkok, Thailand. The multiplex polymerase chain reaction was used to screen the resistance genes. After screening, a disk diffusion assay and the minimum inhibitory concentration were investigated. The draft genome sequence of isolate 2A85589 was obtained using an Illumina HiSeq X-Ten platform. The genome was assembled using SPAdes 3.0.0. Antimicrobial resistance genes were identified using ResFinder 3.1. Results: We reported E. coli ST101 of isolate 2A85589, an mcr-1-carrying resistance gene isolated from the migratory bird species Hirundo rustica in Thailand. The draft genome of 2A85589 was 4,621,016 bp in size. IncHI1A plasmid was identified using PlasmidFinder with high coverage. In silico analysis detected the presence of eight putative acquired resistance genes, namely blaTEM-1B, mcr-1, mef(A), mef(B), QnrS1, sul3, tet(A), and tet(B), which conferred resistance to ß-lactam, colistin, macrolide, quinolone, sulfonamide, and tetracycline. Conclusion: This study underlines the potential risk of the environmental contamination of mcr-1-carrying E. coli isolated from the migratory bird. The long range migration of birds can result in dissemination of mcr-1-carrying bacteria globally. Therefore, plasmid-mediated colistin is an urgent need to be addressed in both human and veterinary medicine for disease control and prevention.

Risk Factors for Extended-Spectrum ß-Lactamase-Producing Enterobacteriaceae Carriage in Patients Admitted to Intensive Care Unit in a Tertiary Care Hospital in Thailand.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) are important causes of serious infections in intensive care unit (ICU). This study aimed to investigate the risk factors for intestinal carriage of ESBL-PE among patients admitted to ICU, subsequent ESBL-PE infections, and outcomes of these patients. This study prospectively collected rectal swabs from 215 ICU patients in Northern Thailand and ESBL-PE were isolated. A high prevalence of ESBL-PE carriage (134/215, 62.3%) at ICU admission was observed, with Escherichia coli representing the predominant organism (67.5%) followed by Klebsiella pneumoniae (19.4%). Multivariate logistic regression analysis identified chronic renal disease as the independent risk factor for ESBL-PE carriage (p = 0.009; adjusted odds ratio = 4.369; 95% confidence interval = 1.455-13.119). Among colonized patients, 2.2% (3/134) developed ESBL-PE infections during ICU stay. Phylogenetic analysis of E. coli (n = 108) showed that the predominant group was group A (38.0%), followed by groups B1 (17.6%), D (15.7%), B2 (14.8%), C (7.4%), and F (6.5%). Multilocus sequence typing analysis of the pathogenic groups B2, D, and F revealed 11 different sequence types (STs), with ST131 (n = 13) as the most prevalent, followed by ST648 (n = 5), ST38 (n = 4), ST393 (n = 3), and ST1193 (n = 3). These results are of concern since ESBL-PE may be a prerequisite for endogenous infections and potentially disseminate within the hospital. This is the first study describing ESBL-PE carriage among patients at ICU admission and subsequent ESBL-PE infections in Thailand.

Extended spectrum ß-lactamase-producing Escherichia coli among backyard poultry farms, farmers, and environments in Thailand.

Food-producing animals, including poultry, have been considered as potential sources of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli. This study investigates the occurrence and dissemination of ESBL-producing E. coli among backyard poultry farms, farmers, and environments in Northern Thailand. Antimicrobial-resistant phenotypes, resistant determinants, genotypic characterizations, and spread of these isolates were studied. Fecal samples from poultry, farmers, and environments were captured from 27 farms. In total, 587 samples were collected and the overall 27.1% (159/587) of ESBL-producing E. coli isolates were obtained. Among these, ESBL-producing E. coli was isolated from 50% (farmers), 25.9% poultry (24.9% chicken and 36.6% duck) of the fecal samples, and 25.0% of the environmental samples. All isolates demonstrated multidrug resistance, most frequently to ≥ 10 different antimicrobial agents. Molecular analysis of ESBL-encoding genes showed that the predominant gene was blaCTX-M-55 (54.1%), followed by blaCTX-M-14 (28.3%), and blaCTX-M-15 (8.8%). blaCTX-M-27 (3.8%) and blaCTX-M-65 (0.6%) were also detected at low frequencies. Conjugation assays demonstrated that blaCTX-M could be transferred to E. coli J53 with the transfer frequencies ranging from 10-7 to 10-2. Pulsed field gel electrophoresis (PFGE) revealed diverse genotypes, however, identical and closely related PFGE profiles were detected among isolates within and between farms, suggesting the clonal transmission. In addition, our study identified 4 blaCTX-M-27-positive E. coli B2-ST131 isolates. Interestingly, two ST131 isolates, obtained from a farmer and chicken in the same area, showed closely related PFGE profiles. Our results suggest the presence and spread of ESBL-producing E. coli between backyard poultry farms, farmers, and environments in Thailand.

Environmental dissemination of mcr-1 positive Enterobacteriaceae by Chrysomya spp. (common blowfly): An increasing public health risk.

Until recently, the role of insects, and particularly flies, in disseminating antimicrobial resistance (AMR) has been poorly studied. In this study, we screened blowflies (Chrysomya spp.) from different areas near the city of Phitsanulok, Northern Thailand, for the presence of AMR genes and in particular, mcr-1, using whole genome sequencing (WGS). In total, 48 mcr-1-positive isolates were recovered, consisting of 17 mcr-1-positive Klebsiella pneumoniae (MCRPKP) and 31 mcr-1-positive Escherichia coli (MCRPEC) strains. The 17 MCRPKP were shown to be clonal (ST43) with few single poly nucleomorphs (SNPs) by WGS analysis. In in-vitro models, the MCRPKP were shown to be highly virulent. In contrast, 31 recovered MCRPEC isolates are varied, belonging to 12 different sequence types shared with those causing human infections. The majority of mcr-1 gene are located on IncX4 plasmids (29/48, 60.42%), sharing an identical plasmid backbone. These findings highlight the contribution of flies to the AMR contagion picture in low- and middle-income countries and the challenges of tackling global AMR.

Risk Factors for Gastrointestinal Colonization and Acquisition of Carbapenem-Resistant Gram-Negative Bacteria among Patients in Intensive Care Units in Thailand.

This study was conducted to investigate the prevalence of and risk factors for colonization and acquisition of carbapenem-resistant (CR) Gram-negative bacteria (GNB) among patients admitted to intensive care units (ICUs) in two tertiary care hospitals in northern Thailand. Screening of rectal swab specimens for CR-GNB was performed on patients at ICU admission and discharge. The phenotypes and genotypes of all isolates were determined. Risk factors were analyzed by logistic regression analysis. The overall carriage rate of CR-GNB at admission was 11.6% (32/275), with the most predominant species carried being Acinetobacter baumannii (n = 15), followed by Klebsiella pneumoniae (n = 9). The risk factor for CR-GNB colonization was hospitalization within the previous 6 months (P = 0.002). During the ICU stay, the rate of CR-GNB acquisition was 25.2% (52/206), with the most predominant species carried being A. baumannii (n = 28) and K. pneumoniae (n = 13). Risk factors associated with CR-GNB acquisition were the use of an enteral feeding tube (P = 0.008) and administration of third-generation cephalosporins (P = 0.032) and carbapenems (P = 0.045). The most common carbapenemase genes in A. baumannii and K. pneumoniae were blaOXA-23/51 and blaNDM, respectively. Patient-to-patient transmission was demonstrated in three cases, resulting in the acquisition of CR A. baumannii (2 cases) and K. pneumoniae (1 case) isolates from other patients who were admitted during the same period of time. This is the first Indochinese study screening patients, examining patients for the carriage of CR-GNB, and further demonstrating the transfer of CR-GNB isolates in ICUs. Our study suggests that effective infection control measures are required to limit the spread of CR-GNB within hospitals.

Occurrence of Extended-Spectrum and AmpC-Type ß-Lactamase Genes in Escherichia coli Isolated from Water Environments in Northern Thailand.

Sixty-eight cefotaxime-resistant Escherichia coli isolates were recovered from different water environments in Northern Thailand. Isolates were mostly resistant to ceftazidime and aztreonam (>90%). The most common extended-spectrum ß-lactamase-encoding gene was blaCTX-M-group 1 (75%) followed by blaCTX-M-group 9 (13.2%). The co-existence of blaCTX-M and AmpC-type ß-lactamase genes was detected in 4 isolates (5.9%). Two E. coli isolates carrying blaCTX-M from canal and river water samples belonged to the phylogenetic group B2-ST131, which is known to be pathogenic. This is the first study on blaCTX-M and blaCMY-2-carrying E. coli and the emergence of ST131 from water environments in Thailand.

Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1.

The polymixin colistin is a "last line" antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.

Nosocomial spread of class 1 integron-carrying extensively drug-resistant Pseudomonas aeruginosa isolates in a Thai hospital.

Fifty non-duplicate multiresistant isolates of Pseudomonas aeruginosa from a regional hospital in Northern Thailand were investigated for their antimicrobial susceptibility, presence of class 1 integrons and arrangement of gene cassettes as well as their genetic relationships. All but one isolate were classified as extensively drug-resistant P. aeruginosa (XDR-PA). Forty-one isolates (82%) were found to carry class 1 integrons. Amplification of the variable regions of class 1 integrons revealed seven diverse bands ranging in size from 0.7 kb to 7.0 kb. Sequence analysis of class 1 integron variable regions revealed the presence of several gene cassettes associated with resistance to aminoglycosides (aac, aad and aph), including the aac(3)-Ic cassette reported for the first time in Thailand. Gene cassettes encoding resistance to chloramphenicol (cmlA), ß-lactams (bla(PSE), bla(OXA) and bla(VEB)) and rifampicin (arr) were found. The putative small multidrug resistance protein (smr) and an open-reading frame with unknown function (orfD) were also detected. The aadA6-orfD cassette array was the most common integron found in this study. Integron-positive isolates had higher frequencies of antimicrobial resistance than isolates lacking integrons. Pulsed-field gel electrophoresis (PFGE) demonstrated the occurrence of horizontal gene transfer. Interestingly, a large number of XDR-PA isolates carrying identical integrons clearly exhibited the same PFGE pattern, indicating nosocomial spread of these isolates. The presence of XDR-PA carrying class 1 integrons is implicated in the possible spread of drug-resistant organisms, therefore screening for integron-positive P. aeruginosa might be necessary for protection against nosocomial infection.

Diffusion and activity of antibiotics against Burkholderia pseudomallei biofilms.

The diffusion and activity of ceftazidime (CAZ), imipenem (IPM) and trimethoprim/sulfamethoxazole (TMP/SMX) against Burkholderia pseudomallei biofilms were comparatively tested using the high biofilm-producing strain B. pseudomallei 377 and the biofilm-defective mutant B. pseudomallei M6. Biofilms were generated by inoculation of bacteria on polycarbonate membranes placed on the surface of tryptic soy agar plates. The results showed that diffusion of TMP/SMX through B. pseudomallei biofilms was similar for both strains. However, diffusion of CAZ and IPM was significantly faster through strain M6 biofilm in comparison with strain 377 biofilm. The viabilities of strain 377 biofilm were significantly higher than those observed with strain M6 for all antibiotics challenged at 4 h, suggesting that the biofilm-forming capacity may be involved in antibiotic susceptibilities in B. pseudomallei. These results re-emphasise the importance of biofilm for antibiotic resistance in B. pseudomallei.

Class 1 integron containing metallo beta-lactamase gene blaIMP-1 in carbapenem-resistant Pseudomonas aeruginosa in Thailand.

We investigated the genetic properties of two carbapenem-resistant Pseudomonas aeruginosa isolates collected from a regional hospital in the north of Thailand. Both isolates demonstrated high-level resistance to extended-spectrum cephalosporins and carbapenems. Detection of the MBL genes was positive for bla (IMP) in both isolates. Pulsed field gel electrophoresis (PFGE) analysis showed that the two P. aeruginosa isolates were nonclonal. Molecular analysis of the bla (IMP) in isolate 837 showed the presence of a bla (IMP-1) gene inserted in a class 1 integron. The bla (IMP-1) was plasmid-mediated according to the transformation assay. This is the first case of the bla (IMP-1) gene in carbapenem-resistant P. aeruginosa in Thailand, suggesting the further dissemination of this gene in Southeast Asia.

SHV-12 extended spectrum beta-lactamase associated with high-level ceftazidime resistance in Enterobacter cloacae isolated from Thailand.

Four Enterobacter cloacae clinical isolates with reduced susceptibility to ceftazidime from two hospitals in Thailand were studied. Production of extended-spectrum beta-lactamase was confirmed by double disk synergy test and combination disk method. All isolates were highly resistant to ceftazidime but retained susceptibility to imipenem. One isolate was able to hydrolyze cefotaxime, ceftazidime and cefepime, the latter being one of the treatment choices for infection by Enterobacter spp. PCR analysis demonstrated the presence of bla(SHV12) in addition to bla(TEM-1) in all isolates suggesting that SHV-12 was associated with high-level resistance to ceftazidime in the E. cloacae isolates.

Dissemination of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli in Thai hospitals.

Fifty clinical isolates of Klebsiella pneumoniae and Escherichia coli with reduced susceptibility to third-generation cephalosporins, collected from 11 hospitals in Thailand, were studied. All isolates were found to produce extended-spectrum beta-lactamase (ESBL), as judged by double-disk synergy and combination disk methods. Most ESBL-producing K. pneumoniae isolates were resistant to ceftazidime (94%) and aztreonam (90%). In contrast, most ESBL-producing E. coli isolates were resistant to ceftriaxone (95%) and cefotaxime (74%). Plasmid DNA was isolated and beta-lactamase genes were identified by PCR and sequencing. We found that SHV-12 and CTX-M-14 were the main ESBLs responsible for resistance in K. pneumoniae and E. coli, respectively. SHV-27, SHV-28, and CTX-M-14 were detected in three, two, and four K. pneumoniae isolates, respectively. A high genetic diversity among ESBL-producing K. pneumoniae and E. coli isolates was observed. In addition, the finding of a few isolates that produced identical restriction patterns on pulsed field gel electrophoresis (PFGE) suggests the clonal spread of resistant bacteria within the hospital.