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.

Carbapenem-resistant Acinetobacter baumannii producing OXA-23 in Thailand.

We investigated the resistance determinant of 13 clinical isolates of carbapenem-resistant Acinetobacter baumannii collected from a regional hospital in the north of Thailand. All isolates were multidrug resistant and produced the OXA-23 carbapenemase. The bla(OXA-23) gene was found adjacent to ISAba1. Furthermore, two isolates carried the metallo beta-lactamase gene, bla(IMP). The bla(OXA-23) and bla(IMP) genes were plasmid-mediated according to the transformation assays. This is the first description of OXA-23-producing A. baumannii from Thailand.

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.

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.

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.

Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms.

MCR-1 is a lipid A modifying enzyme that confers resistance to the antibiotic colistin. Here, we analyse the impact of MCR-1 expression on E. coli morphology, fitness, competitiveness, immune stimulation and virulence. Increased expression of mcr-1 results in decreased growth rate, cell viability, competitive ability and significant degradation in cell membrane and cytoplasmic structures, compared to expression of catalytically inactive MCR-1 (E246A) or MCR-1 soluble component. Lipopolysaccharide (LPS) extracted from mcr-1 strains induces lower production of IL-6 and TNF, when compared to control LPS. Compared to their parent strains, high-level colistin resistance mutants (HLCRMs) show reduced fitness (relative fitness is 0.41-0.78) and highly attenuated virulence in a Galleria mellonella infection model. Furthermore, HLCRMs are more susceptible to most antibiotics than their respective parent strains. Our results show that the bacterium is challenged to find a delicate equilibrium between expression of MCR-1-mediated colistin resistance and minimalizing toxicity and thus ensuring cell survival.

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.

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.