5 research outputs found
Antibiotic Resistance in Gram-negative Bacteria in Brunei Darussalam: Molecular Characterisation, Epidemiology, Surveillance and Virulence
PhDAntimicrobial resistance (AMR) presents a global threat to human and animal health. Southeast Asia (SEA) is seen as a potential hotspot for the emergence and dissemination of new resistance mechanisms. The World Health Organization highlights research on multi-drug resistant Gram-negative bacteria (Enterobacteriaceae, Acinetobacter and Pseudomonas spp) as a critical priority. This study reports findings from Brunei Darussalam. A molecular epidemiological surveillance study conducted using contemporary MDR Escherichia coli, Klebsiella pneumoniae, A. baumannii and P. aeruginosa of human and avian (farmed poultry) origin. Resistance to carbapenems and polymyxins was investigated using phenotypic (susceptibility, serotyping) and genotypic (PCR, sequencing, typing) methods. A selective bacterial culture media was developed and evaluated as a screening media for polymyxin-resistant (PR) strains. Immunochromatographic assays were used in the rapid identification of novel resistance determinants (OXA-48-like, MCR-1). Pathogenicity, fitness and virulence was correlated with host strain background and the carriage of AMR plasmids by resistant isolates. Carbapenem-resistant (CR) A. baumannii were prevalent amongst hospital isolates and produced OXA-23 carbapenemases, similar to those reported worldwide. CR K. pneumoniae (CRKP) with plasmid encoded OXA-232 carbapenemase were found as part of a hospital outbreak. All identified as members of sequence type 231, a hi-risk epidemic clone in SEA. In separate point prevalence studies, polymyxin resistant E. coli were found in 58% of poultry and 41.7% of human faecal samples. All produced phosphoethanolamine transferases encoded by mcr genes supported by diverse plasmid backbones (IncHI2, IncI2, IncX4) and host strain sequence types (n= 40). Additionally, a novel variant, mcr- 1.8, localised on a 63,056 bp IncI2 conjugative plasmid was identified and characterized in E. coli O88:H31 ST101. PR strains were co-resistant to quinolones, aminoglycosides and phenicols. CTX-M β-lactamases (CTX-M-3 and CTX-M-65) were found in 2 strains but none co-produced a carbapenemase. Comparison of human and avian MCR producing E. coli revealed only 6 sequence types were common to both humans and poultry. Polymyxin resistant strains harboured multiple virulence factors (VF) but no Avian Pathogenic (APEC) or Shiga Toxin (STEC) producing strains were found. Virulence of human and poultry isolates assessed in a Galleria mellonella infection model showed differences in survival rates that did not correlate with virulence score. When virulence of polymyxin resistant transconjugants (TC) was assessed only one which encoded MCR-1.8 exhibited heightened virulence. Growth kinetics showed no obvious differences despite plasmid acquisition. This study demonstrates the high rate of resistance to carbapenems and polymyxins in critical Gram-negative bacteria in Brunei Darussalam. Plasmidmediated polymyxin resistance in E. coli was found to be endemic in poultry and highly prevalent in the human samples studied. Unlike carbapenem resistance, polymyxin resistance was not associated with any predominant clone and did not readily correlate with virulence properties of the strain. However, given the high background rates, enhanced surveillance for MCR-1 within more virulent Enterobacterial backgrounds is warranted, particularly in Southeast Asia countries
CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens.
PURPOSE: A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture medium (SuperPolymyxin). METHODOLOGY: The medium was challenged with 84 isolates, including polymyxin B (POL B)-susceptible and -resistant type strains and colistin (COL)-resistant organisms recovered from human and animal samples. Susceptibility to COL and POL B was determined by agar dilution and broth microtitre dilution. The lower limit for the detection of COL-resistant organisms was also calculated for both CHROMagar COL-APSE and SuperPolymyxin media. The ability to isolate and correctly differentiate COL-resistant organisms within mixed cultures was also assessed and compared using both media. RESULTS: Using CHROMagar COL-APSE, Gram-negative pathogens (n=71) with intrinsic (n=8) or acquired COL (n=63) resistance were recovered with 100 % specificity down to the lower limit of detection of 101 colony-forming units (c.f.u.). The growth on SuperPolymyxin was similar, but notably weaker for COL-resistant non-fermentative bacteria (Acinetobacter, Pseudomonas and Stenotrophomonas). CHROMagar COL-APSE was also more sensitive in supporting the growth of Enterobacteriaceae with COL resistance associated with the carriage of mcr-1. CONCLUSION: CHROMagar COL-APSE is a sensitive and specific medium for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (101 c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1 or novel mechanisms of polymyxin resistance
Evaluation of an Immunochromatographic Lateral Flow Assay (OXA-48 K-SeT) for the Rapid Detection of OXA-48-like Carbapenemases in Enterobacteriaceae
We evaluated an immunochromatographic lateral flow assay for the detection of OXA-48-like carbapenemases (OXA-48 K-SeT) in Enterobacteriaceae (n=82). 100% sensitivity and specificity was observed using bacteria recovered from both solid media and spiked blood culture bottles, with the result obtained in less than 10 minutes
In-vitro and in-vivo Activity of ML302F: A Thioenolate Inhibitor of VIM-subfamily Metallo β-lactamses
The thioenol ML302F was recently identified as an inhibitor of class B metallo-β-lactamases (MBLs). We assessed the activity of ML302F when combined with meropenem (MEM) against 31 carbapenem resistant Gram-negative clinical isolates. Minimum inhibitory concentrations of MEM : ML302F were determined at fixed ratios of 1 : 4 and 1 : 8 using strains producing variants of the clinically relevant VIM-like MBL. Toxicity and efficacy in vivo was assessed in a Galleria mellonella invertebrate model against strains producing VIM-1, VIM-2 and VIM-4 variants. At a fixed MEM : ML302F ratio of 1 : 8, 22/31 isolates were rendered either susceptible (MIC ≤ 2 mg L−1), or intermediate (MIC 4–8 mg L−1) to MEM. ML302F alone was not toxic at up to 80 mg kg−1 in G. mellonella and treatment with MEM 0.6 mg kg−1 : ML302F 4.8 mg kg−1 significantly improved the survival of infected larvae. As ML302F was able to successfully restore susceptibility to resistant strains both in vitro and in vivo it represents a structurally interesting inhibitor in the search for new MBL inhibitors
In vitro and in vivo activity of ML302F: a thioenolate inhibitor of VIM-subfamily metallo β-lactamases
The thioenol ML302F was recently identified as an inhibitor of class B metallo-β-lactamases (MBLs). We assessed the activity of ML302F when combined with meropenem (MEM) against 31 carbapenem resistant Gram-negative clinical isolates. Minimum inhibitory concentrations of MEM:ML302F were determined at fixed ratios of 1:4 and 1 8 using strains producing variants of the clinically relevant VIM-like MBL. Toxicity and efficacy in vivo was assessed in a Galleria mellonella invertebrate model against strains producing VIM-1, VIM-2 and VIM-4 variants. At a fixed MEM:ML302F ratio of 1:8, 22/31 isolates were rendered either susceptible (MIC ≤ 2 mg L-1), or intermediate (MIC 4-8 mg L-1) to MEM. ML302F alone was not toxic at up to 80 mg kg-1 in G. mellonella and treatment with MEM 0.6 mg kg-1:ML302F 4.8 mg kg-1 significantly improved the survival of infected larvae. As ML302F was able to successfully restore susceptibility to resistant strains both in vitro and in vivo it represents a structurally interesting inhibitor in the search for new MBL inhibitors