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Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: David P Fairle is an inventor on a patent application (PCT/AU2013/000742, WO2014005194), and Jeffrey YW Mak and David P Fairlie are inventors on another patent application (PCT/AU2015/050148, WO2015149130) involving MR1 ligands for MR1-restricted MAIT cells owned by University of Queensland, Monash University and University of Melbourne. The other authors declare no competing interests.

The ST131 multilocus sequence type (MLST) of Escherichia coli is a globally successful pathogen whose dissemination is increasing rates of antibiotic resistance. Numerous global surveys have demonstrated the pervasiveness of this clone; in some regions ST131 accounts for up to 30% of all E. coli isolates. However, many regions are underrepresented in these published surveys, including Africa, South America, and Asia. We collected consecutive bloodstream E. coli isolates from three countries in Southeast Asia; ST131 was the most common MLST type. As in other studies, the C2/H30Rx clade accounted for the majority of ST131 strains. Clinical risk factors were similar to other reported studies. However, we found that nearly all of the C2 strains in this study were closely related, forming what we denote the SEA-C2 clone. The SEA-C2 clone is enriched for strains from Asia, particularly Southeast Asia and Singapore. The SEA-C2 clone accounts for all of the excess resistance and virulence of ST131 relative to non-ST131 E. coli. The SEA-C2 strains appear to be locally circulating and dominant in Southeast Asia, despite the intuition that high international connectivity and travel would enable frequent opportunities for other strains to establish themselves.

Polymyxin B-based combinations have emerged as a mainstay treatment against carbapenem-resistant Escherichia coli (CREC). We investigated the activity of polymyxin B-based two-antibiotic combinations against CREC using time-kill studies (TKS) and validated the findings in a hollow-fiber infection model (HFIM). TKS were conducted using 5 clinical CREC strains at 5 log 10 CFU/ml against 10 polymyxin B-based two-antibiotic combinations at maximum clinically achievable concentrations. HFIMs simulating dosing regimens with polymyxin B (30,000U/kg/day) and tigecycline (100 mg every 12 h) alone and in combination were conducted against two CREC strains at 5 log 10 CFU/ml over 120 h. Emergence of resistance was quantified using antibiotic-containing media. Phenotypic characterization (growth rate and stability of resistant phenotypes) of the resistant isolates was performed. All five CREC strains harbored carbapenemases. Polymyxin B and tigecycline MICs ranged from 0.5 mg/liter to 2 mg/liter and from 0.25 mg/liter to 8 mg/liter, respectively. All antibiotics alone did not have bactericidal activity at 24 h in the TKS, except for polymyxin B against two strains. In combination TKS, only polymyxin B plus tigecycline demonstrated both bactericidal activity and synergy in two out of five strains. In the HFIM, polymyxin B alone was bactericidal against both CREC strains before regrowth was observed at 8 h. Phenotypically stable polymyxin B-resistant mutants were observed for both strains, with a reduced growth rate observed in one strain. Tigecycline alone resulted in a slow reduction in bacterial counts. Polymyxin B plus tigecycline resulted in rapid and sustained bactericidal killing up to 120 h. Polymyxin B plus tigecycline is a promising combination against CREC. The clinical relevance of our results warrants further investigations., (Copyright © 2016 American Society for Microbiology.)

Introduction: At the time of the study, 3 plasmid-borne qnr determinants (qnrA, qnrB and qnrS) and 1 plasmid-borne aminoglycoside-modifying enzyme determinant that confers quinolone resistance (aac(6')-Ib-cr) had been described in the literature., Materials and Methods: We studied the prevalence of the 3 qnr determinants in a total of 117 nalidixic acid-resistant urinary isolates of Klebsiella pneumoniae (61 isolates) and Escherichia coli (56 isolates) using multiplex polymerase chain reaction (PCR). Further, a subset of the original strains (comprising 14 E. coli and 38 K. pneumoniae) showing reduced susceptibility to the aminoglycosides underwent PCR for aac(6')-Ib, followed by restriction digestion with BtsCI to detect the variant aac(6')-Ib-cr., Results: Twenty-eight of 61 (45.9%) Klebsiella isolates were found to possess at least 1 qnr determinant. Only 1/56 (1.8%) E. coli isolates were found to possess a qnr determinant. Two of the Klebsiella isolates possessed 2 qnr determinants each (qnrB and qnrS). The predominant determinant was qnrB (19 isolates). There were 11 isolates harbouring qnrS, and only 1 with qnrA. 1/14 (7.1%) E. coli and 35/38 K. pneumoniae (92.1%) were found to possess aac(6')-Ib-cr. There was pairwise association between each of qnr, aac(6')-Ib-cr and the presence of an extended-spectrum beta-lactamase., Conclusions: A high prevalence of plasmid-mediated quinolone resistance determinants [i.e., qnrS, qnrB and aac(6')-Ib-cr] was found in quinolone-resistant K. pneumoniae isolated in a large hospital in Singapore.

There are currently no standardized phenotypic methods for the screening and detection of AmpC enzymes. This study aimed to evaluate different methods to detect AmpC enzymes in Escherichia coli, Klebsiella spp., and Proteus spp., comparing the results from two disk-based methods and an agar dilution method. AmpC activity was determined for 255 clinical isolates by use of a three-dimensional enzyme assay combined with a multiplex PCR assay for plasmid-borne ampC genes. These results were compared against a disk-based inhibitor assay using various combinations of cefpodoxime and cefoxitin as antibiotic substrates and boronic acid or cloxacillin as an AmpC inhibitor. The presence of enzyme induction by disk approximation was evaluated using imipenem, cefoxitin, and amoxicillin-clavulanate as inducing agents against ceftazidime. Finally, an agar dilution assay was performed, using cefoxitin with and without added cloxacillin. AmpC activity was present in 49.8% of test isolates, 93.7% of which were positive for plasmid-borne ampC genes. CIT-like enzymes were predominant in E. coli, and DHA-like enzymes were predominant in Klebsiella spp. The disk-based inhibitor tests performed better than the agar dilution assay, while detection of AmpC by disk induction had a poor sensitivity. The cefoxitin-cloxacillin disk combination provided the best overall performance, with a sensitivity and specificity of 95%. This study confirmed the accuracy of disk-based inhibitor screening for AmpC enzymes, which proved reliable at detecting CIT- and DHA-like plasmid-borne ampC genes. The methods are simple enough for introduction into clinical microbiology laboratories.

Introduction: Antibiotic resistance in gram-negative bacilli is an area of increasing importance. This prospective study was performed to survey antibiotic resistance in Escherichia coli (E. coli), Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. over a 1-year period., Materials and Methods: Non-duplicate isolates of E. coli, Klebsiella spp., P. aeruginosa and Acinetobacter spp. were collected from participating Singapore hospitals during defined collection periods in 2006 and 2007. Confirmatory identification and antibiotic susceptibility testing were performed at Changi General Hospital. Minimum inhibitory concentrations (MIC) to a defined panel of antibiotics were determined using microbroth dilution methods. The presence of extended-spectrum beta lactamases and AmpC beta-lactamases in Enterobacteriaceae was determined by phenotypic methods, and susceptibility results were defined using current breakpoints from the Clinical Laboratory Standards Institute (CLSI)., Results: Seven hundred and forty-six gram-negative bacilli were received for testing. Resistance to extended-spectrum cephalosporins was present in a third of Enterobacteriaceae isolates, and extended-spectrum beta-lactamases (ESBL) carriage was present in 19.6% and 30.1% of E. coli and Klebsiella pneumoniae, respectively. AmpC enzymes were also detected in 8.5% and 5.6% of E. coli and K. pneumoniae isolates respectively. All Enterobacteriaceae were susceptible to imipenem and meropenem. The most active antibiotics against P. aeruginosa were amikacin, meropenem and piperacillin-tazobactam. A third of P. aeruginosa showed reduced susceptibility to polymyxin B. Carbapenem resistance was significantly higher in Acinetobacter baumannii (70.5%) than in other Acinetobacter species (25.0%). The most active antibiotic against A. baumannii was polymyxin B., Conclusion: Antibiotic resistance is prevalent in gram-negative bacilli isolated from Singapore hospitals. The MIC testing surveillance programme complemented susceptibility data from wider laboratory-based surveillance, and has revealed emerging mechanisms of antibiotic resistance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]