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.

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.

We have studied the mechanism by which b-lactam challenge leads to b-lactamase induction in Aeromonas hydrophila through transposon-insertion mutagenesis. Disruption of the DDcarboxypeptidases/endopeptidases, penicillin-binding protein 4 or BlrY leads to elevated monomer-disaccharide-pentapeptide levels in A. hydrophila peptidoglycan and concomitant overproduction of b-lactamase through activation of the BlrAB two-component regulatory system. During b-lactam challenge, monomer-disaccharide-pentapeptide levels increase proportionately with b-lactamase production and b-lactamase induction is inhibited by vancomycin, which binds muro-pentapeptides. Taken together, these data strongly suggest that the Aeromonas spp. blactamase regulatory sensor kinase, BlrB, responds to the concentration of monomerdisaccharide-pentapeptide in peptidoglycan.

Aeromonas hydrophila T429125, a human clinical isolate, possesses three coordinately inducible beta-lactamases encoded by ampH (class D beta-lactamase), cepH (class C beta-lactamase) and imiH (class B beta-lactamase). We report that upstream of ampH there are two genes, blrA and blrB, encoding a putative two-component regulatory system. PCR studies revealed the same blrAB-amp gene arrangement in all Aeromonas spp. isolates tested; namely, Aeromonas veronii bv. sobria, Aeromonas jandaei, Aeromonas mediae, Aeromonas salmonicida and Aeromonas trota. A dominant mutation in the predicted BlrB kinase domain results in beta-lactamase overexpression in A. hydrophila T429125, but in other beta-lactamase-overexpressing mutants blrAB remains intact. Relative to the parent strain, A. hydrophila T429125, beta-lactamase- overexpressing mutants show a clear hierarchy of increased beta-lactamase expression: ImiH > CepH > AmpH. The same hierarchy is seen following beta-lactam challenge of A. hydrophila T429125, and correlates with the number of blr-tag sequences (TTCAC) found upstream of each beta-lactamase gene: ampH (one), cepH (two) and imiH (three).

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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 ( = 15), followed by ( = 9). The risk factor for CR-GNB colonization was hospitalization within the previous 6 months ( = 0.002). During the ICU stay, the rate of CR-GNB acquisition was 25.2% (52/206), with the most predominant species carried being ( = 28) and ( = 13). Risk factors associated with CR-GNB acquisition were the use of an enteral feeding tube ( = 0.008) and administration of third-generation cephalosporins ( = 0.032) and carbapenems ( = 0.045). The most common carbapenemase genes in and were and, respectively. Patient-to-patient transmission was demonstrated in three cases, resulting in the acquisition of CR (2 cases) and (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.

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This is the first report of non-beta-lactamase genes being co-ordinately regulated with a normally co-resident beta-lactamase gene, and the first direct evidence for a role of the cre/blr-tag sequence in the regulation of transcription by BlrA.