Mark A. Webber

Molecular mechanisms of antibiotic resistance.

Antibiotic-resistant bacteria that are difficult or impossible to treat are becoming increasingly common and are causing a global health crisis. Antibiotic resistance is encoded by several genes, many of which can transfer between bacteria. New resistance mechanisms are constantly being described, and new genes and vectors of transmission are identified on a regular basis. This article reviews recent advances in our understanding of the mechanisms by which bacteria are either intrinsically resistant or acquire resistance to antibiotics, including the prevention of access to drug targets, changes in the structure and protection of antibiotic targets and the direct modification or inactivation of antibiotics.

Fluoroquinolone resistance: mechanisms, impact on bacteria, and role in evolutionary success

Quinolone and fluoroquinolone antibiotics are potent, broad-spectrum agents commonly used to treat a range of infections. Resistance to these agents is multifactorial and can be via one or a combination of target-site gene mutations, increased production of multidrug-resistance (MDR) efflux pumps, modifying enzymes, and/or target-protection proteins. Fluoroquinolone-resistant clinical isolates of bacteria have emerged readily and recent data have shown that resistance to this class of antibiotics can have diverse, species-dependent impacts on host-strain fitness. Here we outline the impacts of quinolone-resistance mutations in relation to the fitness and evolutionary success of mutant strains.

The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis.

The ability of an isogenic set of mutants of Salmonella enterica serovar Typhimurium L354 (SL1344) with defined deletions in genes encoding components of tripartite efflux pumps, including acrB, acrD, acrF and tolC, to colonize chickens was determined in competition with L354. In addition, the ability of L354 and each mutant to adhere to, and invade, human embryonic intestine cells and mouse monocyte macrophages was determined in vitro. The tolC and acrB knockout mutants were hyper‐susceptible to a range of antibiotics, dyes and detergents; the tolC mutant was also more susceptible to acid pH and bile and grew more slowly than L354. Complementation of either gene ablated the phenotype. The tolC mutant poorly adhered to both cell types in vitro and was unable to invade macrophages. The acrB mutant adhered, but did not invade macrophages. In vivo, both the acrB mutant and the tolC mutant colonized poorly and did not persist in the avian gut, whereas the acrD and acrF mutant colonized and persisted as well as L354. These data indicate that the AcrAB–TolC system is important for the colonization of chickens by S. Typhimurium and that this system has a role in mediating adherence and uptake into target host cells.

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. Methods In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. Conclusion This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies.

Loss of or inhibition of all multidrug resistance efflux pumps of Salmonella enterica serovar Typhimurium results in impaired ability to form a biofilm

OBJECTIVES To investigate the contribution of multidrug efflux pump systems of Salmonella enterica serovar Typhimurium to the formation of a competent biofilm. METHODS Biofilm formation by a wild-type strain and 10 efflux mutant strains was quantified using crystal violet biofilm assays and visualized using scanning electron microscopy. Curli expression was investigated qualitatively and quantitatively by measuring binding of the dye Congo red to polymerized curli and by comparative RT-PCR. RESULTS All efflux mutants of Salmonella Typhimurium were compromised in their ability to form biofilms. Scanning electron microscopy images showed that the mutants were able to adhere to a surface but were unable to form a complex three-dimensional biofilm. Congo red assays demonstrated an inability of the efflux mutants to produce curli, a proteinaceous filament present on the cell surface and an essential component of the Salmonella biofilm extracellular matrix. Mutants expressed significantly less csgB or csgD than wild-type. Chemical inactivation of efflux in wild-type Salmonella Typhimurium with the efflux inhibitors (EIs) phenyl-arginine-β-naphthylamide, carbonyl cyanide m-chlorophenylhydrazone and chlorpromazine also repressed biofilm formation. CONCLUSIONS Our data demonstrates a link between all efflux systems of Salmonella Typhimurium and biofilm formation. Loss of functional efflux gives rise to a lack of curli expression. Biofilm formation was also inhibited by addition of a variety of EIs with differing mechanisms of action, suggesting a novel role for EIs as anti-biofilm compounds.

Absence of Mutations in marRAB or soxRS in acrB-Overexpressing Fluoroquinolone-Resistant Clinical and Veterinary Isolates of Escherichia coli

ABSTRACT The amount of acrB, marA, and soxS mRNA was determined in 36 fluoroquinolone-resistant E. coli from humans and animals, 27 of which displayed a multiple-resistance phenotype. acrB mRNA was elevated in 11 of 36 strains. A mutation at codon 45 (Arg→Cys) in acrR was found in 6 of these 11 strains. Ten of the 36 isolates appeared to overexpresssoxS, and five appeared to overexpress marA. A number of mutations were found in the marR andsoxR repressor genes, correlating with greater amounts ofmarA and soxS mRNA, respectively.

The Global Consequence of Disruption of the AcrAB-TolC Efflux Pump in Salmonella enterica Includes Reduced Expression of SPI-1 and Other Attributes Required To Infect the Host

The mechanisms by which RND pumps contribute to pathogenicity are currently not understood. Using the AcrAB-TolC system as a paradigm multidrug-resistant efflux pump and Salmonella enterica serovar Typhimurium as a model pathogen, we have demonstrated that AcrA, AcrB, and TolC are each required for efficient adhesion to and invasion of epithelial cells and macrophages by Salmonella in vitro. In addition, AcrB and TolC are necessary for Salmonella to colonize poultry. Mutants lacking acrA, acrB, or tolC showed differential expression of major operons and proteins involved in pathogenesis. These included chemotaxis and motility genes, including cheWY and flgLMK and 14 Salmonella pathogenicity island (SPI)-1-encoded type III secretion system genes, including sopE, and associated effector proteins. Reverse transcription-PCR confirmed these data for identical mutants in two other S. Typhimurium backgrounds. Western blotting showed reduced production of SipA, SipB, and SipC. The absence of AcrB or TolC also caused widespread repression of chemotaxis and motility genes in these mutants, and for acrB::aph, this was associated with decreased motility. For mutants lacking a functional acrA or acrB gene, the nap and nir operons were repressed, and both mutants grew poorly in anaerobic conditions. All phenotypes were restored to that of the wild type by trans-complementation with the wild-type allele of the respective inactivated gene. These data explain how mutants lacking a component of AcrAB-TolC are attenuated and that this phenotype is a result of decreased expression of numerous genes encoding proteins involved in pathogenicity. The link between antibiotic resistance and pathogenicity establishes the AcrAB-TolC system as fundamental to the biology of Salmonella.

Triclosan resistance in Salmonella enterica serovar Typhimurium

OBJECTIVES The aim of this study was to characterize the mechanisms of resistance to triclosan in Salmonella enterica serovar Typhimurium. METHODS Mutants resistant to triclosan were selected from nine S. enterica serovar Typhimurium strains. Mutants were characterized by genotyping, mutagenesis and complementation of fabI and analysis of efflux activity. Fitness of triclosan-resistant mutants was determined in vitro and in vivo. RESULTS Three distinct resistance phenotypes were observed: low- (LoT), medium- (MeT) and high-level (HiT) with MICs of 4-8, 16-32 and >32 mg/L of triclosan, respectively, for inhibition. The genotype of fabI did not correlate with triclosan MIC. Artificial overexpression and mutagenesis of fabI in SL1344 each resulted in low-level triclosan resistance, indicating that FabI alone does not mediate high-level triclosan resistance in Salmonella Typhimurium. Active efflux of triclosan via AcrAB-TolC confers intrinsic resistance to triclosan as inactivation of acrB and tolC in wild-type strains and the triclosan-resistant mutants led to large decreases in triclosan resistance, which were reversed by complementation. Exemplars of each phenotype were evaluated for fitness in vivo; no fitness cost was seen and mutants colonized and persisted in chickens throughout a 28 day competitive index experiment. CONCLUSIONS These data show that triclosan resistance can occur via distinct pathways in salmonella and that mutants selected after single exposure to triclosan are fit enough to compete with wild-type strains.

Phenotypic and proteomic characterization of multiply antibiotic-resistant variants of Salmonella enterica serovar Typhimurium selected following exposure to disinfectants.

ABSTRACT In previous work, Salmonella enterica serovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-TolC for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.

A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

OBJECTIVES Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR. METHODS The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format. RESULTS The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins. CONCLUSIONS The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.