Experiences in fosfomycin susceptibility testing and resistance mechanism determination in Escherichia coli from urinary tract infections in the UK

PURPOSE: With an increase in the numbers of bacterial isolates resistant to first-line antibiotics, there has been a revival in the use of older drugs including fosfomycin with novel mechanisms of action. We aimed to investigate the prevalence and genotypic nature of fosfomycin resistance in Escherichia coli from urinary tract infections (UTIs) using the various methods available in the clinical microbiology laboratory. METHODOLOGY: In total, 1000 culture-positive urine samples were assessed for the presence of E. coli and fosfomycin susceptibility was determined using the MAST Uri system, microbroth dilution, agar dilution and E-test strips.Results/Key findings. Initial investigation using breakpoint susceptibility testing on the MAST Uri system identified 62 of 657 (9.5 %) E. coli isolates as fosfomycin-resistant (MIC≥32 µg ml-1). However, on further testing, a lower rate of eight of the 62 (1.3 %) were robustly confirmed to be resistant using microbroth dilution, agar dilution and E-test strips. These true resistant isolates belonged to diverse E. coli multi-locus sequence types and each had a unique set of chromosomal alterations in genes associated with fosfomycin resistance. Fosfomycin-resistant isolates were not multiply drug resistant and did not carry plasmidic fosfomycin resistance genes. Therefore, the use of fosfomycin may be unlikely to drive selection of a particular clone or movement of transferable resistance genes. CONCLUSION: Fosfomycin remains a viable option for the treatment of E. coli in uncomplicated UTIs; different susceptibility testing platforms can give very different results regarding the prevalence of fosfomycin resistance, with false positives being a potential problem that may unnecessarily limit the use of this agent

MarA, RamA, and SoxS as Mediators of the Stress Response:Survival at a Cost

To survive and adapt to changing environments, bacteria have evolved mechanisms to express appropriate genes at appropriate times. Exposure to antimicrobials triggers a global stress response in Enterobacteriaceae, underpinned by activation of a family of transcriptional regulators, including MarA, RamA, and SoxS. These control a program of altered gene expression allowing a rapid and measured response to improve fitness in the presence of toxic drugs. Increased expression of marA, ramA, and soxS up regulates efflux activity to allow detoxification of the cell. However, this also results in trade-offs in other phenotypes, such as impaired growth rates, biofilm formation and virulence. Here, we review the current knowledge regarding the trade-offs that exist between drug survival and other phenotypes that result from induction of marA, ramA, and soxS. Additionally, we present some new findings linking expression of these regulators and biofilm formation in Enterobacteriaceae, thereby demonstrating the interconnected nature of regulatory networks within the cell and explaining how trade-offs can exist between important phenotypes. This has important implications for our understanding of how bacterial virulence and biofilms can be influenced by exposure to antimicrobials

Exposure of Salmonella enterica Serovar Typhimurium to High Level Biocide Challenge Can Select Multidrug Resistant Mutants in a Single Step

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations

Parallel evolutionary pathways to antibiotic resistance selected by biocide exposure

OBJECTIVES: Biocides are widely used to prevent infection. We aimed to determine whether exposure of Salmonella to various biocides could act as a driver of antibiotic resistance. METHODS: Salmonella enterica serovar Typhimurium was exposed to four biocides with differing modes of action. Antibiotic-resistant mutants were selected during exposure to all biocides and characterized phenotypically and genotypically to identify mechanisms of resistance. RESULTS: All biocides tested selected MDR mutants with decreased antibiotic susceptibility; these occurred randomly throughout the experiments. Mutations that resulted in de-repression of the multidrug efflux pump AcrAB-TolC were seen in MDR mutants. A novel mutation in rpoA was also selected and contributed to the MDR phenotype. Other mutants were highly resistant to both quinolone antibiotics and the biocide triclosan. CONCLUSIONS: This study shows that exposure of bacteria to biocides can select for antibiotic-resistant mutants and this is mediated by clinically relevant mechanisms of resistance prevalent in human pathogens

Functional genomics to identify the factors contributing to successful persistence and global spread of an antibiotic resistance plasmid

Background: The spread of bacterial plasmids is an increasing global problem contributing to the widespread dissemination of antibiotic resistance genes including β-lactamases. Our understanding of the details of the biological mechanisms by which these natural plasmids are able to persist in bacterial populations and are able to establish themselves in new hosts via conjugative transfer is very poor. We recently identified and sequenced a globally successful plasmid, pCT, conferring β-lactam resistance. Results: Here, we investigated six plasmid encoded factors (tra and pil loci; rci shufflon recombinase, a putative sigma factor, a putative parB partitioning gene and a pndACB toxin-antitoxin system) hypothesised to contribute to the 'evolutionary success' of plasmid pCT. Using a functional genomics approach, the role of these loci was investigated by systematically inactivating each region and examining the impact on plasmid persistence, conjugation and bacterial host biology. While the tra locus was found to be essential for all pCT conjugative transfer, the second conjugation (pil) locus was found to increase conjugation frequencies in liquid media to particular bacterial host recipients (determined in part by the rci shufflon recombinase). Inactivation of the pCT pndACB system and parB did not reduce the stability of this plasmid. Conclusions: Our findings suggest the success of pCT may be due to a combination of factors including plasmid stability within a range of bacterial hosts, a lack of a fitness burden and efficient transfer rates to new bacterial hosts rather than the presence of a particular gene or phenotype transferred to the host. The methodology used in our study could be applied to other 'successful' globally distributed plasmids to discover the role of currently unknown plasmid backbone genes or to investigate other factors which allow these elements to persist and spread

High level fluoroquinolone resistance in Escherichia coli isolated from animals in Turkey is due to multiple mechanisms

The aim of this study was to determine the molecular mechanisms of fluoroquinolone resistance in E. coli isolated from cattle, goats, sheep, cats, and dogs in Turkey. Twenty nonreplicate E. coli isolates (chosen on the basis of RAPD pattern) from food-producing animals were selected for the study. To identify phenotypic differences between isolates, the sum of the MIC values of 14 antimicrobials was calculated; values ranged from 565 to 2520 mu g/mL, indicating the diversity in antimicrobial resistance present in the panel of isolates. PCR and qRT-PCR were used to characterize the presence and expression levels of known molecular mechanisms of fluoroquinolone resistance. The number of E. coli isolates having single, double, and triple topoisomerase mutations was 2, 10, and 5, respectively. Moreover, the number of qnrA -, qnrS -, oqxB -, and aac(6') Ib-cr-containing E. coli isolates was 1, 4, 1, and 17, respectively. Increased expression of acrB and soxS was detected in 2 and 9 isolates, respectively. The results of this study show a wide range of different mechanisms of fluoroquinolone resistance in E. coli isolates in Turkey.European Cooperation in Science and Technology (COST) - BM070

Self-assembled hydrogels utilizing polymer–nanoparticle interactions

Mouldable hydrogels that flow on applied stress and rapidly self-heal are increasingly utilized as they afford minimally invasive delivery and conformal application. Here we report a new paradigm for the fabrication of self-assembled hydrogels with shear-thinning and self-healing properties employing rationally engineered polymer–nanoparticle (NP) interactions. Biopolymer derivatives are linked together by selective adsorption to NPs. The transient and reversible interactions between biopolymers and NPs enable flow under applied shear stress, followed by rapid self-healing when the stress is relaxed. We develop a physical description of polymer–NP gel formation that is utilized to design biocompatible gels for drug delivery. Owing to the hierarchical structure of the gel, both hydrophilic and hydrophobic drugs can be entrapped and delivered with differential release profiles, both in vitro and in vivo. The work introduces a facile and generalizable class of mouldable hydrogels amenable to a range of biomedical and industrial applications.Wellcome Trust-MIT Postdoctoral FellowshipMisrock Foundation (Cancer Nanotechnology Postdoctoral Fellowship)United States. Dept. of Defense. Congressionally Directed Medical Research Programs (Postdoctoral Fellowship Award W81XWH-13-1-0215)National Institutes of Health (U.S.) (NIH-R01 DE016516

The Escherichia coli MarA protein regulates the ycgZ-ymgABC operon to inhibit biofilm formation

The Escherichia coli marRAB operon is a paradigm for chromosomally encoded antibiotic resistance. The operon exerts its effect via an encoded transcription factor called MarA that modulates efflux pump and porin expression. In this work, we show that MarA is also a regulator of biofilm formation. Control is mediated by binding of MarA to the intergenic region upstream of the ycgZ-ymgABC operon. The operon, known to influence the formation of curli fibres and colanic acid, is usually expressed during periods of starvation. Hence, the ycgZ-ymgABC promoter is recognised by σ38 (RpoS)-associated RNA polymerase (RNAP). Surprisingly, MarA does not influence σ38 -dependent transcription. Instead, MarA drives transcription by the housekeeping σ70 -associated RNAP. The effects of MarA on ycgZ-ymgABC expression are coupled with biofilm formation by the rcsCDB phosphorelay system, with YcgZ, YmgA and YmgB forming a complex that directly interacts with the histidine kinase domain of RcsC

Lemurs in Cacao: Presence and Abundance within the Shade Plantations of Northern Madagascar

© 2019 S. Karger AG, Basel. The recognition that much biodiversity exists outside protected areas is driving research to understand how animals survive in anthropogenic landscapes. In Madagascar, cacao (Theobroma cacao) is grown under a mix of native and exotic shade trees, and this study sought to understand whether lemurs were present in these agroecosystems. Between November 2016 and March 2017, discussions with farmers, nocturnal reconnaissance surveys and camera traps were used to confirm the presence of lemurs in the Cokafa and Mangabe plantations near Ambanja, north-west Madagascar. Four species of lemur were encountered in nocturnal surveys: Mirza zaza, Phaner parienti, Microcebussp. and Cheirogaleussp. with encounter rates of 1.2, 0.4, 0.4 and 0.3 individuals/km, respectively. The presence of Lepilemur dorsalis was confirmed by camera trap. This is the first time lemurs have been studied in cacao plantations, and understanding how these threatened animals use anthropogenic landscapes is vital for their conservation