Publications by authors named "Mark A Webber"

65 Publications

Repeated exposure of nosocomial pathogens to silver does not select for silver resistance but does impact ciprofloxacin susceptibility.

Acta Biomater 2021 Jul 28. Epub 2021 Jul 28.

School of Chemical Engineering, University of Birmingham, Edgbaston B15 2TT, United Kingdom. Electronic address:

The rise of antimicrobial resistant bacteria coupled with a void in antibiotic development marks Antimicrobial Resistance as one of the biggest current threats to modern medicine. Antimicrobial metals are being developed and used as alternative anti-infectives, however, the existence of known resistance mechanisms and limited data regarding bacterial responses to long-term metal exposure are barriers to widespread implementation. In this study, a panel of reference and clinical strains of major nosocomial pathogens were subjected to serial dosage cycles of silver and ciprofloxacin. Populations exposed to silver initially showed no change in sensitivity, however, increasingly susceptibility was observed after the 25th cycle. A control experiment with ciprofloxacin revealed a selection for resistance over time, with silver treated bacteria showing faster adaptation. Morphological analysis revealed filamentation in Gram negative species suggesting membrane perturbation, while sequencing of isolated strains identified mutations in numerous genes. These included those encoding for efflux systems, chemosensory systems, stress responses, biofilm formation and respiratory chain processes, although no consistent locus was identified that correlated with silver sensitivity. These results suggest that de novo silver resistance is hard to select in a range of nosocomial pathogens, although silver exposure may detrimentally impact sensitivity to antibiotics in the long term. STATEMENT OF SIGNIFICANCE: The adaptability of microbial life continuously calls for the development of novel antibiotic molecules, however, the cost and risk associated with their discovery have led to a drying up in the pipeline, causing antimicrobial resistance (AMR) to be a major threat to healthcare. From all available strategies, antimicrobial metals and, more specifically, silver showcase large bactericidal spectrum and limited toxic effect which coupled with a large range of processes available for their delivery made these materials as a clear candidate to tackle AMR. Previous reports have shown the ability of this metal to enact a synergistic effect with other antimicrobial therapies, nevertheless, the discovery of Ag resistance mechanisms since the early 70s and limited knowledge on the long term influence of silver on AMR poses a threat to their applicability. The present study provides quantitative data on the influence of silver based therapies on AMR development for a panel of reference and clinical strains of major nosocomial pathogens, revealing that prolonged silver exposure may detrimentally impact sensitivity to antibiotics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2021.07.056DOI Listing
July 2021

Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management.

Microb Genom 2021 06;7(6)

University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organizations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1565 positive samples (172 per 100 000 population) from 1376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6 % of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. In total, 1035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with six care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients, indicating infection control measures were effective; and found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mgen.0.000589DOI Listing
June 2021

Contribution of Different Mechanisms to Ciprofloxacin Resistance in spp.

Front Microbiol 2021 6;12:663731. Epub 2021 May 6.

Guangdong Key Laboratory of Veterinary Drug Development and Safety Evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.

Development of fluoroquinolone resistance can involve several mechanisms that include chromosomal mutations in genes ( and ) encoding the target bacterial topoisomerase enzymes, increased expression of the AcrAB-TolC efflux system, and acquisition of transmissible quinolone-resistance genes. In this study, 176 isolates from animals with a broad range of ciprofloxacin MICs were collected to analyze the contribution of these different mechanisms to different phenotypes. All isolates were classified according to their ciprofloxacin susceptibility pattern into five groups as follows: highly resistant (HR), resistant (R), intermediate (I), reduced susceptibility (RS), and susceptible (S). We found that the ParC T57S substitution was common in strains exhibiting lowest MICs of ciprofloxacin while increased MICs depended on the type of GyrA mutation. The ParC T57S substitution appeared to incur little cost to bacterial fitness on its own. The presence of PMQR genes represented an route for resistance development in the absence of target-site mutations. Switching of the plasmid-mediated quinolone resistance (PMQR) gene location from a plasmid to the chromosome was observed and resulted in decreased ciprofloxacin susceptibility; this also correlated with increased fitness and a stable resistance phenotype. The overexpression of AcrAB-TolC played an important role in isolates with small decreases in susceptibility and expression was upregulated by MarA more often than by RamA. This study increases our understanding of the relative importance of several resistance mechanisms in the development of fluoroquinolone resistance in from the food chain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2021.663731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137344PMC
May 2021

CoronaHiT: high-throughput sequencing of SARS-CoV-2 genomes.

Genome Med 2021 02 9;13(1):21. Epub 2021 Feb 9.

Quadram Institute Bioscience, Norwich Research Park, Norwich, NR4 7UQ, UK.

We present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-021-00839-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871948PMC
February 2021

Exposure of Salmonella biofilms to antibiotic concentrations rapidly selects resistance with collateral tradeoffs.

NPJ Biofilms Microbiomes 2021 01 11;7(1). Epub 2021 Jan 11.

Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk, NR4 7UQ, UK.

Most bacteria in nature exist in biofilms, which are inherently tolerant to antibiotics. There is currently very limited understanding of how biofilms evolve in response to sub-lethal concentrations of antimicrobials. In this study, we use a biofilm evolution model to study the effects of sub-inhibitory concentrations of three antibiotics on Salmonella Typhimurium biofilms. We show that biofilms rapidly evolve resistance to each antibiotic they are exposed to, demonstrating a strong selective pressure on biofilms from low antibiotic concentrations. While all antibiotics tested select for clinical resistance, there is no common mechanism. Adaptation to antimicrobials, however, has a marked cost for other clinically important phenotypes, including biofilm formation and virulence. Cefotaxime selects mutants with the greatest deficit in biofilm formation followed by azithromycin and then ciprofloxacin. Understanding the impacts of exposure of biofilms to antibiotics will help understand evolutionary trajectories and may help guide how best to use antibiotics in a biofilm context. Experimental evolution in combination with whole-genome sequencing is a powerful tool for the prediction of evolution trajectories associated with antibiotic resistance in biofilms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41522-020-00178-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801651PMC
January 2021

Mobilization of Tn1721-like structure harboring bla between P1-like bacteriophage in Salmonella and plasmids in Escherichia coli in China.

Vet Microbiol 2021 Feb 14;253:108944. Epub 2020 Dec 14.

Guangdong Key Laboratory for Veterinary Drug Development and Safety Evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China. Electronic address:

The aim of this study was to explore the characteristics of bla carriage and mobilization in Salmonella and Escherichia coli isolates from food-producing animals in China. A total of 2280 E. coli and 229 Salmonella isolates collected from food animals from June 2003 to September 2014 were screened for the presence of bla gene. The bla-positive isolates were typed and plasmid DNA sequenced to determine the genetic context of bla and plasmid types present. Bacterial fitness was evaluated by growth curve and plasmid stability in vitro. CTX-M-27-positive E. coli (18, 0.79 %) and Salmonella (34, 14.85 %) were detected. PFGE profiles of CTX-M-27-positive strains revealed a wide variety of genotypes and S. Indiana was the most prevalent serotype. Replicon typing, S1-PFGE and hybridization of CTX-M-27-carrying plasmids confirmed that bla gene was located on IncFII (12/18), IncN (4/18), and non-typeable (2/18) plasmids in E. coli and on P1-like bacteriophage (21/34), IncP (4/34), IncFIB (4/34), IncN (2/34), IncHI2 (2/34), and IncA/C (1/34) plasmids in Salmonella. Comparison and analysis of gene context of bla in P1-like bacteriophage and plasmids revealed they shared the same structure and contained an identical genetic context with the Tn1721-like structure ΔISEcp1B-bla-IS903D-iroN-Δmap-Tn1721. In addition, plasmid stability tests indicated that the bla P1-like bacteriophage were more stable than plasmids in the absence of cefotaxime selective pressure. These results demonstrate that Tn1721-like transposons harboring CTX-M-27 could be mobilized between different plasmids in E. coli and P1-like bacteriophage disseminated among Salmonella.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetmic.2020.108944DOI Listing
February 2021

Donor plasmids for phenotypically neutral chromosomal gene insertions in .

Microbiology (Reading) 2020 12;166(12):1115-1120

Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk, NR4 7UQ, UK.

Recombineering using bacteriophage lambda Red recombinase (λ-Red) uses homologous recombination to manipulate bacterial genomes and is commonly applied to disrupt genes to elucidate their function. This is often followed by the introduction of a wild-type copy of the gene on a plasmid to complement its function. This is often not, however, at a native copy number and the introduction of a chromosomal version of a gene can be a desirable solution to provide wild-type copy expression levels of an allele . Here, we present a simple methodology based on the λ-Red-based 'gene doctoring' technique, where we developed tools used for chromosomal tagging in a conserved locus downstream of and found no impact on a variety of important phenotypes. The tools described provide an easy, quick and inexpensive method of chromosomal modification for the creation of a library of insertion mutants to study gene function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mic.0.000994DOI Listing
December 2020

Bathing babies: current practices in UK neonatal intensive care units.

Arch Dis Child Fetal Neonatal Ed 2021 Mar 10;106(2):227. Epub 2020 Nov 10.

Neonatal Intensive Care Unit, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/archdischild-2020-320355DOI Listing
March 2021

A call for action to the biomaterial community to tackle antimicrobial resistance.

Biomater Sci 2020 Sep;8(18):4951-4974

School of Chemical Engineering, University of Birmingham, Edgbaston B15 2TT, UK.

The global surge of antimicrobial resistance (AMR) is a major concern for public health and proving to be a key challenge in modern disease treatment, requiring action plans at all levels. Microorganisms regularly and rapidly acquire resistance to antibiotic treatments and new drugs are continuously required. However, the inherent cost and risk to develop such molecules has resulted in a drying of the pipeline with very few compounds currently in development. Over the last two decades, efforts have been made to tackle the main sources of AMR. Nevertheless, these require the involvement of large governmental bodies, further increasing the complexity of the problem. As a group with a long innovation history, the biomaterials community is perfectly situated to push forward novel antimicrobial technologies to combat AMR. Although this involvement has been felt, it is necessary to ensure that the field offers a united front with special focus in areas that will facilitate the development and implementation of such systems. This paper reviews state of the art biomaterials strategies striving to limit AMR. Promising broad-spectrum antimicrobials and device modifications are showcased through two case studies for different applications, namely topical and implantables, demonstrating the potential for a highly efficacious physical and chemical approach. Finally, a critical review on barriers and limitations of these methods has been developed to provide a list of short and long-term focus areas in order to ensure the full potential of the biomaterials community is directed to helping tackle the AMR pandemic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0bm01160fDOI Listing
September 2020

A genome-wide analysis of Escherichia coli responses to fosfomycin using TraDIS-Xpress reveals novel roles for phosphonate degradation and phosphate transport systems.

J Antimicrob Chemother 2020 11;75(11):3144-3151

Quadram Institute, Norwich Research Park, Colney Lane, Norwich NR4 7UQ, UK.

Background: Fosfomycin is an antibiotic that has seen a revival in use due to its unique mechanism of action and efficacy against isolates resistant to many other antibiotics. In Escherichia coli, fosfomycin often selects for loss-of-function mutations within the genes encoding the sugar importers, GlpT and UhpT. There has, however, not been a genome-wide analysis of the basis for fosfomycin susceptibility reported to date.

Methods: Here we used TraDIS-Xpress, a high-density transposon mutagenesis approach, to assay the role of all genes in E. coli involved in fosfomycin susceptibility.

Results: The data confirmed known fosfomycin susceptibility mechanisms and identified new ones. The assay was able to identify domains within proteins of importance and revealed essential genes with roles in fosfomycin susceptibility based on expression changes. Novel mechanisms of fosfomycin susceptibility that were identified included those involved in glucose metabolism and phosphonate catabolism (phnC-M), and the phosphate importer, PstSACB. The impact of these genes on fosfomycin susceptibility was validated by measuring the susceptibility of defined inactivation mutants.

Conclusions: This work reveals a wider set of genes that contribute to fosfomycin susceptibility, including core sugar metabolism genes and two systems involved in phosphate uptake and metabolism previously unrecognized as having a role in fosfomycin susceptibility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkaa296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566553PMC
November 2020

Emergence of ciprofloxacin heteroresistance in foodborne Salmonella enterica serovar Agona.

J Antimicrob Chemother 2020 10;75(10):2773-2779

Guangdong Key Laboratory for Veterinary Drug Development and Safety Evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.

Background: Bacterial heteroresistance has been increasingly identified as an important phenomenon for many antibiotic/bacterium combinations.

Objectives: To investigate ciprofloxacin heteroresistance in Salmonella and characterize mechanisms contributing to ciprofloxacin heteroresistance.

Methods: Ciprofloxacin-heteroresistant Salmonella were identified by population analysis profiling (PAP). Target mutations and the presence of PMQR genes were detected using PCR and sequencing. Expression of acrB, acrF and qnrS was conducted by quantitative RT-PCR. Competition ability and virulence were also compared using pyrosequencing, blue/white screening, adhesion and invasion assays and a Galleria model. Two subpopulations were whole-genome sequenced using Oxford Nanopore and Illumina platforms.

Results: PAP identified one Salmonella from food that yielded a subpopulation demonstrating heteroresistance to ciprofloxacin at a low frequency (10-9 to 10-7). WGS and PFGE analyses confirmed that the two subpopulations were isogenic, with six SNPs and two small deletions distinguishing the resistant from the susceptible. Both subpopulations possessed a T57S substitution in ParC and carried qnrS. The resistant subpopulation was distinguished by overexpression of acrB and acrF, a deletion within rsxC and altered expression of soxS. The resistant population had a competitive advantage against the parental population when grown in the presence of bile salts but was attenuated in the adhesion and invasion of human intestinal cells.

Conclusions: We determined that heteroresistance resulted from a combination of mutations in fluoroquinolone target genes and overexpression of efflux pumps associated with a deletion in rsxC. This study warns that ciprofloxacin heteroresistance exists in Salmonella in the food chain and highlights the necessity for careful interpretation of antibiotic susceptibility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkaa288DOI Listing
October 2020

AlbaTraDIS: Comparative analysis of large datasets from parallel transposon mutagenesis experiments.

PLoS Comput Biol 2020 07 17;16(7):e1007980. Epub 2020 Jul 17.

Quadram Institute Bioscience, Norwich Research Park, Colney Lane, Norwich, United Kingdom.

Bacteria need to survive in a wide range of environments. Currently, there is an incomplete understanding of the genetic basis for mechanisms underpinning survival in stressful conditions, such as the presence of anti-microbials. Transposon directed insertion-site sequencing (TraDIS) is a powerful tool to identify genes and networks which are involved in survival and fitness under a given condition by simultaneously assaying the fitness of millions of mutants, thereby relating genotype to phenotype and contributing to an understanding of bacterial cell biology. A recent refinement of this approach allows the roles of essential genes in conditional stress survival to be inferred by altering their expression. These advancements combined with the rapidly falling costs of sequencing now allows comparisons between multiple experiments to identify commonalities in stress responses to different conditions. This capacity however poses a new challenge for analysis of multiple data sets in conjunction. To address this analysis need, we have developed 'AlbaTraDIS'; a software application for rapid large-scale comparative analysis of TraDIS experiments that predicts the impact of transposon insertions on nearby genes. AlbaTraDIS can identify genes which are up or down regulated, or inactivated, between multiple conditions, producing a filtered list of genes for further experimental validation as well as several accompanying data visualisations. We demonstrate the utility of our new approach by applying it to identify genes used by Escherichia coli to survive in a wide range of different concentrations of the biocide Triclosan. AlbaTraDIS identified all well characterised Triclosan resistance genes, including the primary target, fabI. A number of new loci were also implicated in Triclosan resistance and the predicted phenotypes for a selection of these were validated experimentally with results being consistent with predictions. AlbaTraDIS provides a simple and rapid method to analyse multiple transposon mutagenesis data sets allowing this technology to be used at large scale. To our knowledge this is the only tool currently available that can perform these tasks. AlbaTraDIS is written in Python 3 and is available under the open source licence GNU GPL 3 from https://github.com/quadram-institute-bioscience/albatradis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pcbi.1007980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390408PMC
July 2020

Contribution of NDM and OXA-type carbapenemases to carbapenem resistance in clinical from Nigeria.

Infect Dis (Lond) 2020 09 9;52(9):644-650. Epub 2020 Jun 9.

The Quadram Institute, Norwich Research Park, Colney, Norwich, UK.

infections are rarely diagnosed in many hospitals in Nigeria due to a lack of capacity for the identification of the organism in spite of the clinical significance of this opportunistic nosocomial organism. We assembled a panel of presumptive isolates of from tertiary hospitals in Nigeria and analysed mechanisms of resistance phenotypically and by whole genome sequencing. Twenty-one clinical isolates of identified using standard microbiological tests were tested for susceptibility to a panel of antibiotics by the agar dilution method, and production of ESBLs using phenotypic tests. Whole genome sequencing and comparative genomic analysis were used to determine the antimicrobial resistance genes, strain types, phylogenetic relationships and genetic context of resistance genes. The MIC and MIC of most antibiotics were very high with no difference between MIC and MIC values apart for amikacin, meropenem and colistin where MIC and MIC ranged between 1-4 µg/ml and 64->64 µg/ml, respectively. Multiple resistance genes were detected in most of the isolates including , various family alleles and . Interestingly, carriage did not always result in phenotypic carbapenem resistance. Whole genome alignments typing showed strains belonged to three major clades. Strains within these clades had different resistance genes and resistance patterns. This report shows a high level of resistance to important antibiotics and carbapenem resistance in in Nigeria. We hope this work will serve as a reference for future study in the sub-Saharan region of Africa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/23744235.2020.1775881DOI Listing
September 2020

A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility.

J Antimicrob Chemother 2020 09;75(9):2516-2525

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK.

Objectives: A whole-genome screen at sub-gene resolution was performed to identify candidate loci that contribute to enhanced or diminished ciprofloxacin susceptibility in Salmonella enterica serovar Typhi.

Methods: A pool of over 1 million transposon insertion mutants of an S. Typhi Ty2 derivative were grown in a sub-MIC concentration of ciprofloxacin, or without ciprofloxacin. Transposon-directed insertion site sequencing (TraDIS) identified relative differences between the mutants that grew following the ciprofloxacin treatment compared with the untreated mutant pool, thereby indicating which mutations contribute to gain or loss of ciprofloxacin susceptibility.

Results: Approximately 88% of the S. Typhi strain's 4895 annotated genes were assayed, and at least 116 were identified as contributing to gain or loss of ciprofloxacin susceptibility. Many of the identified genes are known to influence susceptibility to ciprofloxacin, thereby providing method validation. Genes were identified that were not known previously to be involved in susceptibility, and some of these had no previously known phenotype. Susceptibility to ciprofloxacin was enhanced by insertion mutations in genes coding for efflux, other surface-associated functions, DNA repair and expression regulation, including phoP, barA and marA. Insertion mutations that diminished susceptibility were predominantly in genes coding for surface polysaccharide biosynthesis and regulatory genes, including slyA, emrR, envZ and cpxR.

Conclusions: A genomics approach has identified novel contributors to gain or loss of ciprofloxacin susceptibility in S. Typhi, expanding our understanding of the impact of fluoroquinolones on bacteria and of mechanisms that may contribute to resistance. The data also demonstrate the power of the TraDIS technology for antibacterial research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkaa204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443733PMC
September 2020

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

Front Microbiol 2020 5;11:828. Epub 2020 May 5.

Quadram Institute Biosciences, Norwich, United Kingdom.

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 , , and 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 , , and . 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.00828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216687PMC
May 2020

Transmission of plasmid-borne and chromosomal blaCTX-M-64 among Escherichia coli and Salmonella isolates from food-producing animals via ISEcp1-mediated transposition.

J Antimicrob Chemother 2020 06;75(6):1424-1427

National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.

Objectives: To clarify the transmission mechanism of the blaCTX-M-64 gene between Escherichia coli and Salmonella isolates from food animals.

Methods: A total of 329 E. coli and 60 Salmonella isolates collected from food animals in 2016 were screened for the presence of blaCTX-M-64 genes. The blaCTX-M-64-positive isolates were typed and plasmid and chromosome DNA was sequenced to determine the genetic context of blaCTX-M-64 and the plasmid types present.

Results: The blaCTX-M-64 gene was identified in only three E. coli isolates but was the predominant gene in the Salmonella isolates (n = 9). These 12 CTX-M-64-positive isolates were all resistant to ampicillin, cefotaxime, ceftiofur, ceftriaxone, ceftazidime and florfenicol and 9 were resistant to ciprofloxacin. The blaCTX-M-64 gene was located on transferable IncI2 plasmids and an IncHI2 plasmid in three E. coli and one Salmonella isolate, respectively. The remaining eight Salmonella isolates contained blaCTX-M-64 integrated into the chromosome. Different genetic contexts of blaCTX-M-64 genes were found among the 12 isolates: ISEcp1-blaCTX-M-64-orf477-A/C on IncI2 plasmids of 3 E. coli isolates; ΔISEcp1-blaCTX-M-64-orf477-A/C in the chromosome of 1 Salmonella isolate; and ISEcp1-blaCTX-M-64-orf477 on the IncHI2 plasmid and chromosome of 8 Salmonella isolates.

Conclusions: To the best of our knowledge, this is the first report of chromosomally encoded CTX-M-64 in Salmonella isolates. ISEcp1-mediated transposition is likely to be responsible for the spread of blaCTX-M-64 between different plasmids and chromosomes in Enterobacteriaceae especially E. coli and Salmonella.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkaa044DOI Listing
June 2020

TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance.

Genome Res 2020 02 12;30(2):239-249. Epub 2020 Feb 12.

Quadram Institute Bioscience, Norwich Research Park, Norwich, NR4 7UQ, United Kingdom.

Understanding the genetic basis for a phenotype is a central goal in biological research. Much has been learnt about bacterial genomes by creating large mutant libraries and looking for conditionally important genes. However, current genome-wide methods are largely unable to assay essential genes which are not amenable to disruption. To overcome this limitation, we developed a new version of "TraDIS" (transposon directed insertion-site sequencing) that we term "TraDIS-Xpress" that combines an inducible promoter into the transposon cassette. This allows controlled overexpression and repression of all genes owing to saturation of inserts adjacent to all open reading frames as well as conventional inactivation. We applied TraDIS-Xpress to identify responses to the biocide triclosan across a range of concentrations. Triclosan is endemic in modern life, but there is uncertainty about its mode of action with a concentration-dependent switch from bacteriostatic to bactericidal action unexplained. Our results show a concentration-dependent response to triclosan with different genes important in survival between static and cidal exposures. These genes include those previously reported to have a role in triclosan resistance as well as a new set of genes, including essential genes. Novel genes identified as being sensitive to triclosan exposure include those involved in barrier function, small molecule uptake, and integrity of transcription and translation. We anticipate the approach we show here, by allowing comparisons across multiple experimental conditions of TraDIS data, and including essential genes, will be a starting point for future work examining how different drug conditions impact bacterial survival mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.254391.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050523PMC
February 2020

Potentiation of curing by a broad-host-range self-transmissible vector for displacing resistance plasmids to tackle AMR.

PLoS One 2020 15;15(1):e0225202. Epub 2020 Jan 15.

Institute of Microbiology & Infection and School of Biosciences, University of Birmingham, Edgbaston, Birmingham, England, United Kingdom.

Plasmids are potent vehicles for spread of antibiotic resistance genes in bacterial populations and often persist in the absence of selection due to efficient maintenance mechanisms. We previously constructed non-conjugative high copy number plasmid vectors that efficiently displace stable plasmids from enteric bacteria in a laboratory context by blocking their replication and neutralising their addiction systems. Here we assess a low copy number broad-host-range self-transmissible IncP-1 plasmid as a vector for such curing cassettes to displace IncF and IncK plasmids. The wild type plasmid carrying the curing cassette displaces target plasmids poorly but derivatives with deletions near the IncP-1 replication origin that elevate copy number about two-fold are efficient. Verification of this in mini IncP-1 plasmids showed that elevated copy number was not sufficient and that the parB gene, korB, that is central to its partitioning and gene control system, also needs to be included. The resulting vector can displace target plasmids from a laboratory population without selection and demonstrated activity in a mouse model although spread is less efficient and requires additional selection pressure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225202PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961859PMC
March 2020

Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance.

PLoS Pathog 2019 12 26;15(12):e1008101. Epub 2019 Dec 26.

Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a predictive model of pump-contacts that could be exploited in the future for the development of targeted therapeutics and efflux pump inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975555PMC
December 2019

Opening Pandora's box: High-level resistance to antibiotics of last resort in Gram-negative bacteria from Nigeria.

J Glob Antimicrob Resist 2020 06 22;21:211-217. Epub 2019 Oct 22.

The Quadram Institute, Norwich Research Park, Colney, Norwich NR4 7UQ, UK; Norwich Medical School, Norwich Research Park, Colney, Norwich NR4 7UA, UK.

Objectives: The aim of this study was to determine the percentage of antimicrobial-resistant isolates and the associated resistance mechanisms in Gram-negative bacteria from South Western Nigeria.

Methods: A total of 306 non-duplicate unbiased Gram-negative isolates were recovered from patients admitted to three teaching hospitals in South Western Nigeria in 2011 and 2013. Isolates were from clinical samples as well as from stool samples of inpatients without infection to assess antimicrobial resistance patterns in carriage isolates. Antimicrobial susceptibility testing was performed, and PCR and sequencing were used to identify genes encoding various known β-lactamases. Based on phenotypic and genotypic results, 10 isolates representing the diversity of phenotypes present were selected for whole-genome sequencing (WGS).

Results: Antimicrobial susceptibility testing revealed the following resistance rates: fluoroquinolones, 78.1%; third-generation cephalosporins, 92.2%; and carbapenems, 52.6%. More resistant isolates were isolated from stools of uninfected patients compared with clinical infection specimens. Klebsiella (10%) and Escherichia coli (7%) isolates produced a carbapenemase. WGS of selected isolates identified the presence of globally disseminated clones.

Conclusion: This study illustrates a crisis for the use of first-line antimicrobial therapy in Nigerian patients. It is likely that Nigeria is playing a significant role in the spread of antimicrobial resistance owing to its large population with considerable global mobility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jgar.2019.10.016DOI Listing
June 2020

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

Mol Microbiol 2019 11 29;112(5):1609-1625. Epub 2019 Sep 29.

School of Biosciences, Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

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 σ (RpoS)-associated RNA polymerase (RNAP). Surprisingly, MarA does not influence σ -dependent transcription. Instead, MarA drives transcription by the housekeeping σ -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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mmi.14386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900184PMC
November 2019

The Emergence of Chromosomally Located in From Foodborne Animals in China.

Front Microbiol 2019 5;10:1268. Epub 2019 Jun 5.

National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.

The emergence and increase in prevalence of resistance to cephalosporins amongst isolates of from food animals imposes a public health threat. The aim of the present study was to investigate the prevalence and characteristics of CTX-M-producing isolates from raw meat and food animals. 27 of 152 (17.76%) isolates were ESBL-positive including 21/70 (30%) from food animals and 6/82 (7.32%) from raw meat. CTX-M-55 was the most prevalent ESBL type observed (12/27, 44.44%). 7 of 12 CTX-M-55-positive isolates were Indiana, 2 were Typhimurium, 2 were Chester, and the remaining isolate was not typeable. Eight CTX-M-55-positive isolates were highly resistant to fluoroquinolones (MIC = 64 ug/mL) and co-harbored and . Most of the CTX-M-55 positive isolates (11/12) carried genes on the chromosome, with the remaining isolate carrying this gene on a transferable 280 kb IncHI2 plasmid. A chromosomal gene from one isolate transferred onto a 250 kb IncHI2 plasmid which was subsequently conjugated into recipient strain J53. PFGE and MLST profiles showed a wide range of strain types were carrying . Our study demonstrates the emergence and prevalence of foodborne harboring a chromosomally located in China. The co-existence of PMQR genes with in isolates suggests co-selection and dissemination of resistance to both fluoroquinolones and cephalosporins in via the food chain in China represents a public health concern.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2019.01268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560199PMC
June 2019

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

J Med Microbiol 2019 Feb 13;68(2):161-168. Epub 2018 Dec 13.

2​Quadram Institute, Norwich Research Park, Colney Lane, Norwich, NR4 7UA, 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). 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 transferrable 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jmm.0.000901DOI Listing
February 2019

Clinically Relevant Plasmid-Host Interactions Indicate that Transcriptional and Not Genomic Modifications Ameliorate Fitness Costs of Carbapenemase-Carrying Plasmids.

mBio 2018 04 24;9(2). Epub 2018 Apr 24.

Antimicrobials Research Group, Institute of Microbiology & Infection, College of Medical & Dental Sciences, University of Birmingham, Birmingham, United Kingdom

The rapid dissemination of antimicrobial resistance (AMR) around the globe is largely due to mobile genetic elements, such as plasmids. They confer resistance to critically important drugs, including extended-spectrum beta-lactams, carbapenems, and colistin. Large, complex resistance plasmids have evolved alongside their host bacteria. However, much of the research on plasmid-host evolution has focused on small, simple laboratory plasmids in laboratory-adapted bacterial hosts. These and other studies have documented mutations in both host and plasmid genes which occur after plasmid introduction to ameliorate fitness costs of plasmid carriage. We describe here the impact of two naturally occurring variants of a large AMR plasmid (pKpQIL) on a globally successful pathogen. In our study, after pKpQIL plasmid introduction, no changes in coding domain sequences were observed in their natural host, However, significant changes in chromosomal and plasmid gene expression may have allowed the bacterium to adapt to the acquisition of the AMR plasmid. We hypothesize that this was sufficient to ameliorate the associated fitness costs of plasmid carriage, as pKpQIL plasmids were maintained without selection pressure. The dogma that removal of selection pressure (e.g., antimicrobial exposure) results in plasmid loss due to bacterial fitness costs is not true for all plasmid/host combinations. We also show that pKpQIL impacted the ability of to form a biofilm, an important aspect of virulence. This study used highly relevant models to study the interaction between AMR plasmids and pathogens and revealed striking differences from results of studies done on laboratory-adapted plasmids and strains. Antimicrobial resistance is a serious problem facing society. Many of the genes that confer resistance can be shared between bacteria through mobile genetic elements, such as plasmids. Our work shows that when two clinically relevant AMR plasmids enter their natural host bacteria, there are changes in gene expression, rather than changes to gene coding sequences. These changes in gene expression ameliorate the potential fitness costs of carriage of these AMR plasmids. In line with this, the plasmids were stable within their natural host and were not lost in the absence of selective pressure. We also show that better understanding of the impact of resistance plasmids on fundamental pathogen biology, including biofilm formation, is crucial for fighting drug-resistant infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.02303-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915730PMC
April 2018

Catheter sepsis and antisepsis: matters of life, death, obscurity and resistance.

Arch Dis Child Fetal Neonatal Ed 2018 Mar 25;103(2):F94-F96. Epub 2018 Jan 25.

Norwich Medical School, University of East Anglia, Norwich, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/archdischild-2017-313150DOI Listing
March 2018

Quinolone-resistant gyrase mutants demonstrate decreased susceptibility to triclosan.

J Antimicrob Chemother 2017 10;72(10):2755-2763

Institute of Microbiology & Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham B152TT, UK.

Objectives: Cross-resistance between antibiotics and biocides is a potentially important driver of MDR. A relationship between susceptibility of Salmonella to quinolones and triclosan has been observed. This study aimed to: (i) investigate the mechanism underpinning this; (ii) determine whether the phenotype is conserved in Escherichia coli; and (iii) evaluate the potential for triclosan to select for quinolone resistance.

Methods: WT E. coli, Salmonella enterica serovar Typhimurium and gyrA mutants were used. These were characterized by determining antimicrobial susceptibility, DNA gyrase activity and sensitivity to inhibition. Expression of stress response pathways (SOS, RpoS, RpoN and RpoH) was measured, as was the fitness of mutants. The potential for triclosan to select for quinolone resistance was determined.

Results: All gyrase mutants showed increased triclosan MICs and altered supercoiling activity. There was no evidence for direct interaction between triclosan and gyrase. Identical substitutions in GyrA had different impacts on supercoiling in the two species. For both, there was a correlation between altered supercoiling and expression of stress responses. This was more marked in E. coli, where an Asp87Gly GyrA mutant demonstrated greatly increased fitness in the presence of triclosan. Exposure of parental strains to low concentrations of triclosan did not select for quinolone resistance.

Conclusions: Our data suggest gyrA mutants are less susceptible to triclosan due to up-regulation of stress responses. The impact of gyrA mutation differs between E. coli and Salmonella. The impacts of gyrA mutation beyond quinolone resistance have implications for the fitness and selection of gyrA mutants in the presence of non-quinolone antimicrobials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkx201DOI Listing
October 2017

Surface Finish has a Critical Influence on Biofilm Formation and Mammalian Cell Attachment to Additively Manufactured Prosthetics.

ACS Biomater Sci Eng 2017 Aug 28;3(8):1616-1626. Epub 2017 Jun 28.

Institute of Food Research, Norwich Research Park, Norwich NR4 7UG, United Kingdom.

Additive manufacturing (AM) technologies enable greater geometrical design freedom compared with subtractive processes. This flexibility has been used to manufacture patient-matched implants. Although the advantages of AM are clear, the optimization at each process stage is often understated. Here we demonstrate that surface finishing of selective laser melted (SLM) implants significantly alters topography, which has implications for cellular and biofilm adhesion. Hot isostatic pressing of as-fabricated Ti-6Al-4V implants was shown to reduce porosity (1.04 to 0.02%) and surface roughness (34 ± 8 to 22 ± 3 μm). Despite these surface changes, preosteoblasts exhibited a similar viability and proliferation after 7 days of culture. Contrastingly, sandblasting and polishing significantly reduced cellular activity and increased cytotoxicity. Bacterial specimens (S and ) adhered more homogeneously to sandblasted implants compared with other treatments. This suggests that sandblasting may place the implant at risk of infection and reduce the strength of interaction with the surrounding soft tissues. The ability to tune the adhesion of cells to additively manufactured Ti-6Al-4V implants using postprocessing methods was demonstrated. Because the degree of tissue integration required of implants is application specific, these methods may be useful to tailor osseointegration. However, surface competition between mammalian and bacterial cells remains a challenge.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsbiomaterials.7b00336DOI Listing
August 2017

Novel approaches to the treatment of bacterial biofilm infections.

Br J Pharmacol 2017 Jul 2;174(14):2237-2246. Epub 2017 Feb 2.

Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK.

Bacterial infection remains a major challenge to healthcare and is responsible for significant morbidity and mortality. This situation is becoming complicated by an increasingly ageing and susceptible population and large numbers of bacterial isolates, which have developed resistance to antibiotics. Bacteria that form biofilms and colonize or infect medical devices or wounds are particularly hard to treat as biofilms are inherently highly antibiotic resistant. Most infections have a component where bacteria exist as a biofilm and as a result, prevention or treatment of biofilm-associated infections is highly important. A number of novel strategies to kill biofilms have been in development; these include the use of weak organic acids, photo irradiation and the application of bacteriophage. All have promise and are able to effectively kill biofilms in model systems, but for each there are still unanswered questions. This review summarizes the main features of biofilm infections, each of these novel approaches and the evidence that is still lacking before these potential treatments can be incorporated into clinical usage.

Linked Articles: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bph.13706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481657PMC
July 2017

Response to Letter to Editor: 'Limitations of In Vitro Antimicrobial Dressings Study'.

Authors:
Mark A Webber

Burns 2016 08 6;42(5):1148. Epub 2016 Jun 6.

Institute for Microbiology and Infection University of Birmingham, Birmingham, B15 2TT, U.K. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.burns.2016.02.015DOI Listing
August 2016
-->