A novel hemA mutation is responsible for a small-colony-variant phenotype in Escherichia coli

We identified a small colony variant (SCV) of an amoxicillin/clavulanic acid-resistant derivative of a clinical isolate of Escherichia coli from Malawi, which was selected for in vitro in a subinhibitory concentration of gentamicin. The SCV was auxotrophic for hemin and had impaired biofilm formation compared to the ancestral isolates. A single novel nucleotide polymorphism (SNP) in hemA, which encodes a glutamyl-tRNA reductase that catalyses the initial step of porphyrin biosynthesis leading to the production of haem, was responsible for the SCV phenotype. We showed the SNP in hemA resulted in a significant fitness cost to the isolate, which persisted even in the presence of hemin. However, the phenotype quickly reverted during sequential sub-culturing in liquid growth media. As hemA is not found in mammalian cells, and disruption of the gene results in a significant fitness cost, it represents a potential target for novel drug development specifically for the treatment of catheter-associated urinary tract infections caused by E. coli

Antibiotic Resistance Is Associated with Integrative and Conjugative Elements and Genomic Islands in Naturally Circulating Streptococcus pneumoniae Isolates from Adults in Liverpool, UK

Pneumonia is the sixth largest cause of death in the UK. It is usually caused by Streptococcus pneumoniae, which healthy individuals can carry in their nose without symptoms of disease. Antimicrobial resistance further increases mortality and morbidity associated with pneumococcal infection, although few studies have analysed resistance in naturally circulating pneumococcal isolates in adult populations. Here, we report on the resistome and associated mobile genetic elements within circulating pneumococcus isolated from adult volunteers enrolled in the experimental human pneumococcal colonisation (EHPC) research program at the Liverpool School of Tropical Medicine, UK. Pneumococcal isolates collected from 30 healthy asymptomatic adults who had volunteered to take part in clinical research were screened for antibiotic susceptibility to erythromycin and tetracycline, and whole-genome sequenced. The genetic context of resistance to one or both antibiotics in four isolates was characterised bioinformatically, and any association of the resistance genes with mobile genetic elements was determined. Tetracycline and macrolide resistance genes [tet(M), erm(B), mef(A), msr(D)] were detected on known Tn916-like integrative and conjugative elements, namely Tn6002 and Tn2010, and tet(32) was found for the first time in S. pneumoniae located on a novel 50 kb genomic island. The widespread use of pneumococcal conjugate vaccines impacts on serotype prevalence and transmission within the community. It is therefore important to continue to monitor antimicrobial resistance (AMR) genes present in both vaccine types and non-vaccine types in response to contemporary antimicrobial therapies and characterise the genetic context of acquired resistance genes to continually optimise antibiotic therapies

The molecular basis of antibiotic treatment failure in chronic urinary tract infections

Urinary tract infections (UTIs) are amongst the most common infections worldwide, and are becoming increasingly difficult to treat. In addition to the acceleration of classic antimicrobial resistance, recurrence after initial resolution is common. Our clinical experience is that chronically infected patients sometimes fail to respond to antibiotics predicted to be effective from culture-based sensitivity testing, while antibiotics predicted to be unsuitable can succeed. We hypothesized that the bladder environment could lead to differential bacterial gene expression, resulting in differences in minimum inhibitory concentration (MICs) compared with standard culture. Here, using strains of Escherichia coli evolved in the lab to be resistant to amoxicillin–clavulanic acid, we present data that MICs differ depending on which media the assay is performed in (M9, ISO, LB, human urine), as well as in urine-containing supernatant enriched from urothelial organoids. Next, we examined the behaviour of patient-derived Enterococcus faecalis, one of the main causative agents of chronic UTIs in the elderly. We are in the process of evaluating the MIC of first-line UTI antibiotics using growth media supplemented with urine, to more closely mimic the native uropathogen environment. Moreover, we are characterising the resistance genes expressed in those differing environments using next generation sequencing technology and comparing the results with those obtained from bacteria grown on standard diagnostic media. Our work demonstrates the danger of extrapolating biological behaviour from artificial culture substrates and may lead to better diagnostic tests and treatments for chronic UTI

The pH-dependence of lipid-mediated antimicrobial peptide resistance in a model Staphylococcal plasma membrane: a two-for-one mechanism of epithelial defence circumvention.

The mechanisms of membrane defence by lysylphosphatidylglycerol (LPG), were investigated using synthetic biomimetic mono- and bilayer models of methicillin resistant S. aureus ST239 TW, based on its lipid composition in both pH 7.4 (28% LPG) and pH 5.5 (51% LPG) cultures. These models incorporated a stable synthetic analogue of LPG (3adLPG) to facilitate long-duration biophysical studies, which were previously limited by the lability native LPG. Both increased 3adLPG content and full headgroup ionization at pH 5.5, increased bilayer order and dampened overall charge, via the formation of neutral ion pairs with anionic lipids. Ion pair formation in air/liquid interface lipid monolayers elicited a significant condensing effect, which correlated with the inhibition of subphase-injected magainin 2 F5W partitioning. In fluid phase lipid vesicles, increasing the proportion of 3adLPG from 28 to 51 mol% completely inhibited the adoption of the membrane-active ?-helical conformation of the peptide, without the need for full headgroup ionization. Neutron reflectivity measurements performed on biomimetic PG/3adLPG fluid floating bilayers, showed a significant ordering effect of mild acidity on a bilayer containing 30 mol% 3adLPG, whilst peptide binding/partitioning was only fully inhibited in a bilayer with 55 mol% 3adLPG at pH 5.5. These findings are discussed with respect to the roles of LPG in resistance to human epithelial defences in S. aureus and the continued evolution of this opportunistic pathogen’s virulence

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

Shotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~ 200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high- quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open- source software pipeline, ‘ResPipe’

First identification of blaNDM-5 producing Escherichia coli from neonates and HIV infected adult in Tanzania.

Introduction: Carbapenem-resistant Enterobacteriaceae are emerging as a global public-health threat and cause substantial challenges in clinical practice. Gap Statement: There is a need for increased and continued genomic surveillance of antimicrobial resistance genes globally in order to detect outbreaks and dissemination of clinically important resistance genes and their associated mobile genetic elements in human pathogens. Aim: To describe the resistance mechanisms of carbapenem-resistant Escherichia coli. Methods: Rectal swabs from neonates and newly diagnosed human immunodeficiency virus (HIV) infected adults were collected between April 2017 and May 2018 and screened for fecal carriage of carbapenamases and OXA-48 producing Enterobacteriaceae. Bacterial isolates were identified using matrix assisted laser desorption ionization time of flight mass spectrometry. Antimicrobial susceptibility testing was performed by E-test. Whole genomes of carbapenem resistant E. coli were investigated using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads. Results: Three carbapenem-resistant E. coli were detected, two from neonates and one from an HIV infected adult. All three isolates carried blaNDM-5. Two E. coli from neonates belong to ST167 and blaNDM-5 co-existed with blaCTX-M-15 and blaOXA-01, and all were carried on IncFIA type plasmids. The E. coli from the HIV infected adult belongs to ST2083, and carried blaNDM-5 on an IncX3 type plasmid and blaCMY-42 on an IncI type plasmid. All blaNDM-5 carrying plasmids contained conjugation related genes. In addition, E. coli from the HIV infected adult carried three more plasmid types; IncFIA, IncFIB and Col(BS512). One E. coli from a neonate also carried one extra plasmid Col(BS512). All three E. coli harbored resistance genes to fluoroquinolone, aminoglycosides, sulfamethoxazole, trimethoprim, macrolides and tetracycline, carried on the IncFIA type plasmid. Furthermore, E. coli from the neonates carried a chloramphenicol resistance gene (catB3) also on the IncFIA plasmid. All three isolates were susceptible to colistin. Conclusion: This is the first report from Tanzania detecting blaNDM-5 producing E. coli. The carbapenemase gene was carried on an IncFIA and IncX3 type plasmids. Our findings highlight the urgent need for a robust antimicrobial resistance (AMR) surveillance system to monitor and rapidly report on the incidence and spread of emerging resistant bacteria in Tanzania

Evaluation of the Antimicrobial Activity of Cationic Polymers against Mycobacteria: Toward Antitubercular Macromolecules.

Antimicrobial resistance is a global healthcare problem with a dwindling arsenal of usable drugs. Tuberculosis, caused by Mycobacterium tuberculosis, requires long-term combination therapy and multi- and totally drug resistant strains have emerged. This study reports the antibacterial activity of cationic polymers against mycobacteria, which are distinguished from other Gram-positive bacteria by their unique cell wall comprising a covalently linked mycolic acid-arabinogalactan-peptidoglycan complex (mAGP), interspersed with additional complex lipids which helps them persist in their host. The present study finds that poly(dimethylaminoethyl methacrylate) has particularly potent antimycobacterial activity and high selectivity over two Gram-negative strains. Removal of the backbone methyl group (poly(dimethylaminoethyl acrylate)) decreased antimycobacterial activity, and poly(aminoethyl methacrylate) also had no activity against mycobacteria. Hemolysis assays revealed poly(dimethylaminoethyl methacrylate) did not disrupt red blood cell membranes. Interestingly, poly(dimethylaminoethyl methacrylate) was not found to permeabilize mycobacterial membranes, as judged by dye exclusion assays, suggesting the mode of action is not simple membrane disruption, supported by electron microscopy analysis. These results demonstrate that synthetic polycations, with the correctly tuned structure are useful tools against mycobacterial infections, for which new drugs are urgently required

Molecular characterisation of the first New Delhi metallo-β-lactamase 1-producing Acinetobacter baumannii from Tanzania

Background We aimed to characterise the genetic determinants and context of two meropenem-resistant clinical isolates of Acinetobacter baumannii isolated from children hospitalised with bloodstream infections in Dar es Salaam, Tanzania. Methods Antimicrobial susceptibility was determined by disc diffusion E-test and broth microdilution. Genomes were completed using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads and characterisation of the genetic context of resistance genes, multi-locus sequence types (ST) and phylogenetic analysis were determined bioinformatically. Results Twelve Acinetobacter baumannii were isolated from 2226 blood cultures and two of which were meropenem resistant. The two meropenem resistant isolates, belonging to distinct STs; ST374 and ST239, were found to harbour blaNDM-1, which was chromosomally located in isolate DT0544 and plasmid located in isolate DT01139. The genetic environment of blaNDM- 1 shows the association of insertion sequence ISAba125 with blaNDM-1 in both isolates. Both isolates also harboured genes conferring resistance to other β-lactams, aminoglycosides and cotrimoxazole. Conclusions This is the first report of NDM-1 producing isolates of A. baumannii from Tanzania. The genetic context of the blaNDM-1 provides further evidence of the importance of ISAba125 in 44 the spread of blaNDM-1 in A. baumannii. Local surveillance should be strengthened to keep clinicians updated on the incidence of these and other multidrug-resistant and difficult-to- treat bacteria. Accession numbers: The chromosomal and plasmid sequences of DT0544 and DT01139 were submitted to GenBank with accession numbers PRJNA679703 and PRJNA679704, respectively

Piperacillin/tazobactam resistance in a clinical isolate of Escherichia colidue to IS26-mediated amplification of blaTEM-1B

We identified a clinical isolate of Escherichia coli displaying an unusual, emerging phenotype; piperacillin/tazobactam (TZP)-resistant, 3rd generation cephalosporin-susceptible. Prior to treatment with TZP, a TZP-susceptible E. coli isolate was isolated from the same patient. Hyperproduction of a class A β-lactamase has previously been linked to this phenotype, but the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood. Clonality of the two isolates was initially assessed with RFLP, and β-lactamase activity was determined using a nitrocefin assay. Both isolates were sequenced on Illumina and Oxford Nanopore Technology platforms and fitness assessed competitively. A plasmid construct containing the insertion sequence IS26was used to capture a translocatable unit (TU) in vitro. The two E. coli clinical isolates were confirmed to be clonal, with the TZP-resistant isolate hyperproducing blaTEM-1. However, no promoter region mutations were identified in the TZP-resistant isolate. Hybrid assembly revealed that an ~11kb segment of DNA was excised from a IS26 flanked pseudo-compound transposon in the TZP-resistant isolate, forming a circular TU containing blaTEM-1. Multiple re-insertion events of the TU, mediated by IS26, led to tandem repeats of the TU within the chromosome, increasing the copy number of blaTEM-1. Excision and insertion events were confirmed via capture of the TU. Amplification of the TU in the TZP-resistant isolate incurred no significant change in fitness in different environmental conditions. This study improves the understanding of the TZP-resistant, 3rd generation cephalosporin-susceptible phenotype in E. coli and antimicrobial resistance prediction of this phenotype from genotypic data

A genomic epidemiological study shows prevalence of antimicrobial resistance in Enterobacterales is associated with the livestock host, as well as antimicrobial usage

Enterobacterales from livestock are potentially important reservoirs for antimicrobial resistance (AMR) to pass through the food chain to humans, thereby increasing the AMR burden and affecting our ability to tackle infections. In this study 168 isolates from four Enterobacterales genera, primarily Escherichia coli, were purified from livestock (cattle, pigs, and sheep) faeces from 14 farms in the United Kingdom. Their genomes were resolved using long and short read sequencing to analyse AMR genes and their genetic context, as well as to explore the relationship between AMR burden and on-farm antimicrobial usage (AMU), in the three months prior to sampling. Although E. coli isolates were genomically diverse, phylogenetic analysis using a core-genome SNP tree indicated pig isolates to generally be distinct from sheep, with cattle isolates being intermediates. Approximately 28% of isolates harboured AMR genes, with the greatest proportion detected in pigs, followed by cattle then sheep; pig isolates also harboured the highest number of AMR genes per isolate. Although 90% of sequenced isolates harboured diverse plasmids, only 11% of plasmids (n=58/522) identified contained AMR genes, with 91% of AMR plasmids being from pig, 9% from cattle and none from sheep isolates; these results indicated pigs were a principle reservoir of AMR genes harboured by plasmids likely involved in their horizontal transfer. Significant associations were observed between AMU (mg/kg) and AMR. As both the total and the numbers of different antimicrobial classes used on-farm increased, the risk of multi-drug resistance (MDR) in isolates rose. However, even when AMU on pig farms was comparatively low, pig isolates had increased likelihood of being MDR; harbouring relatively more resistances than other livestock species. Therefore, our results show that AMR prevalence in livestock is not only impacted by recent AMU on-farm but also livestock related factors, which can influence the AMR burden in these reservoirs and its plasmid mediated transmissio