Activity-based protein profiling of serine hydrolases and penicillin-binding proteins in Enterococcus faecium.

Enterococcus faecium is a gut commensal bacterium which is gaining increasing relevance as an opportunistic, nosocomial pathogen. Its high level of intrinsic and acquired antimicrobial resistance is causing a lack of treatment options, particularly for infections with vancomycin-resistant strains, and prioritizes the identification and functional validation of novel druggable targets. Here, we use activity-based protein profiling (ABPP), a chemoproteomics approach using functionalized covalent inhibitors, to detect active serine hydrolases across 11 E. faecium and Enterococcus lactis strains. Serine hydrolases are a big and diverse enzyme family, that includes known drug targets such as penicillin-binding proteins (PBPs), whereas other subfamilies are underexplored. Comparative gel-based ABPP using Bocillin-FL revealed strain- and growth condition-dependent variations in PBP activities. Profiling with the broadly serine hydrolase-reactive fluorescent probe fluorophosphonate-TMR showed a high similarity across E. faecium clade A1 strains, but higher variation across A2 and E. lactis strains. To identify these serine hydrolases, we used a biotinylated probe analog allowing for enrichment and identification via liquid chromatography-mass spectrometry. We identified 11 largely uncharacterized targets (α,ß-hydrolases, SGNH-hydrolases, phospholipases, and amidases, peptidases) that are druggable and accessible in live vancomycin-resistant E. faecium E745 and may possess vital functions that are to be characterized in future studies.

Interactions between commensal Enterococcus faecium and Enterococcus lactis and clinical isolates of Enterococcus faecium.

Enterococcus faecium (Efm) is a versatile pathogen, responsible for multidrug-resistant infections, especially in hospitalized immunocompromised patients. Its population structure has been characterized by diverse clades (A1, A2, and B (reclassified as E. lactis (Ela)), adapted to different environments, and distinguished by their resistomes and virulomes. These features only partially explain the predominance of clade A1 strains in nosocomial infections. We investigated in vitro interaction of 50 clinical isolates (clade A1 Efm) against 75 commensal faecal isolates from healthy humans (25 clade A2 Efm and 50 Ela). Only 36% of the commensal isolates inhibited clinical isolates, while 76% of the clinical isolates inhibited commensal isolates. The most apparent overall differences in inhibition patterns were presented between clades. The inhibitory activity was mainly mediated by secreted, proteinaceous, heat-stable compounds, likely indicating an involvement of bacteriocins. A custom-made database targeting 76 Bacillota bacteriocins was used to reveal bacteriocins in the genomes. Our systematic screening of the interactions between nosocomial and commensal Efm and Ela on a large scale suggests that, in a clinical setting, nosocomial strains not only have an advantage over commensal strains due to their possession of AMR genes, virulence factors, and resilience but also inhibit the growth of commensal strains.

Deciphering Staphylococcus aureus-host dynamics using dual activity-based protein profiling of ATP-interacting proteins.

The utilization of ATP within cells plays a fundamental role in cellular processes that are essential for the regulation of host-pathogen dynamics and the subsequent immune response. This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus, we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type-specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts. These findings offer valuable insights into the dynamics of S. aureus-host cell interactions, shedding light on modulating host immune responses to S. aureus, which could involve developing immunomodulatory therapies. IMPORTANCE: This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.

Comprehensive virulence profiling and evolutionary analysis of specificity determinants in Staphylococcus aureus two-component systems.

In the Staphylococcus aureus genome, a set of highly conserved two-component systems (TCSs) composed of histidine kinases (HKs) and their cognate response regulators (RRs) sense and respond to environmental stimuli, which drive the adaptation of the bacteria. This study investigates the complex interplay between TCSs in S. aureus USA300, a predominant methicillin-resistant S. aureus strain, revealing shared and unique virulence regulatory pathways and genetic variations mediating signal specificity within TCSs. Using TCS-related mutants from the Nebraska Transposon Mutant Library, we analyzed the effects of inactivated TCS HKs and RRs on the production of various virulence factors, in vitro infection abilities, and adhesion assays. We found that the TCSs' influence on virulence determinants was not associated with their phylogenetic relationship, indicating divergent functional evolution. Using the co-crystallized structure of the DesK-DesR from Bacillus subtilis and the modeled structures of the four NarL TCSs in S. aureus, we identified interacting residues, revealing specificity determinants and conservation within the same TCS, even from different strain backgrounds. The interacting residues were highly conserved within strains but varied between species due to selection pressures and the coevolution of cognate pairs. This study unveils the complex interplay and divergent functional evolution of TCSs, highlighting their potential for future experimental exploration of phosphotransfer between cognate and non-cognate recombinant HK and RRs.IMPORTANCEGiven the widespread conservation of two-component systems (TCSs) in bacteria and their pivotal role in regulating metabolic and virulence pathways, they present a compelling target for anti-microbial agents, especially in the face of rising multi-drug-resistant infections. Harnessing TCSs therapeutically necessitates a profound understanding of their evolutionary trajectory in signal transduction, as this underlies their unique or shared virulence regulatory pathways. Such insights are critical for effectively targeting TCS components, ensuring an optimized impact on bacterial virulence, and mitigating the risk of resistance emergence via the evolution of alternative pathways. Our research offers an in-depth exploration of virulence determinants controlled by TCSs in S. aureus, shedding light on the evolving specificity determinants that orchestrate interactions between their cognate pairs.

Adaptive cell wall thickening in Enterococcus faecalis is associated with decreased vancomycin susceptibility.

OBJECTIVES: Enterococcus faecalis can adopt both a commensal and a nosocomial lifestyle, resisting numerous antibiotics. In this study, we aim to investigate the relationship between the cell wall (CW) thickness and decreased susceptibility to vancomycin (VD) in van-gene negative clinical isolates of E. faecalis (nMIC 8 = 2, nMIC 4 = 3, ST30, ST40, and ST59). METHODS: The CW thickness was assessed in VD strains and compared with vancomycin susceptible isolates of the same sequence type (ST) (Vancomycin susceptible [VS]; nMIC 2 = 5). The VD and VS strains were subjected to serial passage (evolved [ev]) with and without vancomycin selection. Subsequent measurements of CW thickness and vancomycin MICs were performed. RESULTS: The VD strains exhibited increased CW thickness when compared with ST-related VS strains (ΔCW thickness VD vs. VS ST30 25 nm, ST59 15 nm, and ST40 1 nm). Serial passages without vancomycin selection led to a decrease in CW thickness and vancomycin MIC in VD strains (ΔCW thickness VD vs. evVD ST30 22 nm, ST59 3 nm, and ST40 2 nm). Serial passages with vancomycin selection caused an increase in CW thickness and vancomycin MIC in ST-related VS strains (ΔCW thickness VS vs. evVS ST30 22 nm, ST59 16 nm, and ST40 1 nm). DISCUSSION: Adaptive changes in CW thickness were observed in response to vancomycin exposure. Increased CW thickness correlated with decreased vancomycin susceptibility, whereas decreased CW thickness correlated with increased vancomycin susceptibility. Core single nucleotide polymorphisms in the evolved mutants were mostly found in genes encoding proteins associated with the cytoplasm or the cytoplasmic membrane. The potential relevance of these adaptive changes is underlined by the observed phenotypes in clinical isolates. Our findings emphasize the importance of monitoring adaptive changes, as vancomycin-resistant enterococci infections are a growing concern.

The first tigecycline resistant Enterococcus faecium in Norway was related to tigecycline exposure.

OBJECTIVES: We describe the first tigecycline resistant enterococcal isolate in Norway and the mechanisms involved. MATERIAL AND METHODS: The Norwegian National Advisory Unit on Detection of Antimicrobial Resistance (K-res). received in 2022 an Enterococcus faecium blood culture isolate with decreased susceptibility to tigecycline from a hospitalized patient in the South-Eastern Norway Health region for confirmatory testing. K-res verified a tigecycline-resistant E. faecium (TigR) with broth microdilution MIC of 0.5 mg/L. The patient had received treatment with tigecycline because of an infection with a linezolid- and vancomycin-resistant but tigecycline susceptible E. faecium (TigS) 47 days prior to the detection of the corresponding tigecycline-resistant isolate. Whole-genome comparisons, cgMLST and SNP analyses revealed that the two ST117 strains were closely related. RESULTS: The TigR isolate showed a novel deletion of 2 amino acids (K57Y58) in a polymorphic region of ribosomal protein S10 previously associated with tigecycline resistance and a deletion of the tet(M) leader peptide previously related to increased expression of tet(M) and tigecycline resistance in enterococci. CONCLUSIONS: Genomic and epidemiological analyses confirm that the two E. faecium (TigR and TigS) are closely related isolates of the same strain and that the two deletions (in rpsJ and of tet(M) leader peptide) account for the tigecycline resistance in TigR.

Emergence of vancomycin-resistant enterococci from vancomycin-susceptible enterococci in hospitalized patients under antimicrobial therapy.

OBJECTIVES: Enterococci are opportunistic pathogens with plastic genomes that evolve, acquire, and transmit antimicrobial-resistant determinants such as vancomycin resistance clusters. While vancomycin-resistant enterococci (VRE) have emerged as successful nosocomial pathogens, the mechanism by which vancomycin-susceptible enterococci (VSE) transform to VRE in hospitalized patients remains understudied. METHODS: Genomes of Enterococcus faecium from two critically ill hospitalized patients subjected to multiple antibiotic therapies, including broad-spectrum antibiotics, were investigated. To identify mechanisms of resistance evolution, genomes of vancomycin-susceptible and -resistant isolates were compared. RESULTS: While VSE isolates were initially identified, VRE strains emerged post-vancomycin therapy. Comparative genomics revealed horizontal transmission of mobile genetic elements containing the Tn1549 transposon, which harbours the vanB-type vancomycin resistance gene cluster. This suggests that broad-spectrum antibiotic stress promoted the transfer of resistance-conferring elements, presumably from another gut inhabitant. CONCLUSION: This is one of the first studies investigating VSE and VRE isolates from the same patient. The mechanism of transmission and the within-patient evolution of vancomycin resistance via mobile genetic elements under antibiotic stress is illustrated. Our findings serve as a foundation for future studies building on this knowledge which can further elucidate the dynamics of antibiotic stress, resistance determinant transmission, and interactions within the gut microbiota.

The first vanE-type vancomycin resistant Enterococcus faecalis isolates in Norway - phenotypic and molecular characteristics.

OBJECTIVES: We aimed to characterize the vanE cluster and its genetic support in the first Norwegian vanE-type isolates and assess genetic relatedness to other vanE isolates. METHODS: Two vanE-type vancomycin resistant Enterococcus faecalis (vanE-VREfs) isolates (E1 and E2) recovered from the same patient 30 months apart were examined for antimicrobial susceptibility, genome sequence, vancomycin resistance induction, vanE transferability, genome mutation rate, and phylogenetic relationship to E. faecalis closed genomes and two vanE-VREfs from North America. RESULTS: The ST34 E1 and E2 strains expressed low-level vancomycin resistance and susceptibility to teicoplanin. Their vanE gene clusters were part of a non-transferable Tn6202. The histidine kinase part of vanSE was expressed although a premature stop codon (E1) and insertion of a transposase (E2) truncated their vanSE gene. The vancomycin resistance phenotype in E1 was inducible while constitutive in E2. E1 showed a 125-fold higher mutation rate than E2. Variant calling showed 60 variants but nearly identical chromosomal gene content and synteny between the isolates. Their genomes also showed high similarity to another ST34 vanE-VREfs from Canada. CONCLUSION: In-depth genomic analyses of the first two vanE-VREfs found in Europe identified a single chromosomal insertion site of two variants of vanE-conferring Tn6202. Single nucleotide polymorphism (SNP) and core genome multilocus sequence type (cgMLST) analyses show the genomes are different. This can be explained by the high mutation rate of E1 and acquisition of different mobile genetic elements; thus, we believe the two isolates from the same patient are genetically related. Genome similarities also suggest relatedness between the Canadian and Norwegian vanE-VREfs.

Genomic insights into Enterococcus faecium isolates from marine bivalves highlight One Health concerns and healthcare linkages.

Enterococci, especially Enterococcus faecium, are one of today's leading causes of multidrug-resistant infections in hospital settings. The marine environment may harbour enterococci, but its role as an evolutionary niche and as a vector for the spread of enterococci is sparsely investigated. Hence, by applying enterococci in bivalves as a sentinel tool, this study aimed to describe the prevalence of enterocooci along the Norwegian coast and in addition the phylogeny of E. faecium in particular. Enterococci in batch samples of marine bivalves, harvested from 86 different locations, were quantitatively examined by a culture-dependent most probable number (MPN) method. Isolates were identified by MALDI-TOF-MS prior to antimicrobial susceptibility testing by broth microdilution. In-detail analyses of a representative selection of E. faecium isolates (n=148) were done by Illumina whole-genome sequencing, and assembled genomes were compared to closed E. faecium genomes in the public databases and to genomes from commensal and clinical isolates from Norway. Diversity among E. faecium within the same batch sample of bivalves was also explored. Enterococci were detected in 287 of the 471 examined bivalve samples, but in low concentrations with a median value of <18 MPN /100 g. From positive samples, 479 isolates of enterococci were identified belonging to ten different species, where E. faecium (n=247), Enterococcus hirae (n=114) and Enterococcus faecalis (n=66) were most frequently found. Resistance towards one or more antimicrobial agents was observed in 197 isolates (41 %), none of the isolates showed acquired resistance to vancomycin or linezolid. Phylogenetic analyses revealed high diversity among the E. faecium isolates and showed that the marine niche is dominated by strains from the non-clinical setting belonging to clade A2 (n=85) and B (E. lactis) (n=60). Only three isolates belonged to the hospital-associated clade A1 (ST80 and ST117). Two of these clustered with one isolate from a hospitalized patient and one from a non-hospitalized person. This study demonstrated a high prevalence, but low concentrations of enterococci in bivalves, and low levels of antimicrobial resistance. E. faecium genomes showed high population diversity and that very few E. faecium isolates in bivalves may have arisen from the human healthcare system. A systematic surveillance of target micro-organisms applying methods examining multiple isolates from the same bivalve sample provides important data to assess the enterococcal phylogeny, antimicrobial resistance and the level of faecal pollution in the marine environment.

The population structure of vancomycin-resistant and -susceptible Enterococcus faecium in a low-prevalence antimicrobial resistance setting is highly influenced by circulating global hospital-associated clones.

Between 2010 and 2015 the incidence of vancomycin-resistant Enterococcus faecium (VREfm) in Norway increased dramatically. Hence, we selected (1) a random subset of vancomycin-resistant enterococci (VRE) from the Norwegian Surveillance System for Communicable Diseases (2010-15; n=239) and (2) Norwegian vancomycin-susceptible E. faecium (VSEfm) bacteraemia isolates from the national surveillance system for antimicrobial resistance in microbes (2008 and 2014; n=261) for further analysis. Whole-genome sequences were collected for population structure, van gene cluster, mobile genetic element and virulome analysis, as well as antimicrobial susceptibility testing. Comparative genomic and phylogeographical analyses were performed with complete genomes of global E. faecium strains from the National Center for Biotechnology Information (NCBI) (1946-2022; n=272). All Norwegian VREfm and most of the VSEfm clustered with global hospital-associated sequence types (STs) in the phylogenetic subclade A1. The vanB2 subtype carried by chromosomal Tn1549 integrative conjugative elements was the dominant van type. The major Norwegian VREfm cluster types (CTs) were in accordance with concurrent European CTs. The dominant vanB-type VREfm CTs, ST192-CT3/26 and ST117-CT24, were mostly linked to a single hospital in Norway where the clones spread after independent chromosomal acquisition of Tn1549. The less prevalent vanA VRE were associated with more diverse CTs and vanA carrying Inc18 or RepA_N plasmids with toxin-antitoxin systems. Only 5 % of the Norwegian VRE were Enterococcus faecalis, all of which contained vanB. The Norwegian VREfm and VSEfm isolates harboured CT-specific virulence factor (VF) profiles supporting biofilm formation and colonization. The dominant VREfm CTs in general hosted more virulence determinants than VSEfm. The phylogenetic clade B VSEfm isolates (n=21), recently classified as Enterococcus lactis, harboured fewer VFs than E. faecium in general, and particularly subclade A1 isolates. In conclusion, the population structure of Norwegian E. faecium isolates mirrors the globally prevalent clones and particularly concurrent European VREfm/VSEfm CTs. Novel chromosomal acquisition of vanB2 on Tn1549 from the gut microbiota, however, formed a single major hospital VREfm outbreak. Dominant VREfm CTs contained more VFs than VSEfm.

Enterococcal Membrane Vesicles as Vaccine Candidates.

Enterococcus faecium is a leading cause of nosocomial infections, particularly in immunocompromised patients. The rise of multidrug-resistant E. faecium, including Vancomycin-Resistant Enterococci (VRE), is a major concern. Vaccines are promising alternatives to antibiotics, but there is currently no vaccine available against enterococci. In a previous study, we identified six protein vaccine candidates associated with extracellular membrane vesicles (MVs) produced by nosocomial E. faecium. In this study, we immunized rabbits with two different VRE-derived MV preparations and characterized the resulting immune sera. Both anti-MV sera exhibited high immunoreactivity towards the homologous strain, three additional VRE strains, and eight different unrelated E. faecium strains representing different sequence types (STs). Additionally, we demonstrated that the two anti-MV sera were able to mediate opsonophagocytic killing of not only the homologous strain but also three unrelated heterologous VRE strains. Altogether, our results indicate that E. faecium MVs, regardless of the purification method for obtaining them, are promising vaccine candidates against multidrug-resistant E. faecium and suggest that these naturally occurring MVs can be used as a multi-antigen platform to elicit protective immune responses against enterococcal infections.

Reversible vancomycin susceptibility within emerging ST1421 Enterococcus faecium strains is associated with rearranged vanA-gene clusters and increased vanA plasmid copy number.

Vancomycin variable enterococci (VVE) are van-positive enterococci with a vancomycin-susceptible phenotype (VVE-S) that can convert to a resistant phenotype (VVE-R) and be selected for during vancomycin exposure. VVE-R outbreaks have been reported in Canada and Scandinavian countries. The aim of this study was to examine the presence of VVE in whole genome sequenced (WGS) Australian bacteremia Enterococcus faecium (Efm) isolates collected through the Australian Group on Antimicrobial resistance (AGAR) network. Eight potential VVEAus isolates, all identified as Efm ST1421, were selected based on the presence of vanA and a vancomycin-susceptible phenotype. During vancomycin selection, two potential VVE-S harboring intact vanHAX genes, but lacking the prototypic vanRS and vanZ genes, reverted to a resistant phenotype (VVEAus-R). Spontaneous VVEAus-R reversion occurred at a frequency of 4-6 × 10-8 resistant colonies per parent cell in vitro after 48 h and led to high-level vancomycin and teicoplanin resistance. The S to R reversion was associated with a 44-bp deletion in the vanHAX promoter region and an increased vanA plasmid copy number. The deletion in the vanHAX promoter region enables an alternative constitutive promoter for the expression of vanHAX. Acquisition of vancomycin resistance was associated with a low fitness cost compared with the corresponding VVEAus-S isolate. The relative proportion of VVEAus-R vs. VVEAus-S decreased over time in serial passages without vancomycin selection. Efm ST1421 is one of the predominant VanA-Efm multilocus sequence types found across most regions of Australia, and has also been associated with a major prolonged VVE outbreak in Danish hospitals.

Taking hospital pathogen surveillance to the next level.

High-throughput bacterial genomic sequencing and subsequent analyses can produce large volumes of high-quality data rapidly. Advances in sequencing technology, with commensurate developments in bioinformatics, have increased the speed and efficiency with which it is possible to apply genomics to outbreak analysis and broader public health surveillance. This approach has been focused on targeted pathogenic taxa, such as Mycobacteria, and diseases corresponding to different modes of transmission, including food-and-water-borne diseases (FWDs) and sexually transmitted infections (STIs). In addition, major healthcare-associated pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and carbapenemase-producing Klebsiella pneumoniae are the focus of research projects and initiatives to understand transmission dynamics and temporal trends on both local and global scales. Here, we discuss current and future public health priorities relating to genome-based surveillance of major healthcare-associated pathogens. We highlight the specific challenges for the surveillance of healthcare-associated infections (HAIs), and how recent technical advances might be deployed most effectively to mitigate the increasing public health burden they cause.

Transiently silent acquired antimicrobial resistance: an emerging challenge in susceptibility testing.

Acquisition and expression of antimicrobial resistance (AMR) mechanisms in bacteria are often associated with a fitness cost. Thus, evolutionary adaptation and fitness cost compensation may support the advance of subpopulations with a silent resistance phenotype when the antibiotic selection pressure is absent. However, reports are emerging on the transient nature of silent acquired AMR, describing genetic alterations that can change the expression of these determinants to a clinically relevant level of resistance, and the association with breakthrough infections causing treatment failures. This phenomenon of transiently silent acquired AMR (tsaAMR) is likely to increase, considering the overall expansion of acquired AMR in bacterial pathogens. Moreover, the augmented use of genotypic methods in combination with conventional phenotypic antimicrobial susceptibility testing (AST) will increasingly enable the detection of genotype and phenotype discrepancy. This review defines tsaAMR as acquired antimicrobial resistance genes with a corresponding phenotype within the wild-type distribution or below the clinical breakpoint for susceptibility for which genetic alterations can mediate expression to a clinically relevant level of resistance. References to in vivo resistance development and therapeutic failures caused by selected resistant subpopulations of tsaAMR in Gram-positive and Gram-negative pathogens are given. We also describe the underlying molecular mechanisms, including alterations in the expression, reading frame or copy number of AMR determinants, and discuss the clinical relevance concerning challenges for conventional AST.

Characterization and Fitness Cost of Tn7100, a Novel Integrative and Conjugative Element Conferring Multidrug Resistance in Haemophilus influenzae.

A multidrug-resistant (MDR) strain of Haemophilus influenzae, Hi-228, with phenotypic resistance toward ampicillin, cefotaxime, chloramphenicol, gentamicin, and azithromycin, was isolated in Oslo, Norway. The strain was part of a clonal outbreak (2016-2017) comprising five ST143 strains with identical resistotypes. Hi-228 carries a novel integrative and conjugative element (ICE), Tn7100, contributing to this remarkable and previously unreported MDR profile. Tn7100 contains the following resistance genes: bla TEM-1B, catA2, aac(6')-Im, aph(2″)-Ib, mef (E), and mel. The latter four are previously unreported or rarely reported in H. influenzae. In this study, we investigated the genetic environment, mechanisms of transfer, impact on phenotypic susceptibility, and fitness cost of this ICE. We found that Tn7100 has an overall structure similar to the previously described ICE Tn6686, with bla TEM-1B and catA2 carried by Tn3 and Tn10, respectively. The major difference between Tn7100 and Tn6686 is that Tn7100 lacks tet(B) but carries the resistance gene pairs aac(6')-Im and aph(2″)-Ib and mef (E) and mel. The gene pairs are located on the novel transposable elements Tn7470 and Tn7471, which have high sequence identities to a plasmid in Enterobacterales and an ICE in streptococcal species, respectively. Tn7100 does circularize and is transferable, however, at a low frequency. Head-to-head competition experiments showed that uptake of Tn7100 reduces bacterial fitness. Our study shows that MDR strains are capable of clonal spread and that the H. influenzae supragenome comprises an increasingly wide range of transferable resistance genes, with evidence of transfer from unrelated genera. The findings offer a glimpse into the genome dynamics of H. influenzae, highlighting the importance of rational antibiotic usage to contain antimicrobial resistance and the emergence of MDR strains in this important pathogen.

Structural basis for PoxtA-mediated resistance to phenicol and oxazolidinone antibiotics.

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Šout of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site.

The interplay between community and hospital Enterococcus faecium clones within health-care settings: a genomic analysis.

BACKGROUND: The genomic relationships among Enterococcus faecium isolates are the subject of ongoing research that seeks to clarify the origins of observed lineages and the extent of horizontal gene transfer between them, and to robustly identify links between genotypes and phenotypes. E faecium is considered to form distinct groups-A and B-corresponding to isolates derived from patients who were hospitalised (A) and isolates from humans in the community (B). The additional separation of A into the so-called clades A1 and A2 remains an area of uncertainty. We aimed to investigate the relationships between A1 and non-A1 groups and explore the potential role of non-A1 isolates in shaping the population structure of hospital E faecium. METHODS: We collected short-read sequence data from invited groups that had previously published E faecium genome data. This hospital-based isolate collection could be separated into three groups (or clades, A1, A2, and B) by augmenting the study genomes with published sequences derived from human samples representing the previously defined genomic clusters. We performed phylogenetic analyses, by constructing maximum-likelihood phylogenetic trees, and identified historical recombination events. We assessed the pan-genome, did resistome analysis, and examined the genomic data to identify mobile genetic elements. Each genome underwent chromosome painting by use of ChromoPainter within FineSTRUCTURE software to assess ancestry and identify hybrid groups. We further assessed highly admixed regions to infer recombination directionality. FINDINGS: We assembled a collection of 1095 hospital E faecium sequences from 34 countries, further augmented by 33 published sequences. 997 (88%) of 1128 genomes clustered as A1, 92 (8%) as A2, and 39 (4%) as B. We showed that A1 probably emerged as a clone from within A2 and that, because of ongoing gene flow, hospital isolates currently identified as A2 represent a genetic continuum between A1 and community E faecium. This interchange of genetic material between isolates from different groups results in the emergence of hybrid genomes between clusters. Of the 1128 genomes, 49 (4%) hybrid genomes were identified: 33 previously labelled as A2 and 16 previously labelled as A1. These interactions were fuelled by a directional pattern of recombination mediated by mobile genetic elements. By contrast, the contribution of B group genetic material to A1 was limited to a few small regions of the genome and appeared to be driven by genomic sweep events. INTERPRETATION: A2 and B isolates coming into the hospital form an important reservoir for ongoing A1 adaptation, suggesting that effective long-term control of the effect of E faecium could benefit from strategies to reduce these genomic interactions, such as a focus on reducing the acquisition of hospital A1 strains by patients entering the hospital. FUNDING: Wellcome Trust.

Bacterial genomic epidemiology with mixed samples.

Genomic epidemiology is a tool for tracing transmission of pathogens based on whole-genome sequencing. We introduce the mGEMS pipeline for genomic epidemiology with plate sweeps representing mixed samples of a target pathogen, opening the possibility to sequence all colonies on selective plates with a single DNA extraction and sequencing step. The pipeline includes the novel mGEMS read binner for probabilistic assignments of sequencing reads, and the scalable pseudoaligner Themisto. We demonstrate the effectiveness of our approach using closely related samples in a nosocomial setting, obtaining results that are comparable to those based on single-colony picks. Our results lend firm support to more widespread consideration of genomic epidemiology with mixed infection samples.

Bacterial Membrane-Derived Vesicles Attenuate Vancomycin Activity against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures.

Novel genomic islands and a new vanD-subtype in the first sporadic VanD-type vancomycin resistant enterococci in Norway.

BACKGROUND: Vancomycin-resistant enterococci (VRE) represent several types of transferable vancomycin resistance gene clusters. The vanD type, associated with moderate to high level vancomycin resistance, has only sporadically been described in clinical isolates. The aim of this study was to perform a genetic characterization of the first VanD-type VRE strains detected in Norway. METHODS: The VanD-type VRE-strains (n = 6) from two patient cases were examined by antimicrobial susceptibility testing and whole genome sequencing (WGS) to uncover Van-phenotype, strain phylogeny, the vanD gene clusters, and their genetic surroundings. The putative transferability of vanD was examined by circularization PCR and filter mating. RESULTS: The VanD-type Enterococcus faecium (n = 4) and Enterococcus casseliflavus (n = 2) strains recovered from two cases (A and B), expressed moderate to high level vancomycin resistance (MIC 64->256 mg/L) and various levels of teicoplanin susceptibility (MIC 2->256 mg/L). WGS analyses revealed phylogenetically different E. faecium strains (A1, A2, and A3 of case A and B1 from case B) as well as vanD gene clusters located on different novel genomic islands (GIs). The E. casseliflavus strains (B2 and B3 of case B) were not clonally related, but harbored nearly identical novel GIs. The vanD cluster of case B strains represents a novel vanD-subtype. All the vanD-GIs were integrated at the same chromosomal site and contained genes consistent with a Clostridiales origin. Circular forms of the vanD-GIs were detected in all strains except B1. Transfer of vanD to an E. faecium recipient was unsuccessful. CONCLUSIONS: We describe the first VanD-type E. casseliflavus strains, a novel vanD-subtype, and three novel vanD-GIs with a genetic content consistent with a Clostridiales order origin. Despite temporal occurrence, case A and B E. faecium strains were phylogenetically diverse and harbored different vanD subtypes and vanD-GIs.