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Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen. Identifying VREfm transmission dynamics permits targeted interventions, and while genomics is increasingly being utilised, methods are not yet standardised or optimised for accuracy. We aimed to develop a standardized genomic method for identifying putative VREfm transmission links. Using comprehensive genomic and epidemiological data from a cohort of 308 VREfm infection or colonization cases, we compared multiple approaches for quantifying genetic relatedness. We showed that clustering by core genome multilocus sequence type (cgMLST) was more informative of population structure than traditional MLST. Pairwise genome comparisons using split k-mer analysis (SKA) provided the high-level resolution needed to infer patient-to-patient transmission. The more common mapping to a reference genome was not sufficiently discriminatory, defining more than three times more genomic transmission events than SKA (3729 compared to 1079 events). Here, we show a standardized genomic framework for inferring VREfm transmission that can be the basis for global deployment of VREfm genomics into routine outbreak detection and investigation., Vancomycin-resistant Enterococcus faecium is an important healthcare-associated pathogen and genomic analyses could inform targeted interventions. Here, the authors optimise an analysis pipeline for identification of putative transmission events using core genome multilocus sequence type clustering and split kmer analysis.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) is an important agent of hospital-acquired infection. VanA phenotype is characterized by resistance to high levels of vancomycin and teicoplanin and is encoded by the vanA gene, whereas VanD phenotype is characterized by resistance to vancomycin and susceptibility or intermediate resistance to teicoplanin; however, some isolates carry a VanD phenotype with a vanA genotype, but there are many gaps in the knowledge about the genetic mechanisms behind this pattern. OBJECTIVE: To characterize the genetic structure, clonality, and mobile genetic elements of VRE isolates that display a VanD-vanA phenotype. RESULTS: All vanA VRE-fm isolates displayed minimum inhibitory concentration (MIC) for vancomycin > 32µg/mL and intermediate or susceptible MIC range for teicoplanin (8–16µg/mL). The isolates were not clonal, and whole-genome sequencing analysis showed that they belonged to five different STs (ST478, ST412, ST792, ST896, and ST1393). The absence of some van complex genes were observed in three isolates: Ef5 lacked vanY and vanZ, Ef2 lacked vanY, and Ef9 lacked orf1 and orf2; moreover, another three isolates had inverted positions of orf1, orf2, vanR, and vanS genes. IS1542 was observed in all isolates, whereas IS1216 in only five. Moreover, presence of other hypothetical protein-encoding genes located downstream the vanZ gene were observed in six isolates. CONCLUSION: VRE isolates can display some phenotypes associated to vanA genotype, including VanA and VanB, as well as VanD; however, further studies are needed to understand the exact role of genetic variability, rearrangement of the transposon Tn1546, and presence of insertion elements in isolates with this profile.

Two dimensional molybdenum disulfide (MoS

In this paper, we evaluated the drug-receptor interactions responsible for the antimicrobial activity of thymol, the major compound present in the essential oil (EO) of

The bacterial nitroreductases (NRs) NfsB and NfsA are conserved homodimeric FMN-dependent flavoproteins that are responsible for the reduction of nitroaromatic substrates. Berberine (BBR) is a plant-derived isoquinoline alkaloid with a large conjugated ring system that is widely used in the treatment of various diseases. It was recently found that the gut microbiota convert BBR into dihydroberberine (dhBBR, the absorbable form) mediated by bacterial NRs. The molecular basis for the transformation of BBR by the gut microbiota remains unclear. Here, kinetic studies showed that NfsB from Escherichia coli (EcNfsB), rather than EcNfsA, is responsible for the conversion of BBR to dhBBR in spite of a low reaction rate. The crystal structure of the EcNfsB–BBR complex showed that BBR binds into the active pocket at the dimer interface, and its large conjugated plane stacks above the plane of the FMN cofactor in a nearly parallel orientation. BBR is mainly stabilized by π-stacking interactions with both neighboring aromatic residues and FMN. Structure-based mutagenesis studies further revealed that the highly conserved Phe70 and Phe199 are important residues for the conversion of BBR. The structure revealed that the C6 atom of BBR (which receives the hydride) is ∼7.5 Å from the N5 atom of FMN (which donates the hydride), which is too distant for hydride transfer. Notably, several well ordered water molecules make hydrogen-bond/van der Waals contacts with the N1 atom of BBR in the active site, which probably donate protons in conjunction with electron transfer from FMN. The structure–function studies revealed the mechanism for the recognition and binding of BBR by bacterial NRs and may help to understand the conversion of BBR by the gut microbiota.

Objectives The increasing prevalence of VRE necessitates their reliable detection, especially for low-level resistance mediated by vanB in Enterococcus faecium. In this prospective study we analysed if vanB-mediated vancomycin resistance can be reliably detected by Vitek2. Methods One thousand, three hundred and forty-four enterococcal isolates from routine clinical specimens were tested by Vitek2 (bioMérieux, Nürtingen, Germany). Additionally, a bacterial suspension (with a turbidity equivalent to that of a 0.5 McFarland standard) was inoculated on chromID VRE screening agar (bioMérieux) and incubated for 48 h. If vancomycin tested susceptible by Vitek2 but growth was detected on the screening agar, PCR for vanA/vanB was performed (GeneXpert vanA/B test, Cepheid, Frankfurt, Germany). For isolates that tested susceptible to vancomycin by Vitek2 but were vanA/B positive, MICs were determined before and after cultivation in broth with increasing concentrations of vancomycin. Results One hundred and fifty-six out of 491 E. faecium were VRE and were predominantly vanB positive (81.0%). Of these, Vitek2 did not identify 14 as VRE (sensitivity 91.0%). By broth microdilution 9/14 isolates demonstrated high MICs (≥32 mg/L) and 5/14 showed low vancomycin MICs, which did not increase despite vancomycin exposure. Three of the 14 isolates demonstrated growth on chromID VRE; after vancomycin exposure seven additional isolates were able to grow on chromID VRE. Conclusions Vitek2 fails to detect vanB-mediated vancomycin resistance consistently, especially, but not limited to, low-level resistance. As this may lead to treatment failure and further dissemination of vanB VRE, additional methods (e.g. culture on VRE screening agar or PCR) are necessary to reliably identify vanB-positive enterococci in clinical routine.

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.\ud \ud

Background Wheat yellow dwarf virus disease is infected by barley yellow dwarf virus (BYDV), which causes leaf yellowing and dwarfing symptoms in wheat, thereby posing a serious threat to China's food production. The infection of plant viruses can produce large numbers of vsiRNAs, which can target host transcripts and cause symptom development. However, few studies have been conducted to explore the role played by vsiRNAs in the interaction between BYDV-GAV and host wheat plants. Methods In this study, small RNA sequencing was conducted to profile vsiRNAs in BYDV-GAV-infected wheat plants. The putative targets of vsiRNAs were predicted by the bioinformatics software psRNATarget. RT-qPCR and VIGS were employed to identify the function of selected target transcripts. To confirm the interaction between vsiRNA and the target, 5′ RACE was performed to analyze the specific cleavage sites. Results From the sequencing data, we obtained a total of 11,384 detected vsiRNAs. The length distribution of these vsiRNAs was mostly 21 and 22 nt, and an A/U bias was observed at the 5′ terminus. We also observed that the production region of vsiRNAs had no strand polarity. The vsiRNAs were predicted to target 23,719 wheat transcripts. GO and KEGG enrichment analysis demonstrated that these targets were mostly involved in cell components, catalytic activity and plant-pathogen interactions. The results of RT-qPCR analysis showed that most chloroplast-related genes were downregulated in BYDV-GAV-infected wheat plants. Silencing of a chlorophyll synthase gene caused leaf yellowing that was similar to the symptoms exhibited by BYDV-GAV-inoculated wheat plants. A vsiRNA from an overlapping region of BYDV-GAV MP and CP was observed to target chlorophyll synthase for gene silencing. Next, 5′ RACE validated that vsiRNA8856 could cleave the chlorophyll synthase transcript in a sequence-specific manner. Conclusions This report is the first to demonstrate that BYDV-GAV-derived vsiRNAs can target wheat transcripts for symptom development, and the results of this study help to elucidate the molecular mechanisms underlying leaf yellowing after viral infection.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

The aim of our study was to characterize the epidemiological situation concerning nosocomial vancomycin-resistant Enterococcus faecalis of VanA-phenotype (VREfs-VanA) in Poland by investigating their clonal relationships and the vanA-associated mobilome. One-hundred twenty-five clinical isolates of VREfs-VanA collected between 2004 and 2016 were studied by phenotypic assays, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR detection of plasmid-specific genes, and Tn1546 structure and localization mapping. Selected isolates were subjected to PFGE-S1, Southern hybridization, genomic sequencing and conjugation experiments. The majority of isolates (97.6%) belonged to clonal complexes CC2 and CC87 of E. faecalis. All isolates were resistant to vancomycin and teicoplanin, and resistance to ciprofloxacin and aminoglycosides (high level) was very prevalent in this group. VanA phenotype was associated with 16 types of Tn1546, carrying insertion sequences IS1216, ISEfa4, IS1251 and IS1542, located on repUS1pVEF1, rep1pIP501, rep2pRE25, rep9pAD1/pTEF2/pCF10 and rep6pS86 replicons. The most common Tn1546 B- and BB-type transposons, harbouring one or two copies of IS1216, were inserted between rep18ap200B and repUS1pVEF1 genes and located on ~ 20 kb and 150–200 kb plasmids. VREfs-VanA in Poland represent a polyclonal group, indicating a number of acquisitions of the vanA determinant. The repUS1pVEF1-vanA plasmids, unique for Poland, were the main factor beyond the acquisition of vancomycin resistance by E. faecalis, circulating in Polish hospitals.

Purpose Vancomycin-resistant enterococci infection is a worrying worldwide clinical problem. To evaluate the accuracy of GeneXpert vanA/vanB in the diagnosis of VRE, we conducted a systematic review in the study. Methods Experimental data were extracted from publications until May 03 2021 related to the diagnostic accuracy of GeneXpert vanA/vanB for VRE in PubMed, Embase, Web of Science and the Cochrane Library. The accuracy of GeneXpert vanA/vanB for VRE was evaluated using summary receiver to operate characteristic curve, pooled sensitivity, pooled specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio. Results 8 publications were divided into 3 groups according to two golden standard references, vanA and vanB group, vanA group, vanB group, including 6 researches, 5 researches and 5 researches, respectively. The pooled sensitivity and specificity of group vanA and vanB were 0.96 (95% CI, 0.93–0.98) and 0.90 (95% CI, 0.88–0.91) respectively. The DOR was 440.77 (95% CI, 37.92–5123.55). The pooled sensitivity and specificity of group vanA were 0.86 (95% CI, 0.81–0.90) and 0.99 (95% CI, 0.99–0.99) respectively, and those of group vanB were 0.85 (95% CI, 0.63–0.97) and 0.82 (95% CI, 0.80–0.83) respectively. Conclusion GeneXpert vanA/vanB can diagnose VRE with high-accuracy and shows greater accuracy in diagnosing vanA., Author summary Vancomycin-resistant enterococci (VRE), firstly identified in the mid-1980, is a type of antimicrobial resistance bacteria. In recent years, they were found more colonization in patients with critical diseases, showing new resistance to many antibacterial drugs, which is a worrisome clinical problem worldwide. Traditionally, VRE testing is performed mainly by culture which is a standard reference but requires complex steps and takes a long time. Currently, GeneXpert vanA/vanB were approved as a rapid and sensitive molecular assay for detecting VRE. However, the accuracy of GeneXpert vanA/vanB is without systematic-analyses in evidence-based medicine. Therefore, we conducted a data integration and analysis in this study. Finally, we draw a conclusion that GeneXpert vanA/vanB has a high accuracy diagnosing VRE in comparation with conventional culture and PCR. Furthermore, GeneXpert vanA/vanB shows more accuracy in diagnosing vanA. In addition, we suggest that an additional test is needed for further detecting vanB. This finding provides a promising direction for the diagnosis of VRE to a certain extent.

Resistance in VanA-type vancomycin-resistant Enterococcus faecium (VREfm) is due to an inducible gene cassette encoding seven proteins (vanRSHAXYZ). This provides for an alternative peptidoglycan (PG) biosynthesis pathway whereby D-Ala–D-Ala is replaced by D-Ala–d-lactate (Lac), to which vancomycin cannot bind effectively. This study aimed to quantify cytoplasmic levels of normal and alternative pathway PG intermediates in VanA-type VREfm by liquid chromatography-tandem mass spectrometry before and after vancomycin exposure and to correlate these changes with changes in vanA operon mRNA levels measured by real-time quantitative PCR (RT-qPCR). Normal pathway intermediates predominated in the absence of vancomycin, with low levels of alternative pathway intermediates. Extended (18-h) vancomycin exposure resulted in a mixture of the terminal normal (UDP-N-acetylmuramic acid [NAM]–l-Ala–D-Glu–l-Lys–D-Ala–D-Ala [UDP-Penta]) and alternative (UDP-NAM–l-Ala–γ-D-Glu–l-Lys–D-Ala–D-Lac [UDP-Pentadepsi]) pathway intermediates (2:3 ratio). Time course analyses revealed normal pathway intermediates responding rapidly (peaking in 3 to 10 min) and alternative pathway intermediates responding more slowly (peaking in 15 to 45 min). RT-qPCR demonstrated that vanA operon mRNA transcript levels increased rapidly after exposure, reaching maximal levels in 15 min. To resolve the effect of increased van operon protein expression on PG metabolite levels, linezolid was used to block protein biosynthesis. Surprisingly, linezolid dramatically reduced PG intermediate levels when used alone. When used in combination with vancomycin, linezolid only modestly reduced alternative UDP-linked PG intermediate levels, indicating substantial alternative pathway presence before vancomycin exposure. Comparison of PG intermediate levels between VREfm, vancomycin-sensitive Enterococcus faecium, and methicillin-resistant Staphylococcus aureus after vancomycin exposure demonstrated substantial differences between S. aureus and E. faecium PG biosynthesis pathways. IMPORTANCE VREfm is highly resistant to vancomycin due to the presence of a vancomycin resistance gene cassette. Exposure to vancomycin induces the expression of genes in this cassette, which encode enzymes that provide for an alternative PG biosynthesis pathway. In VanA-type resistance, these alternative pathway enzymes replace the D-Ala–D-Ala terminus of normal PG intermediates with D-Ala–D-Lac terminated intermediates, to which vancomycin cannot bind. While the general features of this resistance mechanism are well known, the details of the choreography between vancomycin exposure, vanA gene induction, and changes in the normal and alternative pathway intermediate levels have not been described previously. This study comprehensively explores how VREfm responds to vancomycin exposure at the mRNA and PG intermediate levels.

We report the occurrence and genomic features of multidrug-resistant vancomycin-resistant Enterococcus faecium vanA belonging to a novel sequence type (designated ST1336), carrying a Tn1546-like element, in marine brown mussels (Perna perna) from anthropogenically affected coastal waters of the Atlantic coast of Brazil, highlighting a potential source of dissemination for related ecosystems, with additional consequences for seafood safety and quality.

Streptococcus thoraltensis was first isolated from pigs and rabbits. Later, isolation from human oral and nasal cavities and from throat and oropharynx was documented. S. thoraltensis was isolated from patients with periodontitis, tonsillopharyngitis, and chorioamnionitis suggesting a possible pathological role in human infections. All S. thoraltensis isolates of animal and human origins were sensitive to vancomycin.Standard microbiological identification methods, biochemical analysis, and antibiotic susceptibility testing using disk diffusion and E methods were used. Automatic species identification and antibiotic susceptibility testing were carried out using the Vitek 2 compact system. Molecular analysis of vancomycin resistance gene was carried out using a PCR with specific primers for vanA.We report a healthy young female adult, aged 19 years, with history of exposure to pet rabbit who had nasal colonization with S. thoraltensis. Identification of S. thoraltensis was based on traditional microbiological methods (culture, Gram stain, and biochemical tests), and the Vitek 2 compact system with 97% confidence rate. Antibiotic susceptibility testing of the isolate indicated resistance to most antibiotics, including penicillins, cephalosporins, methicillin, and glycopeptides. The minimal inhibitory concentration for vancomycin and teicoplanin was exceptionally high (256 μg/mL). Molecular analysis indicated the absence of vanA gene in S. thoraltensis.We report for the first time the isolation of a fully vancomycin-resistant S. thoraltensis independent of vanA from a healthy human anterior nasal cavity. The pathological role of this newly identified organism with an exceptionally rare resistance pattern in human infections is yet to be identified.

Objectives Vancomycin resistance in Enterococcus spp., mediated mainly by the vanA resistance gene, has become a major health concern as it has spread worldwide. Therefore, a rapid method is urgently required to detect the vanA gene for timely and appropriate antimicrobial control of resistant Enterococcus infections. Methods The loop-mediated isothermal amplification (LAMP) assay was optimised for vanA detection in Enterococcus spp. isolates. Results The LAMP primer set designed in this study could reliably recognise seven distinct regions of the vanA gene and amplify the gene within 25 min at an isothermal temperature of 65 °C with high specificity. The sensitivity of the optimised assay was high, with a detection limit for vanA as low as 100 pg/μL, which is 100-fold more sensitive than the PCR assay. A special advantage of this optimised LAMP method is that the vanA gene could be detected directly from clinical specimens. Conclusion This optimised LAMP assay has great application potential for efficient detection of vanA in clinical diagnosis and epidemiological studies.

Introduction. The majority of vancomycin-resistant Enterococcus faecium (VREfm) in Australia is of the vanB genotype. An outbreak of vanA VREfm emerged in our haematology/oncology unit between November 2014 and May 2015. The first case of daptomycin non-susceptible E. faecium (DNSEfm) detected was a patient with vanA VREfm bacteraemia who showed clinical failure of daptomycin therapy, prompting microbiologic testing confirming daptomycin non-susceptibility. Objectives. To describe the patient profiles, antibiotic susceptibility and genetic relatedness of vanA VREfm isolates in the outbreak. Methods. Chart review of vanA VREfm colonized and infected patients was undertaken to describe the demographics, clinical features and outcomes of therapy. Whole genome sequencing of vanA VREfm isolates involved in the outbreak was conducted to assess clonality. Results. In total, 29 samples from 24 patients tested positive for vanA VREfm (21 screening swabs and 8 clinical isolates). Five isolates were DNSEfm (four patients colonized, one patient with bacteraemia), with only one patient exposed to daptomycin previously. In silico multi-locus sequence typing of the isolates identified 25/26 as ST203, and 1/26 as ST796. Comparative genomic analysis revealed limited core genome diversity amongst the ST203 isolates, consistent with an outbreak of a single clone of vanA VREfm. Conclusions. Here we describe an outbreak of vanA VREfm in a haematology/oncology unit. Genomic analysis supports transmission of an ST203 vanA VRE clone within this unit. Daptomycin non-susceptibility in 5/24 patients left linezolid as the only treatment option. Daptomycin susceptibility cannot be assumed in vanA VREfm isolates and confirmatory testing is recommended.

Armeniaspirols are potent antibiotics containing an unusual spiro[4.4]non-8-ene moiety. Herein, we describe the cloning and functional analysis of the armeniaspirol biosynthetic gene cluster. Gene-inactivation studies and subsequent isolation of previously unknown biosynthetic intermediates shed light on intriguing biosynthetic details. Remarkably, deletion of ams15, which encodes a protein bearing a flavin-binding domain, led to the accumulation of several non-spiro intermediates with various numbers of chlorine substitutions on the pyrrole moiety. The di- and trichloropyrrole species were converted by Streptomyces albus expressing Ams15 into mono- and dichlorinated spiro derivatives, respectively. In addition, in vitro conversion of these non-spiro intermediates into des-N-methyl spiro intermediates by the cell lysate of the same recombinant strain proved Ams15 to be responsible for spiro formation through oxidative dehalogenation.

The discovery of beta1-adrenoceptor (β

Migrating birds have been implicated in pathogen dissemination over long distances. The lack of data on the intestinal microbiota of birds makes these animals a promising path in order to understand their potential role in the transmission of antibiotic-resistant bacteria. This study aimed to investigate the diversity of enterococcal species, and to analyse the antimicrobial-resistant phenotypes/genotypes, as well as the genetic lineages of isolates obtained from faecal and pellet samples of colonial wild birds in Tunisia. Seventy-nine enterococci were recovered from 150 wild birds, after inoculation of samples in Slanetz-Bartley agar, and were identified as E. faecalis (n = 53), E. faecium (n = 19) and E. casseliflavus (n = 7). Antimicrobial susceptibility was tested, and the following rates of resistance were found: tetracycline (46.8%); erythromycin (34.2%); chloramphenicol (8.8%); gentamicin and streptomycin (2.5-3.8%); ciprofloxacin, trimethoprim-sulfamethoxazole and kanamycin (12.7-21%); and ampicillin and linezolid (0%). The tet(M), tet(L), erm(B), erm(C), aac(6')-Ie-aph(2″)-Ia and cat genes were detected in most tetracycline-, erythromycin-, gentamicin- and chloramphenicol-resistant enterococci, respectively. Three vancomycin-resistant E. faecalis isolates were detected, two with the vanA gene (into Tn1546) and one with the vanB2 gene (into Tn5382); these isolates showed different sequence types determined by multi-locus sequence typing (ST9, ST16 and a new ST848). Seven E. casseliflavus isolates harbouring the intrinsic vancomycin resistance mechanism vanC2 were obtained. The gelE, ace, agg, esp and hyl virulence genes were detected among vanA/vanB2 enterococci. This study provides insight into the possible role of wild birds in the spread of certain antimicrobial resistance genes, particularly vanA/vanB2. To the authors' knowledge, this is the first report of vanB2-containing enterococci in Africa.

The spread of vancomycin-resistant enterococci (VRE) is a major threat in nosocomial settings. A large-scale multiclonal VRE outbreak has rarely been reported in Japan due to low VRE prevalence. We evaluated the transmission of vancomycin resistance in a multiclonal VRE outbreak, conducted biological and genomic analyses of VRE isolates, and assessed the implemented infection control measures. In total, 149 patients harboring VanA-type VRE were identified from April 2017 to October 2019, with 153 vancomycin-resistant Enterococcus faecium isolated being grouped into 31 pulsotypes using pulsed-field gel electrophoresis, wherein six sequence types belonged to clonal complex 17. Epidemic clones varied throughout the outbreak; however, they all carried vanA-plasmids (pIHVA). pIHVA is a linear plasmid, carrying a unique structural Tn1546 containing vanA; it moves between different Enterococcus spp. by genetic rearrangements. VRE infection incidence among patients in the “hot spot” ward correlated with the local VRE colonization prevalence. Local prevalence also correlated with vancomycin usage in the ward. Transmission of a novel transferrable vanA-plasmid among Enterococcus spp. resulted in genomic diversity in VRE in a non-endemic setting. The prevalence of VRE colonization and vancomycin usage at the ward level may serve as VRE cross-transmission indicators in non-intensive care units for outbreak control.

Background VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Objectives To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth. Methods NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller–Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested. Results All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates. Conclusions NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE.

Objective Vancomycin resistant enterococci (VRE) are of significant public health concern. The identification of VRE in livestock and food has increased. The objective of this study was to determine if the transferrable vancomycin resistance genes vanA and vanB were present in probiotics marketed for use in animals that claimed to contain Enterococcus spp. Results Of the 40 products selected, Enterococcus spp. DNA was successfully extracted from 36 products. Of these 36 products with enterococcal DNA, 2 (6%) had a PCR product consistent with vanA which was confirmed by sequencing. None of the products appeared to contain vanB.

Increasing multidrug resistance, including vancomycin resistance, in enterococci is a major concern in clinical settings. Horizontal gene transfer, such as via plasmids, has been shown to play a crucial role in the acquisition of vancomycin resistance. Among vancomycin resistance types, the VanA type is one of the most prevalent, and outbreaks caused by VanA-type vancomycin-resistant enterococci (VRE) have occurred worldwide. Here, we describe an enterococcal linear plasmid responsible for multispecies local spread of VanA-type VRE. Such a study is important because although hospital outbreaks caused by mixed enterococcal species have been reported, this particular spread indicates plasmid transfer across species. This is a crucial finding because the high risk for such a spread of antimicrobial resistance calls for regular monitoring and surveillance., Vancomycin-resistant enterococci pose a threat in the clinical setting and have been linked to hospital outbreaks worldwide. In 2017, a local spread of VanA-type vancomycin-resistant enterococci (VRE) occurred in Japan, and 25 enterococcal isolates, including 14 Enterococcus faecium, 8 E. raffinosus, and 3 E. casseliflavus isolates, were identified from four inpatients. Molecular analysis of the multispecies of VanA-type VRE revealed the involvement of both the dissemination of clonally related VRE strains between patients and the horizontal transfer of plasmids harboring the vanA gene cluster between Enterococcus spp. Pulsed-field gel electrophoresis showed that the plasmid DNAs without S1 nuclease treatment were able to migrate into the gel, suggesting that the topology of the plasmid was linear. Whole-genome sequencing revealed that this plasmid, designated pELF2, was 108,102 bp long and encoded multiple antimicrobial resistance genes, including ermA and ant(9). The amino acid sequences of putative replication- and transfer-related genes were highly conserved between pELF2 and pELF1, the latter of which was the first identified enterococcal conjugative linear plasmid. On comparing the genomic structure, pELF2 showed the presence of a backbone similar to that of pELF1, especially with respect to the nucleotide sequences of both terminal ends, indicating a hybrid-type linear plasmid, possessing two different terminal structures. pELF2 possessed a broad host range and high conjugation frequencies for enterococci. The easy transfer of pELF2 to different Enterococcus spp. in vitro might explain this local spread of multiple species, highlighting the clinical threat from the spread of antimicrobial resistance by an enterococcal linear plasmid. IMPORTANCE Increasing multidrug resistance, including vancomycin resistance, in enterococci is a major concern in clinical settings. Horizontal gene transfer, such as via plasmids, has been shown to play a crucial role in the acquisition of vancomycin resistance. Among vancomycin resistance types, the VanA type is one of the most prevalent, and outbreaks caused by VanA-type vancomycin-resistant enterococci (VRE) have occurred worldwide. Here, we describe an enterococcal linear plasmid responsible for multispecies local spread of VanA-type VRE. Such a study is important because although hospital outbreaks caused by mixed enterococcal species have been reported, this particular spread indicates plasmid transfer across species. This is a crucial finding because the high risk for such a spread of antimicrobial resistance calls for regular monitoring and surveillance.

The purpose of the study was to investigate the molecular characteristics and genetic relatedness of the first reported cases of vancomycin-resistant enterococci (VRE) from the Tripoli Medical Center, Libya. In total, 43 VRE isolates were obtained from various clinical sites throughout the years 2013-2014, including 40

National audience; Failure to understand the microbial ecology driving the proliferation of antibiotic resistance in the environment prevents us from developing strategies to limit the spread of antibiotic resistant infectious disease. In this study, we developed for the first time a tyramide signal amplification-fluorescence in situ hybridization-fluorescence-activated cell sorting protocol (TSA-FISH-FACS) for the characterization of all vanA carrying bacteria in wastewater samples. Firstly, we validated the TSA-FISH protocol through microscopy in pure cultures and wastewater influent. Then, samples were sorted and quantified by FACS and qPCR. Significantly higher percentage tagging of cells was detected in vanA carrying pure cultures and wastewater samples spiked with vanA carrying cells as compared to vanA negative Gram positive strains and non-spiked wastewater samples respectively. qPCR analysis targeting vanZ, a regulating gene in the vanA cluster, showed its relative abundance was significantly greater in Enterococcus faecium ATCC 700221-spiked and positively sorted samples compared to the E. faecium spiked and negatively sorted samples. Phylogenetic analysis was then performed. Although further efforts are needed to overcome technical problems, we have, for the first time, demonstrated sorting bacterial-cells carrying antibiotic resistance genes from wastewater samples through a TSA-FISH-FACS protocol and provided insight into the microbial ecology of vancomycin resistant bacteria. Future potential applications using this approach will include the separation of members of an environmental microbial community (cultured and hard-to-culture) to allow for metagenomics on single cells or, in the case of clumping, targeting a smaller portion of the community with a priori knowledge that the target gene is present.

Objective To evaluate the sensitivity and specificity of the Allplex™ Entero-DR, a quantitative PCR-based method, for the detection of β-lactamase-encoding genes and vancomycin-resistance determinants in 156 previously characterized Gram-negative bacilli and Enterococcus spp. from bacterial cultures. Result The method had 100% sensitivity and between 92 and 100% of specificity for identifying blaKPC, blaVIM, blaIMP, blaNDM, blaOXA-48-like, blaCTX-M and vanA. In nine isolates, unspecific amplifications were detected. The Ct of these false positives was above 33. The Ct of the correctly identified bla and van genes did not surpass 28 and 30, respectively. None of the clinical isolates included as negative controls yielded any amplification. Therefore, the Allplex™ Entero-DR assay is a highly accurate test for the detection of important antibiotic resistance determinants. With this assay, reliable results can be obtained within 3 h. However, according to our data, samples with Ct values greater than 33 should be considered with caution.

Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus. We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.

Flavophospholipol (FPL) is an antimicrobial feed additive that has been approved for use in livestock animals and has the potential to decrease horizontal dissemination of antimicrobial resistance genes. Since previous studies showed that FPL has an inhibitory effect on plasmid transfer, in vitro experiments have proven the efficacy of FPL in reducing the conjugative transfer of plasmids encoding the extended-spectrum β-lactamase (ESBL) and vanA genes. These are among the most important antimicrobial resistance loci known. ESBL-producing Escherichia coli and vancomycin-resistant Enterococcus faecalis (VRE) were exposed to several concentrations of FPL, and transfer frequency and plasmid curing activity were determined. FPL inhibited the conjugative transfer of plasmids harboring ESBL and vanA genes in a concentration-dependent manner in all strains. Further transfer experiments revealed that FPL could decrease or increase transfer frequency depending on plasmid type when transfer frequency was at low levels. The plasmid curing activity of FPL was also observed in ESBL-producing E. coli in a concentration-dependent manner, suggesting that they partially contribute to the inhibition of conjugative transfer. These results suggest that the use of FPL as a feed additive might decrease the dissemination of ESBL and vanA genes among livestock animals.

Objectives The aim of this study was to evaluate the frequency as well as the phenotypic and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) isolates from clinical specimens at three university teaching hospitals in Urmia, Northwest Iran, from 2012–2015. Methods Following identification of the isolates, antibiotic susceptibility testing was performed. The presence of the mecA, vanA and pvl genes was evaluated, and staphylococcal cassette chromosome mec (SCCmec) typing was performed. Results A total of 177 S. aureus isolates were collected from various clinical specimens. Antibiotic susceptibility testing revealed high resistance rates to penicillin (98.9%), followed by erythromycin (61.6%). A total of 95 isolates (53.7%) were confirmed as MRSA. Among the initially screened vancomycin-intermediate S. aureus (VISA) isolates, one isolate with a minimum inhibitory concentration (MIC) of 6 μg/mL harboured the vanA gene. Eleven MRSA isolates (11.6%) were also VRSA. A majority (23/95; 24.2%) of MRSA were classified as SCCmec type III. Only 6 MRSA isolates (6.3%) harboured the pvl gene. Conclusions This study highlights the presence of MRSA along with VISA and VRSA in our setting. To our knowledge, this is the first report showing that a strain can be defined as VISA phenotypically and as VRSA by molecular analysis. Such a finding raises major concerns with regard to control measures and reliable laboratory tests for screening of resistant strains.

The aim of this study was to characterize virulence determinants and antibiotic resistance profiles in enterococci obtained from various clinical sources in the northwest of Iran. A total of 160 enterococcal clinical isolates from various wards of University Teaching Hospitals were collected and specified by biochemical test, from September 2014 to July 2015. Identification of enterococci was confirmed by multiplex PCR in the genus and species level. Antibiotic resistance properties and virulence determinants were examined by phenotypic and molecular methods. Of 160 enterococcal isolates, 125 (78.12%) and 35 (21.88%) isolates were identified as Enterococcus faecalis and Enterococcus faecium, respectively. The most common antibiotic nonsusceptible pattern observed was resistance toward rifampicin [n = 122 (76.25%)] followed by erythromycin [n = 117 (73.12%)]. Among all isolates, gelE [n = 140 (87.5%)], cpd [n = 137 (85.6%)], and asa1 [n = 118 (73.8%)] were the most prevalent virulence genes studied. Thirty isolates (11 E. faecalis, 19 E. faecium) were found to be resistant to vancomycin, with minimum inhibitory concentration of ≥256 μg/ml. Twenty-seven isolates carried the vanA gene, whereas none of the isolates carried vanB. E. faecalis had a considerable ability to show virulence genes and drug resistance. Emergence of antibiotic-resistant enterococci and the high prevalence of virulence traits in our study could be regarded as an alarming situation.

Vancomycin-resistant enterococci (VRE) are pathogens of increasing medical importance. In Brno, Czech Republic, we collected 37 samples from the effluent of a wastewater treatment plant (WWTP), 21 surface swabs from hospital settings, and 59 fecal samples from hospitalized patients and staff. Moreover, we collected 284 gull cloacal swabs from the colony situated 35km downstream the WWTP. Samples were cultured selectively. Enterococci were identified using MALDI-TOF MS, phenotypically tested for susceptibility to antibiotics, and by PCR for occurrence of resistance and virulence genes. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used to examine genotypic diversity. VRE carrying the vanA gene were found in 32 (86%, n=37) wastewater samples, from which we obtained 49 isolates: Enterococcus faecium (44) and Enterococcus gallinarum (2), Enterococcus casseliflavus (2), and Enterococcus raffinosus (1). From 33 (69%) of 48 inpatient stool samples, we obtained 39 vanA-carrying VRE, which belonged to E. faecium (33 isolates), Enterococcus faecalis (4), and Enterococcus raffinosus (2). Nearly one-third of the samples from hospital surfaces contained VRE with the vanA gene. VRE were not detected among gulls. Sixty-seven (84%, n=80) E. faecium isolates carried virulence genes hyl and/or esp. Virulence of E. faecalis was encoded by gelE, asa1, and cylA genes. A majority of the E. faecium isolates belonged to the clinically important sequence types ST17 (WWTP: 10 isolates; hospital: 4 isolates), ST18 (9;8), and ST78 (5;0). The remaining isolates belonged to ST555 (2;0), ST262 (1;6), ST273 (3;0), ST275 (1;0), ST549 (2;0), ST19 (0;1), ST323 (3;0), and ST884 (7;17). Clinically important enterococci carrying the vanA gene were almost continually detectable in the effluent of the WWTP, indicating insufficient removal of VRE during wastewater treatment and permanent shedding of these antibiotic resistant pathogens into the environment from this source. This represents a risk of their transmission to the environment.

Maternal docosahexaenoic acid (DHA) is required during pregnancy to supply for normal fetal growth and development. This pilot study aimed to assess the unknown fatty acid (FA) composition in a cohort of non-pregnant and pregnant Israeli women at term and their offspring on a normal diet without n-3 FA supplementation. The fatty acid profile, analyzed using gas chromatography, showed significantly higher plasma monounsaturated (MUFA) and lower n-6 FA percent distribution with similar n-3 index, in pregnant compared to non-pregnant women. RBC exhibited significantly higher MUFA with similar n-3 index, in pregnant compared to non-pregnant women. N-3 FA significantly correlated between neonates’ plasma, with higher n-3 index, and pregnant women's DHA. Conclusion: DHA levels in non-pregnant and pregnant Israeli women at term were comparable and the DHA in pregnant women's plasma positively correlated with their neonate's level, suggesting an efficient mother-fetus FA transfer and/or fetal fatty acid metabolism to longer FA products.

This study aimed at determining the prevalence, antibiotic resistance patterns, and genetic linkage of Vancomycin Resistant Enterococcus faecium (VREfm) from different sources in the southwest of Iran. A total of 51 VREfm isolates were obtained and subjected to antibiotic susceptibility testing, carriage of virulence genes, and pulsed-field gel electrophoresis (PFGE) method. All the VRE isolates exhibited a high level of resistance to teicoplanin, ampicillin, erythromycin, ciprofloxacin, and gentamicin, also carried the vanA gene. A total of 59% and 34% of the VREfm strains harbored esp and hyl genes, respectively. The results from PFGE showed 31 PFGE patterns including 10 common types (CT) and 21 single types (ST) among the VRE isolates. Furthermore, isolates from different sources in each common type revealed cross transmission between clinical and environmental sources. Overall, the study showed a high prevalence of diverse VRE faecium strains with threatening resistance phenotypes in the environment and clinical sections among different ICU wards of Ahvaz hospitals.

The ability to obtain bacterial genomes from the same host has allowed for comparative studies that help in the understanding of the molecular evolution of specific pathotypes. Avian pathogenic Escherichia coli (APEC) is a group of extraintestinal strains responsible for causing colibacillosis in birds. APEC is also suggested to possess a role as a zoonotic agent. Despite its importance, APEC pathogenesis still has several cryptic pathogenic processes that need to be better understood. In this work, a genome-wide survey of eight APEC strains for genes with evidence of recombination revealed that ∼14% of the homologous groups evaluated present signs of recombination. Enrichment analyses revealed that nine Gene Ontology (GO) terms were significantly more represented in recombinant genes. Among these GO terms, several were noted to be ATP-related categories. The search for positive selection in these APEC genomes revealed 32 groups of homologous genes with evidence of positive selection. Among these groups, we found several related to cell metabolism, as well as several uncharacterized genes, beyond the well-known virulence factors ompC, lamB, waaW, waaL, and fliC. A GO term enrichment test showed a prevalence of terms related to bacterial cell contact with the external environment (e.g., viral entry into host cell, detection of virus, pore complex, bacterial-type flagellum filament C, and porin activity). Finally, the genes with evidence of positive selection were retrieved from genomes of non-APEC strains and tested as were done for APEC strains. The result revealed that none of the groups of genes presented evidence of positive selection, confirming that the analysis was effective in inferring positive selection for APEC and not for E. coli in general, which means that the study of the genes with evidence of positive selection identified in this study can contribute for the better understanding of APEC pathogenesis processes.

Increased enterococcal infections in hospitals and multidrug-resistant and vancomycin-resistant enterococci (VRE) isolated from humans, animals, and food sources raised public health concern on the presence of VRE in multiple sources. We performed a comparative analysis of the antimicrobial resistance and genetics of VRE isolates derived from fresh produce and human fecal samples. Of 389 Enterococcus isolates, 8 fecal and 3 produce isolates were resistant to vancomycin and teicoplanin; all harbored vanA gene. The VRE isolates showed multidrug-resistant properties. The isolates from fresh produce in this study showed to have the common shared characteristics with the isolates from humans by the results of antimicrobial resistance, multilocus sequence typing, and Tn 1546 transposon analysis. Therefore, VRE isolates from fresh produce are likely related to VRE derived from humans. The results suggested that VRE may contaminate vegetables through the environment, and the contaminated vegetables could then act as a vehicle for human infections. Ongoing nationwide surveillance of antibiotic resistance and the promotion of the proper use of antibiotics are necessary.

Background/Purpose During the routine screening of vancomycin-resistant Enterococci faecium (VREfm) we found that VanA phenotype– vanA genotype and VanB phenotype– vanA genotype isolates had an identical Tn 1546 -like element structure. This study aimed to evaluate the genetic background and vanA gene expression to identify the mechanisms of the development of the VanB phenotype– vanA genotype VREfm. Methods Twelve VREfm isolates were collected from a 1500-bed tertiary-care teaching hospital in Beijing. Genetic variations of the Tn 1546 -like element were determined by an overlapping polymerase chain reaction assay and sequencing. The genetic background was determined by pulsed-field gel electrophoresis and mutilocus sequence typing. vanA gene expression was evaluated using a TaqMan quantitative real-time polymerase chain reaction. Results For the 12 isolates, six isolates with the VanA phenotype– vanA genotype and six with the VanB phenotype– vanA genotype were identified. According to the structure analysis of the Tn 1546 -like elements, our isolates were divided into two types. In the four isolates of type A, IS 1542 and IS 1216V were inserted into the orf2-vanR and vanX-vanY regions, respectively. In the eight isolates of type D, a similar insertion as type A occurred, except for an ISE fa4 insertion into IS 1542 . A significant difference in vanA gene expression was observed between the VanA and VanB phenotype isolates in type A, but not in type D. Mutilocus sequence typing and pulsed-field gel electrophoresis analysis showed that these isolates have a different genetic background. Conclusion Our results indicated that the occurrence of the VanB phenotype– vanA genotype might not completely depend on the structure of Tn 1546 -like elements and vanA gene expression.

Summary Background Vancomycin-resistant Enterococcus raffinosus has rarely been associated with nosocomial infection and outbreaks. Aim To report the successful control of a nosocomial outbreak of vanA -type vancomycin-resistant E. raffinosus in a surgical intensive care unit. Methods The investigation of the outbreak is reported with control measures taken. Molecular typing of vancomycin-resistant E. raffinosus isolates was performed by repetitive sequence-based polymerase chain reaction (PCR). Findings Between September and October 2014, vancomycin-resistant E. raffinosus isolates were isolated from four patients. The index patient had been hospitalized previously in Portugal, and was not found to be colonized by vancomycin-resistant enterococci on screening cultures obtained at admission. However, vancomycin-resistant E. raffinosus was isolated from a bile sample 19 days after hospital admission. All four isolates were resistant to both vancomycin and teicoplanin due to the presence of the vanA gene, while remaining susceptible to daptomycin and linezolid. Repetitive sequence-based PCR confirmed the spread of a single vanA -positive E. raffinosus clone. Infection control measures including direct PCR screening on rectal specimens, contact precautions, and cohorting of patients and personnel led to successful control of the outbreak. Conclusion This is the first reported outbreak of vanA -type vancomycin-resistant E. raffinosus in France in both clinical and screening specimens among hospitalized patients. The inability of routine selective screening media to detect the vancomycin-resistant E. raffinosus in the index case likely contributed to the outbreak.

We describe clonal shifts in vanA Enterococcus faecium isolates from clinical samples obtained from patients in Denmark from 2015 to the first quarter (Q1) of 2019. During Q1 2019, the vancomycin-variable enterococci (VVE) ST1421-CT1134 vanA E. faecium became the most dominant vanA E. faecium clone and has spread to all five regions in Denmark. Among 174 E. faecium isolates with vanA, vanB or vanA/vanB genes in Q1 2019, 44% belonged to this type. We describe the clonal shift for vanA Enterococcus faecium during the last 4 years and the national spread of a vancomycin-variable vanA E. faecium ST1421-CT1134 clone in Denmark. The aim is to highlight the importance of using molecular methods for detecting vancomycin-variable enterococci (VVE), and to alert other countries about this emerging nosocomial clone.

Background The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) represents a challenge for the treatment of staphylococcal infections in both human and animals worldwide. Although VRSA has been detected in several animal species worldwide, data on the bacterial prevalence in dromedary camels and workers in camel slaughterhouses are scarce. Methods We investigated meat samples from 200 dromedary camel carcasses from three different abattoirs that were being prepared to be sent to the markets. Twenty hand swabs were voluntarily collected from the workers in the same abattoirs. Isolation and identification of the bacterial specimens from the samples were performed using conventional cultural techniques and biochemical identification and were confirmed by PCR amplification of the nuc gene. Antimicrobial susceptibility against nine antimicrobial agents commonly used in human and camels was tested using the disc diffusion method, and genetic analysis was performed by evaluating the mecA gene in phenotypically oxacillin (OXA)- and cefoxitin (FOX)-resistant isolates. The resistance of S. aureus to vancomycin (VAN) was tested by broth microdilution and confirmed by PCR targeting the vanA and vanB genes. The vanA and vanB genes were sequenced. Result S. aureus was detected in both camel meat (29/200, 14.5%) and in abattoir workers (11/20, 55%). Of the collected samples, 27% (8/29, camel) and 54% (6/11, human) were identified as VRSA. All VRSA isolates carried both the vanA and vanB genes. Additionally, all VRSA isolates were also classified as methicillin-resistant S. aureus (MRSA). The vanA amplicons of the isolates from human and camel meat were homologous and clustered with a Chinese reference isolate sequence. Conclusion This study demonstrated that VRSA is present in camel abattoirs in Egypt. Zoonotic transmission between animals and human is probable and reflects both a public health threat and a food safety concern.

Chemiresistive platforms are best suited for developing DNA hybridization detection systems, owing to their ease of fabrication, simple detection methodology and amenability towards electronics. In this work, we report development of a generic, robust, electrospun nanofiber based interdigitated chemiresistive platform for DNA hybridization detection. The platform comprises of interdigitated metal electrodes decorated with electrospun nanofibers on the top. Two approaches viz., drop casting of graphene doped Mn2O3 nanofibers (GMnO) and direct electrospinning of polyaniline/polyethylene oxide (PANi/PEO) composite nanofibers, have been utilized to decorate these electrodes. In both approaches, inter-device variability, a key challenge for converting this proof-of-concept into a tangible prototype/product, has been addressed using a shadow masking technique. Consequently, the relative standard deviation for multiple PANi/PEO nanofiber based chemiresistors has been brought down from 17.82% (without shadow masking) to 4.41% (with shadow masking). The nanofibers are further modified with single-stranded probe DNAs, to capture a desired hybridization event. To establish the generic nature of the platform, detection of multiple target DNAs has been successfully demonstrated. These targets include dengue virus specific consensus primer (DENVCP) and four DNAs corresponding to Staphylococcus aureus specific genes, namely nuc, mecA, vanA and protein A. The chemiresistive detection of DENVCP has been performed in the concentration range of 10 fM – 1 µM, whereas the detection of the other targets has been carried out in the range of 1 pM – 1 µM. Using a 3σ method, we have estimated the limit of detection for the chemiresistive detection of DENVCP to be 1.9 fM.

Vancomycin-resistant Enterococcus spp. is a major health problem worldwide and livestock have been implicated in constituting a reservoir for the transmission of vancomycin resistance to zoonotic pathogens. Vancomycin resistance determinants can be situated on mobile genetic elements and transferred between bacterial species The livestock reservoir must therefore be included in a risk assessment of the vancomycin resistance burden. Avoparcin, a vancomycin analogue, has not been used in Danish pig production for over 20 years and vancomycin has never been used. The objective of this study was to screen faecal samples from Danish pig farms for nine selected vancomycin resistance determinants. We found at least four different vancomycin resistance determinants in all screened Danish pig farms (665 finisher farms and 78 sow farms). The vancomycin resistance determinants present in vanB or vanG clusters were found at significantly different levels in sow and finisher farms. However, vanA was not detected in any of the farms. In conclusion, vancomycin resistance determinants are still present in Danish pig production 25 years after the ban on avoparcin use.

The quality of PCR to detect vancomycin-resistant enterococci (VRE) was evaluated by analysing their performance in six consecutive external quality assessment (EQA) schemes, organized annually since 2013 by Quality Control for Molecular Diagnostics. VRE EQA panels consisted of 12–14 heat-inactivated samples. Sensitivity was tested with vanA-positive Enterococcus faecium (E. faecium), vanB-positive E. faecium, E. faecalis or E. gallinarum or vanC-positive E. gallinarum in different concentrations. Vancomycin-susceptible enterococci, Staphylococcus aureus or sample matrix was used to study the specificity. Participants were asked to report the VRE resistance status of each sample. The detection rate of vanA-positive samples was already 95% in the 2013 EQA panel (range 94–97%) and remained stable over the years. The 2013 detection rate of vanB-positive samples was 82% but increased significantly by more than 10% in subsequent years (96% in 2014, 95% in 2015, 92% in 2016 and 93% in 2017/2018, p

Objectives An Enterococcus faecium isolate (Efa-125) carrying both the vanA and vanB genes was recovered from a patient with bacteraemia treated in a Greek hospital. Since this is the first description in Europe of E. faecium carrying both vanA and vanB genes, the isolate was further studied. Methods Susceptibility to several antibiotics was determined using the VITEK®2 automated system. The isolate was typed by multilocus sequence typing (MLST). To define the genetic units of the vanA and vanB genes, the plasmid content of Efa-125 was analysed by pulsed-field gel electrophoresis (PFGE) of total DNA digested with S1 nuclease followed by hybridisation with digoxigenin-labelled vanA and vanB probes. In addition, plasmids and chromosomes were sequenced using the Illumina MiSeq platform. Results E. faecium Efa-125 belonged to ST117 and expressed resistance both to vancomycin and teicoplanin, with minimum inhibitory concentrations (MICs) for both of 256 mg/L. The vanA gene was carried on a 29 320-bp plasmid exhibiting high similarity to pA6981 previously characterised from Enterococcus gallinarum A6981, whereas vanB was part of a Tn1549-like transposon integrated into the chromosome. Expression of the VanA phenotype was correlated with the presence of intact vanZ and vanS genes. Conclusions This is the first detection in Greece of vanA–vanB genotype/VanA phenotype E. faecium and indicates an evolving epidemiology of vancomycin-resistant enterococci.