Fitness costs of Tn1546-type transposons harboring the vanA operon by plasmid type and structural diversity in Enterococcus faecium.

BACKGROUND: This study analyzed the genetic traits and fitness costs of vancomycin-resistant Enterococcus faecium (VREfm) blood isolates carrying Tn1546-type transposons harboring the vanA operon. METHODS: All E. faecium blood isolates were collected from eight general hospitals in South Korea during one-year study period. Antimicrobial susceptibility testing and vanA and vanB PCR were performed. Growth rates of E. faecium isolates were determined. The vanA-positive isolates were subjected to whole genome sequencing and conjugation experiments. RESULTS: Among 308 E. faecium isolates, 132 (42.9%) were positive for vanA. All Tn1546-type transposons harboring the vanA operon located on the plasmids, but on the chromosome in seven isolates. The plasmids harboring the vanA operon were grouped into four types; two types of circular, nonconjugative plasmids (Type A, n = 50; Type B, n = 46), and two types of putative linear, conjugative plasmids (Type C, n = 16; Type D, n = 5). Growth rates of vanA-positive E. faecium isolates were significantly lower than those of vanA-negative isolates (P < 0.001), and reduction in growth rate under vancomycin pressure was significantly larger in isolates harboring putative linear plasmids than in those harboring circular plasmids (P = 0.020). CONCLUSIONS: The possession of vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination. Fitness burden to bacterial hosts was varied by type and size of the vanA operon-harboring plasmid.

Surveillance of vancomycin-resistant enterococci reveals shift in dominating clusters from vanA to vanB Enterococcus faecium clusters, Denmark, 2015 to 2022.

BackgroundVancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible.AimThe aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE.MethodsFrom 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance.ResultsIn total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E. faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm.ConclusionFrom 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.

[Silencing of Resistance Mechanism in Vancomycin-Resistance Enterococci Using Antisense RNA of vanA Gene]. / vanA Geninin Antisens RNA'si Kullanilarak Vankomisine Dirençli Enterokoklarda Direnç Mekanizmasinin Susturulmasi.

The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.

The epidemiology of enterococci in a tertiary hospital and primary healthcare facilities in Fiji (2019-2022).

OBJECTIVES: We analysed 4 y of laboratory data to characterise the species and determine the antimicrobial susceptibility profiles of enterococci as human pathogens in Fiji. The study also investigated the molecular epidemiology amongst the subset of vancomycin-resistant enterococci (VRE). METHODS: This retrospective study reviewed bacteriological data from Colonial War Memorial Hospital (CWMH) and other healthcare facilities in the Central and Eastern divisions of Fiji. Phenotypic, antimicrobial susceptibility and vanA and vanB PCR testing were performed using locally approved protocols. The first clinical isolates per patient with antimicrobial susceptibility testing results in a single year were included in the analysis. Data was analysed using WHONET software and Microsoft Excel. RESULTS: A total of 1817 enterococcal isolates were reported, 1415 from CWMH and 402 from other healthcare facilities. The majority of isolates, 75% (n = 1362) were reported as undifferentiated Enterococcus spp., 17.8% (n = 324) were specifically identified as Enterococcus faecalis and 6.7% (n = 122) as E. faecium. Overall, 10% of the enterococci isolates were from blood cultures. Among isolates from CWMH, <15% of E. faecium were susceptible to ampicillin, and 17.2% were vancomycin resistant. Overall, 874 enterococcal isolates (including the undifferentiated species) were tested against vancomycin, of which 4.8% (n = 42) were resistance. All of the VRE isolates tested (n = 15) expressed vanA genes. CONCLUSIONS: This study demonstrates the clinical importance of VRE, particularly van A E. faecium in the national referral hospital in Fiji. Enhanced phenotypic and molecular surveillance data are needed to better understand enterococci epidemiology and help guide specific infection prevention and control measures and antibiotic prescribing guidelines.

Mechanisms of acquisition of the vanA operon among vancomycin-resistant Staphylococcus aureus genomes: The tip of the iceberg?

BACKGROUND: Vancomycin is frequently used as a last line of defence against infections due to multidrug-resistant Staphylococcus aureus (S. aureus). A recent finding described the acquisition of vancomycin-resistant S. aureus strains by the integration of an enterococcal plasmid containing the vanA operon into the S. aureus chromosome via homologous recombination involving a specific integration site called locus L2. METHODS: To characterise all mechanisms of acquisition of vanA, this study analysed the 15 706 S. aureus genomes to look for vanA and described its genetic environment. RESULTS: A complete vanA operon was found in 25 S. aureus strains isolated from 12 patients, including nine co-isolated with vancomycin-resistant Enterococcus strains. VanA was found within transposon Tn1546-like elements on 17 plasmids and eight chromosomes. VanA might be acquired through conjugation of enterococcal and staphylococcal plasmids, transposition of Tn1546 carrying vanA and plasmid integration into the chromosome. Further, L2 was detected in 2087 genomes (13.3%) of S. aureus strains across different continents. Six potential chromosomal hotspots for integration of the entire vanA-containing enterococcal plasmid were identified by homologous recombination via L2. CONCLUSIONS: These findings suggest that the recently described scenario in a New York patient could be reproduced anywhere. Surveillance of this possibility is mandatory, especially in patients with vancomycin-resistant Enterococcus infection or colonisation.

Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Prevalence and Molecular Characterization of Vancomycin Variable Enterococcus faecium Isolated From Clinical Specimens.

Vancomycin variable Enterococcus (VVE) bacteria are phenotypically susceptible to vancomycin, but they harbor the vanA gene. We aimed to ascertain the prevalence of VVE among clinically isolated vancomycin-susceptible Enterococcus faecium (VSE) isolates, as well as elucidate the molecular characteristics of the vanA gene cluster within these isolates. Notably, we investigated the prevalence and structure of the vanA gene cluster of VVE. Between June 2021 and May 2022, we collected consecutive, non-duplicated vancomycin-susceptible Enterococcus faecium (VSE) samples. Real-time PCR was performed to detect the presence of vanA, vanB, and vanC. Overlapping PCR with sequencing and whole-genome sequencing were performed for structural analysis. Sequence types (STs) were determined by multilocus sequence typing. Exposure testing was performed to assess the ability of the isolates to acquire vancomycin resistance. Among 282 VSE isolates tested, 20 isolates (7.1%) were VVE. Among them, 17 isolates had partial deletions in the IS1216 or IS1542 sequences in vanS (N=10), vanR (N=5), or vanH (N=2). All VVE isolates belonged to the CC17 complex and comprised five STs, namely ST17 (40.0%), ST1421 (25.0%), ST80 (25.0%), ST787 (5.0%), and ST981 (5.0%). Most isolates were related to three hospital-associated clones (ST17, ST1421, and ST80). After vancomycin exposure, 18 of the 20 VVEs acquired vancomycin resistance. Considering the high reversion rate, detecting VVE by screening VSE for vanA is critical for appropriate treatment and infection control.

A Review of Detection Methods for Vancomycin-Resistant Enterococci (VRE) Genes: From Conventional Approaches to Potentially Electrochemical DNA Biosensors.

Vancomycin-resistant Enterococci (VRE) genes are bacteria strains generated from Gram-positive bacteria and resistant to one of the glycopeptides antibiotics, commonly, vancomycin. VRE genes have been identified worldwide and exhibit considerable phenotypic and genotypic variations. There are six identified phenotypes of vancomycin-resistant genes: VanA, VanB, VanC, VanD, VanE, and VanG. The VanA and VanB strains are often found in the clinical laboratory because they are very resistant to vancomycin. VanA bacteria can pose significant issues for hospitalized patients due to their ability to spread to other Gram-positive infections, which changes their genetic material to increase their resistance to the antibiotics used during treatment. This review summarizes the established methods for detecting VRE strains utilizing traditional, immunoassay, and molecular approaches and then focuses on potential electrochemical DNA biosensors to be developed. However, from the literature search, no information was reported on developing electrochemical biosensors for detecting VRE genes; only the electrochemical detection of vancomycin-susceptible bacteria was reported. Thus, strategies to create robust, selective, and miniaturized electrochemical DNA biosensor platforms to detect VRE genes are also discussed.

Check the melting temperature of the FilmArray BCID panel to avoid missed detection of vanM-type enterococci.

PURPOSE: We aimed to evaluate whether the FilmArray blood culture identification (BCID) panel holds the ability to detect vanM-type vancomycin-resistant enterococci (VRE) clinical isolates effectively. METHODS: Twenty VRE clinical strains, including 10 vanA-type VRE and 10 vanM-type VRE, were collected from patients in five tertiary hospitals, Shanghai, China. By conventional PCR and sequencing, the strains were identified and van genotypes were confirmed. All VRE strains were investigated using the FilmArray BCID panel. All results, including enterococcus assay, vanA/B assay, DNA melting curves and melting temperature (Tm), were recorded. We also compared these results with those obtained via the conventional PCR and sequencing. RESULTS: According to the instructions of the FilmArray BCID panel, the Enterococcus assay is used to identify species and vanA/B assay is used to detect van genes. In all vanA-type VRE, the Enterococcus assay and vanA/B assay were positive. The results correctly showed that the tested strains were VRE. However, in 10 vanM-type VRE, the Enterococcus assay was positive and vanA/B assay were negative. The results mistakenly showed that the tested strains were vancomycin-sensitive enterococci (VSE). In the vanA/B assay, the melting curves of vanM-type VRE were similar to that of vanA-type VRE, but the Tm values were lower. The Tm values were then compared against the expected Tm range for the vanA/B assay. The Tm values of vanM-type VRE fall outside the assay-specific Tm range, resulting in negative reports. Thus, by adjusting the expected Tm range for the Enterococcus assay, the FilmArray BCID panel holds the ability to detect vanM-type VRE. CONCLUSIONS: The vanM-type VRE isolates can be effectively detected by optimizing the expected Tm range for the vanA/B assay.

Structural basis for the transformation of the traditional medicine berberine by bacterial nitroreductase.

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 Šfrom 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.

MoS2 nanosheet induced destructive alterations in the Escherichia coli bacterial membrane.

Two dimensional molybdenum disulfide (MoS2) nanosheets have recently gained wide recognition for their efficient broad-spectrum antibacterial activity complemented with great biocompatibility and minimal bacterial resistance inducing capabilities. However, despite the numerous investigations, the molecular level interactions at the nano-bio interface responsible for their bactericidal activity remain obscure. Herein, through an atomistic molecular dynamics study, we attempt to seek an in-depth understanding of the atomic level details of the underlying mechanism of their antibacterial action against the Escherichia coli (E. coli) bacterial membrane. Our study reveals a two-step MoS2 nanosheet interaction pathway with the bacterial membrane. The nanosheets spontaneously adhere to the membrane surface and prompt vigorous phospholipid extraction majorly via strong van der Waals interactions with lipid hydrophobic tails. The lipid extraction process originates a significant water intrusion in the bilayer hydrophobic region, signifying the onset of cytoplasmic leakage under realistic conditions. Further, a synergistic effect of lipid-lipid self-interactions and lipid-MoS2 dispersion interactions drags the nanosheet to completely immerse in the bilayer hydrophobic core. The embedded nanosheets induce a layerwise structural rearrangement of the membrane lipids in their vicinity, thus altering the structural and dynamic features of the membrane in a localized manner by (i) increasing the lipid fatty acyl tail ordering and (ii) alleviating the lipid lateral dynamics. The detrimental efficacy of the nanosheets can be magnified by enlarging the nanosheet size or by increasing the nanosheet concentration. Our study concludes that the MoS2 nanosheets can exhibit their antibacterial action through destructive phospholipid extraction as well as by altering the morphology of the membrane by embedding in the membrane core.

VanA-Enterococcus faecalis in Poland: hospital population clonal structure and vanA mobilome.

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.

Development of immobilized beta1-adrenoceptor chromatography for rapid discovery of ligands specifically binding to the receptor from herbal extract.

The discovery of beta1-adrenoceptor (ß1-AR) ligands is viewed as an enormous demand for fighting ailments mediated by the receptor including cardiovascular diseases. Such pursuit is gravely challenged due to the lack of lead screening methods with high efficiency. This work developed a chromatographic method for pursuing ß1-AR ligand from the herbal extract by fusing epidermal growth factor receptor (EGFR) as a tag at its C-terminus to stably express the fusion receptor in E. coli, immobilizing the expressed EGFR-tagged ß1-AR onto ibrutinib-derivatized amino microspheres, and applying the immobilized receptor in the analysis of ligand-receptor interaction and herbal extract. Comprehensive characterizations like X-ray photoelectron spectroscopy and retention behaviors of canonical drugs demonstrated high specificity and good stability of the immobilized ß1-AR prepared through the covalent reaction between the EGFR and ibrutinib decorated on the microsphere surface. Frontal analysis of atenolol, metoprolol, and esmolol confirmed their bindings to ß1-AR with association constants of 1.07 × 104, 6.54 × 103, and 1.45 × 104 M-1. The thermodynamic analysis provided proof of electrostatic interaction, hydrogen bonds, and van der Waals force driving those interactions. Pulegone was recognized as a bioactive compound that specifically binding to ß1-AR from the extract of Ziziphora clinopodioides Lam by analyzing the retention peak through reverse-phase high performance liquid chromatography coupled with tandem mass spectrometry. These results, taken together, indicated that the current method is possible to provide an alternative for discovering ß1-AR ligands with high efficiency from complex matrices like herbal extract.

In Silico Evaluation of the Antimicrobial Activity of Thymol-Major Compounds in the Essential Oil of Lippia thymoides Mart. & Schauer (Verbenaceae).

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 Lippia thymoides (L. thymoides) Mart. & Schauer (Verbenaceae). It was previously reported that this EO exhibits antimicrobial activity against Candida albicans (C. albicans), Staphylococcus aureus (S. aureus), and Escherichia coli (E. coli). Therefore, we used molecular docking, molecular dynamics simulations, and free energy calculations to investigate the interaction of thymol with pharmacological receptors of interest to combat these pathogens. We found that thymol interacted favorably with the active sites of the microorganisms' molecular targets. MolDock Score results for systems formed with CYP51 (C. albicans), Dihydrofolate reductase (S. aureus), and Dihydropteroate synthase (E. coli) were -77.85, -67.53, and -60.88, respectively. Throughout the duration of the MD simulations, thymol continued interacting with the binding pocket of the molecular target of each microorganism. The van der Waals (ΔEvdW = -24.88, -26.44, -21.71 kcal/mol, respectively) and electrostatic interaction energies (ΔEele = -3.94, -11.07, -12.43 kcal/mol, respectively) and the nonpolar solvation energies (ΔGNP = -3.37, -3.25, -2.93 kcal/mol, respectively) were mainly responsible for the formation of complexes with CYP51 (C. albicans), Dihydrofolate reductase (S. aureus), and Dihydropteroate synthase (E. coli).

Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

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.

Optimising genomic approaches for identifying vancomycin-resistant Enterococcus faecium transmission in healthcare settings.

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.

Genetic description of VanD phenotype vanA genotype in vancomycin-resistant Enterococcus faecium isolates from a Bone Marrow Transplantation Unit.

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.

Evaluation of GeneXpert vanA/vanB in the early diagnosis of vancomycin-resistant enterococci infection.

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.

Blood fatty acid analysis reveals similar n-3 fatty acid composition in non-pregnant and pregnant women and their neonates in an Israeli pilot study.

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.