Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA-MurG interaction.

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

Asp50Glu mutation in MurA results in fosfomycin resistance in Enterococcus faecium.

OBJECTIVES: Enterococcus faecium is one of the important pathogens causing nosocomial infection, which can be resistant to fosfomycin by obtaining the plasmid-encoded fosfomycin resistance genes, and the mutation of MurA protein encoded by chromosome is a newly discovered fosfomycin resistance mechanism in recent years. METHODS: In this study, we found a fosfomycin-resistant clinical isolate of E. faecium Efm_1415 with fosfomycin MIC of 512 mg/L, carrying Asp50Glu mutant of MurA protein, which was never reported before. To study the role and mechanism of this mutant protein in fosfomycin resistance, we used gene cloning, protein expression, and purification, steady-state kinetic, fosfomycin inhibition assay, and next-generation sequencing (NGS) to investigate the functions, characters, and enzymatic kinetic properties of MurA protein. RESULTS: The results revealed that the Asp50Glu MurA can mediate a 4-fold increase in the fosfomycin MIC of the host bacteria. Compared with the wild-type MurA, the affinity of the Asp50Glu MurA to the substrates was increased, and the enzyme activity cannot be inhibited by the concentration of fosfomycin less than 100 mg/L. CONCLUSIONS: The research on the mutant MurA had gained a new understanding of the fosfomycin resistance mechanisms and helped to find new antibiotics with MurA enzyme as the target of action.

Rare transmission of commensal and pathogenic bacteria in the gut microbiome of hospitalized adults.

Bacterial bloodstream infections are a major cause of morbidity and mortality among patients undergoing hematopoietic cell transplantation (HCT). Although previous research has demonstrated that pathogens may translocate from the gut microbiome into the bloodstream to cause infections, the mechanisms by which HCT patients acquire pathogens in their microbiome have not yet been described. Here, we use linked-read and short-read metagenomic sequencing to analyze 401 stool samples collected from 149 adults undergoing HCT and hospitalized in the same unit over three years, many of whom were roommates. We use metagenomic assembly and strain-specific comparison methods to search for high-identity bacterial strains, which may indicate transmission between the gut microbiomes of patients. Overall, the microbiomes of patients who share time and space in the hospital do not converge in taxonomic composition. However, we do observe six pairs of patients who harbor identical or nearly identical strains of the pathogen Enterococcus faecium, or the gut commensals Akkermansia muciniphila and Hungatella hathewayi. These shared strains may result from direct transmission between patients who shared a room and bathroom, acquisition from a common hospital source, or transmission from an unsampled intermediate. We also identify multiple patients with identical strains of species commonly found in commercial probiotics, including Lactobacillus rhamnosus and Streptococcus thermophilus. In summary, our findings indicate that sharing of identical pathogens between the gut microbiomes of multiple patients is a rare phenomenon. Furthermore, the observed potential transmission of commensal, immunomodulatory microbes suggests that exposure to other humans may contribute to microbiome reassembly post-HCT.

In vitro activities of thiazolidione derivatives combined with daptomycin against clinical Enterococcus faecium strains.

BACKGROUND: Previous reports have demonstrated two thiazolidione derivatives (H2-60 and H2-81) can robustly inhibit the planktonic growth and biofilm formation of S. epidermidis and S. aureus by targeting the histidine kinase YycG. Whereas the antibacterial and anti-biofilm activity of these two thiazolidione derivatives (H2-60 and H2-81) against Enterococcus faecium remains elusive. Here, the pET28a-YycG recombinant plasmid were in vitro expressed in E. coli competent cell BL21 (DE3) and induced to express YycG' protein (conding HisKA and HATPase_c domain) by 0.5 mM IPTG and was purified by Ni - NTA agarose and then for the autophosphorylation test. Antimicrobial testing and time-killing assay were also be determined. Anti-biofilm activity of two derivatives with sub-MIC concentration towards positive biofilm producers of clinical E. faecium were detected using polystyrene microtiter plate and CLSM. RESULTS: The MICs of H2-60 and H2-81 in the clinical isolates of E. faecium were in the range from 3.125 mg/L to 25 mg/L. Moreover, either H2-60 or H2-81 showed the excellent bactericidal activity against E. faecium with monotherapy or its combination with daptomycin by time-killing assay. E. faecium planktonic cells can be decreased by H2-60 or H2-81 for more than 3 × log10 CFU/mL after 24 h treatment when combined with daptomycin. Furthermore, over 90% of E. faecium biofilm formation could markedly be inhibited by H2-60 and H2-81 at 1/4 × MIC value. In addition, the frequency of the eradicated viable cells embedded in mature biofilm were evaluated by the confocal laser microscopy, suggesting that of H2-60 combined with ampicillin or daptomycin was significantly high when compared with single treatment (78.17 and 74.48% vs. 41.59%, respectively, P < 0.01). CONCLUSION: These two thiazolidione derivatives (H2-60 and H2-81) could directly impact the kinase phosphoration activity of YycG of E. faecium. H2-60 combined with daptomycin exhibit the excellent antibacterial and anti-biofilm activity against E. faecium by targeting YycG.

Establishment and Persistence of Glycopeptide-Resistant Enterococcus faecium ST80 and ST117 Clones Within a Health Care Facility Located in a Low-Prevalence Geographical Region.

Vancomycin-resistant Enterococcus faecium (VREfm) is one of the most important nosocomial pathogens with limited therapeutic alternatives. In this study, we followed the trends of VREfm and E. faecium causing bloodstream infections (BSIs) in a Spanish hospital, from 2011 to 2020. During this period, 832 E. faecium strains were isolated and 121 (14.5%) were vancomycin resistant. Nineteen of 101 BSIs (18.8%) caused by E. faecium were due to VREfm. The number of BSI-producing E. faecium isolates increased significantly over the past 5 years, with the percentage of invasive VREfm isolates being substantially higher than the average values in Europe and especially in Spain (<3%). VREfm isolates recovered in 2018 (28) and BSI-producing isolates from 2019 (3) and 2020 (2) were molecularly characterized. All were positive for vanA and belonged to sequence type (ST) 80 (28) or ST117 (5), within clonal complex 17. The isolates were only susceptible to linezolid, although most of them were also susceptible (dose dependent) to daptomycin. We report for the first time the establishment and persistence of the VREfm ST80 and ST117 clones in a Spanish hospital. The spread and establishment of hospital-adapted, multidrug-resistant VREfm clones in health care settings are cause for concern and may precede an increment in the BSIs caused by them.

Substantial Decrease in Vancomycin-Resistant Enterococcus faecium Outbreak Duration and Number of Patients During the Danish COVID-19 Lockdown: A Prospective Observational Study.

Vancomycin-resistant Enterococcus faecium (VREfm) is a globally significant nosocomial pathogen with a rapidly increasing prevalence. The objectives were to investigate VREfm outbreak duration and study the additional impact that infection control bundle strategies (ICBSs) set up to curb coronavirus disease 2019 (COVID-19) spreading had on VREfm outbreaks. Outbreak data set were collected prospectively from April 2, 2014 to August 13, 2020 at Copenhagen University Hospital Bispebjerg, Denmark. All VREfm samples had polymerase chain reaction performed for vanA/vanB genes before whole genome sequencing using the Illumina MiSeq platform. The relatedness of isolates was studied by core genome multilocus sequence typing (cgMLST) using Ridom SeqSphere. Eighty-one outbreaks had a median outbreak duration of 32.5 days (range 5-204 days) and 1,161 VREfm isolates were sequenced. The same cgMLST cluster types reappeared after outbreaks were terminated. When comparing the first 5 months of the COVID-19 pandemic with the corresponding period in 2019, we found a 10-fold decrease in VREfm outbreak patients and median outbreak duration decreased from 56 to 7 days (88%). Several COVID-19 ICBSs were implemented from March 13 through summer 2020. VREfm outbreaks lasted up to 204 days, but our findings suggest that outbreaks might last longer since the same cgMLST persisted in the same wards for years implying an endemic situation with recurrent outbreaks caused by hospital reservoirs or readmittance of unknown VREfm carriers. The sharp decline in VREfm outbreaks during the COVID-19 pandemic was most likely due to the ICBSs, resulting in a decrease in VREfm transmission.

Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea.

The use of phenicol antibiotics in animals has increased. In recent years, it has been reported that the transferable gene mediates phenicol-oxazolidinone resistance. This study analyzed the prevalence and characteristics of phenicol-oxazolidinone resistance genes in Enterococcus faecalis and Enterococcus faecium isolated from food-producing animals and meat in Korea in 2018. Furthermore, for the first time, we reported the genome sequence of E. faecalis strain, which possesses the phenicol-oxazolidinone resistance gene on both the chromosome and plasmid. Among the 327 isolates, optrA, poxtA, and fexA genes were found in 15 (4.6%), 8 (2.5%), and 17 isolates (5.2%), respectively. Twenty E. faecalis strains carrying resistance genes belonged to eight sequence types (STs), and transferability was found in 17 isolates. The genome sequences revealed that resistant genes were present in the chromosome or plasmid, or both. In strains EFS17 and EFS108, optrA was located downstream of the ermA and ant(9)-1 genes. The strains EFS36 and EFS108 harboring poxtA-encoding plasmid cocarried fexA and cfr(D). These islands also contained IS1216E or the transposon Tn554, enabling the horizontal transfer of the phenicol-oxazolidinone resistance with other antimicrobial-resistant genes. Our results suggest that it is necessary to promote the prudent use of antibiotics through continuous monitoring and reevaluation.

Three New Hydroxyphenylacetic Acid Derivatives and A New Alkaloid from Endophytic Fungus Mortierella sp. in Epimedium acuminatum Franch. and Their Antibacterial Activity.

Three new hydroxyphenylacetic acid derivatives, stachylines E-G (1-3), and a new alkaloid, mortieridinone (4), along with six known compounds (5-10), were isolated from endophytic fungus Mortierella sp. in Epimedium acuminatum Franch. Their structures were determined by their spectroscopic analyses and by comparison with the literature data. Compounds 7 and 10 showed selective antibacterial activity against tested multidrug-resistant bacteria with minimum inhibitory concentration (MIC) values ranging from 25 to 3.13 µg/mL.

Clinically Applicable System for Rapidly Predicting Enterococcus faecium Susceptibility to Vancomycin.

Enterococcus faecium is a clinically important pathogen that can cause significant morbidity and death. In this study, we aimed to develop a machine learning (ML) algorithm-based rapid susceptibility method to distinguish vancomycin-resistant E. faecium (VREfm) and vancomycin-susceptible E. faecium (VSEfm) strains. A predictive model was developed and validated to distinguish VREfm and VSEfm strains by analyzing the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) spectra of unique E. faecium isolates from different specimen types. The algorithm used 5,717 mass spectra, including 2,795 VREfm and 2,922 VSEfm mass spectra, and was externally validated with 2,280 mass spectra of isolates (1,222 VREfm and 1,058 VSEfm strains). A random forest-based algorithm demonstrated overall good classification performances for the isolates from the specimens, with mean accuracy, sensitivity, and specificity of 0.78, 0.79, and 0.77, respectively, with 10-fold cross-validation, timewise validation, and external validation. Furthermore, the algorithm provided rapid results, which would allow susceptibility prediction prior to the availability of phenotypic susceptibility results. In conclusion, an ML algorithm designed using mass spectra obtained from the routine workflow may be able to rapidly differentiate VREfm strains from VSEfm strains; however, susceptibility results must be confirmed by routine methods, given the demonstrated performance of the assay. IMPORTANCE A modified binning method was incorporated to cluster MS shifting ions into a set of representative peaks based on a large-scale MS data set of clinical VREfm and VSEfm isolates, including 2,795 VREfm and 2,922 VSEfm isolates. Predictions with the algorithm were significantly more accurate than empirical antibiotic use, the accuracy of which was 0.50, based on the local epidemiology. The algorithm improved the accuracy of antibiotic administration, compared to empirical antibiotic prescription. An ML algorithm designed using MALDI-TOF MS spectra obtained from the routine workflow accurately differentiated VREfm strains from VSEfm strains, especially in blood and sterile body fluid samples, and can be applied to facilitate the rapid and accurate clinical testing of pathogens.

Sintered and 3D-Printed Bulks of MgB2-Based Materials with Antimicrobial Properties.

Pristine high-density bulk disks of MgB2 with added hexagonal BN (10 wt.%) were prepared using spark plasma sintering. The BN-added samples are machinable by chipping them into desired geometries. Complex shapes of different sizes can also be obtained by the 3D printing of polylactic acid filaments embedded with MgB2 powder particles (10 wt.%). Our present work aims to assess antimicrobial activity quantified as viable cells (CFU/mL) vs. time of sintered and 3D-printed materials. In vitro antimicrobial tests were performed against the bacterial strains Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Enterococcus faecium DSM 13590, and Enterococcus faecalis ATCC 29212; and the yeast strain Candida parapsilosis ATCC 22019. The antimicrobial effects were found to depend on the tested samples and microbes, with E. faecium being the most resistant and E. coli the most susceptible.

Bacteriophage Rescue Therapy of a Vancomycin-Resistant Enterococcus faecium Infection in a One-Year-Old Child following a Third Liver Transplantation.

Phage therapy is an experimental therapeutic approach used to target multidrug-resistant bacterial infections. A lack of reliable data with regard to its efficacy and regulatory hurdles hinders a broad application. Here we report, for the first time, a case of vancomycin-resistant Enterococcus faecium abdominal infection in a one-year-old, critically ill, and three times liver transplanted girl, which was successfully treated with intravenous injections (twice per day for 20 days) of a magistral preparation containing two Enterococcus phages. This correlated with a reduction in baseline C-reactive protein (CRP), successful weaning from mechanical ventilation and without associated clinical adverse events. Prior to clinical use, phage genome was sequenced to confirm the absence of genetic determinants conferring lysogeny, virulence or antibiotic resistance, and thus their safety. Using a phage neutralization assay, no neutralizing anti-phage antibodies in the patient's serum could be detected. Vancomycin-susceptible E. faecium isolates were identified in close relation to phage therapy and, by using whole-genome sequencing, it was demonstrated that vancomycin-susceptible E. faecium emerged from vancomycin-resistant progenitors. Covering a one year follow up, we provide further evidence for the feasibility of bacteriophage therapy that can serve as a basis for urgently needed controlled clinical trials.

Clonal spread and horizontal transfer mediate dissemination of phenicol-oxazolidinone-tetracycline resistance gene poxtA in enterococci isolates from a swine farm in China.

The emergence of the phenicol-oxazolidinone-tetracycline resistance gene poxtA becomes a significant challenge for public health, since it confers a decreased susceptibility not only to the last resort drug linezolid, but also to florfenicol and doxycycline widely used in veterinary medicine. To determine the dissemination mechanism of poxtA in enterococci isolates from different healthy pigs in the swine farm, a total of 178 florfenicol-resistant enterococci isolates were collected from 400 fresh faecal swabs in a swine farm in China. The poxtA gene was detected in 11 (6.18 %) enterococci isolates, including 8 E. faecium, 2 E. hirae and 1 E. casseliflavus isolates. Whole genome sequencing indicated that the eight poxtA-harbouring E. faecium strains belonged to four different sequence types, including ST156 and three new STs, ST1818, ST1819 and ST1820. Five out of the 11 poxtA-positive enterococci isolates also harboured optrA gene. Moreover, E. casseliflavus strain DY31 co-harboured poxtA, optrA and cfr. Seven different poxtA-harbouring plasmids were obtained through Nanopore combined with Illumina sequencing. The poxtA-harbouring plasmids exhibited high genetic variation, six out of which belonged to rep2 plasmid of Inc18 family. The poxtA gene was flanked by IS1216E in the left and/or right ends.The optrA and cfr genes were located on different plasmids, respectively, but those genes could be co-transferred with poxtA gene into the recipient E. faecalis strain by electrotransformation. Our study highlights that both clonal spread and horizontal transfer mediated by Inc18 plasmid and IS1216E promote the dissemination of poxtA in enterococci isolates from different healthy pigs in the swine farm.

The epidemiology of bloodstream infections and antimicrobial susceptibility patterns in Thuringia, Germany: a five-year prospective, state-wide surveillance study (AlertsNet).

BACKGROUND: Monitoring pathogens of bloodstream infections (BSI) and their antibiotic susceptibility is important to guide empiric antibiotic treatment strategies and prevention programs. This study assessed the epidemiology of BSI and antibiotic resistance patterns at the German Federal State of Thuringia longitudinally. METHODS: A surveillance network consisting of 26 hospitals was established to monitor BSIs from 01/2015 to 12/2019. All blood culture results, without restriction of age of patients, of the participating hospitals were reported by the respective microbiological laboratory. A single detection of obligate pathogens and a repeated detection of coagulase-negative staphylococci, Bacillus spp., Corynebacterium spp., Micrococcus spp. and Propionibacterium spp., within 96 h were regarded as a relevant positive blood culture. If one of the aforementioned non-obligate pathogens has been detected only once within 96 h, contamination has been assumed. Logistic regression models were applied to analyse the relationship between resistance, year of BSI and hospital size. Generalized estimating equations were used to address potential clustering. RESULTS: A total of 343,284 blood cultures (BC) of 82,527 patients were recorded. Overall, 2.8% (n = 9571) of all BCs were classified as contaminated. At least one relevant pathogen was identified in 13.2% (n = 45,346) of BCs. Escherichia coli (25.4%) was the most commonly detected pathogen, followed by Staphylococcus aureus (15.2%), Staphylococcus epidermidis (8.1%) and Klebsiella pneumoniae (4.6%). In S. aureus, we observed a decline of methicillin resistance (MRSA) from 10.4% in 2015 to 2.5% in 2019 (p < 0.001). The rate of vancomycin resistance in Enterococcus faecium (VRE) has increased from 16.7% in 2015 to 26.9% in 2019 (p < 0.001), with a peak in 2018 (42.5%). In addition, we observed an increase of Cefotaxime (3GC) resistance in E. coli from 10.7% in 2015 to 14.5% in 2019 (p = 0.007) whereas 3GC resistance in K. pneumoniae was stable (2015: 9.9%; 2019: 7.4%, p = 0.35). Carbapenem resistance was less than 1% for both pathogens. These patterns were robustly observed across sensitivity analyses. CONCLUSIONS: We observed evidence for a decline in MRSA, an increase in VRE and a very low rate of carbapenem resistance in gram-negative bacteria. 3GC resistance in E. coli increased constantly over time.

Sequence type 17 is a predictor of subsequent bacteremia in vancomycin-resistant Enterococcus faecium-colonized patients: a retrospective cohort study.

BACKGROUND: Sequence type (ST) 17 vancomycin-resistant Enterococcus faecium (VREF) is frequently isolated in nosocomial settings. The aim of this study was to identify whether ST17 contributes to subsequent bacteremia more often than other STs among hospitalized patients carrying VREF. METHODS: A retrospective cohort study was conducted in patients carrying ST17 VREF and those with non-ST17 VREF. Rectal screening according to hospital policy was used to identify patients with VREF. Subsequent VREF bacteremia events within a year of detection of colonization were recorded. Cox regression analysis was used to adjust the covariates involved in determining the association between ST17 and subsequent bacteremia events. RESULTS: The cohorts comprised 52 patients with ST17 and 169 patients with non-ST17 VREF. One-year VREF bacteremia-free rates were 85.9% and 90.2%, respectively. In multivariate analysis, ST17 was associated with subsequent bacteremia at an adjusted hazard risk (aHR) of 4.02 (95% confidence interval [CI], 1.32-12.29). Liver transplantation (aHR, 40.08; 95% CI, 4.87-329.76) and hematologic malignancy (aHR, 20.97; 95% CI, 4.87-87.82) were also significant. All cases of subsequent bacteremia in ST17 VREF carriers were caused by ST17; however, subsequent bacteremia in non-ST17 carriers was often caused by ST17 or another ST variant. CONCLUSIONS: A specific genotype, ST17 is a predictor of subsequent bacteremia in hospitalized patients carrying VREF. Patients with a hematologic malignancy and those receiving a liver transplant are also at high risk. More targeted strategies may be needed to prevent VREF infection in hospitals.

In vitro Antimicrobial Activity of Fosfomycin, Rifampin, Vancomycin, Daptomycin Alone and in Combination Against Vancomycin-Resistant Enterococci.

PURPOSE: The emergence of vancomycin resistant Enterococci (VRE) is shortening the choices for clinical anti-infective therapy. The aim of this study was to investigate the mechanism of vancomycin resistance and evaluate the effect of fosfomycin (FM), rifampin (RIF), vancomycin (VAN), linezolid (LNZ), daptomycin (DAP) alone or in combination against VRE. METHODS: Eight VRE isolates were collected. A total of 18 antibiotics susceptibility tests were further done for VRE. Whole genome sequencing and bioinformatics analysis were performed. The effect of FM, RIF, VNA, LNZ, DAP alone or in combination was determined using anti-biofilm testing and the time-kill assay. RESULTS: All isolates were susceptible to LNZ and DPA. The high-level resistance determinant of VAN in these strains was due to VanA-type cassette. MLST revealed two different STs for vancomycin-resistant Enterococcus faecium (VREm) and four different STs for vancomycin-resistant E. faecalis (VREs). Virulence genes in VREs were more than VREm, especially for 4942 isolated from blood. Gene acm and uppS were only identified in VREm, while virulence genes related to cytolysin were only found in E. faecalis. Further in vitro studies indicated FM (83 mg/L) combined with DAP (20.6 mg/L) and DAP monotherapy (47.1 mg/L) had bactericidal effect against VRE isolates at 24h. CONCLUSION: High-level resistance determinant of VAN in tested isolates was due to VanA-type cassette. FM combined with DAP is a potential therapeutic option for VRE infections.

Detection of Linezolid-Resistant Enterococcus faecalis and Enterococcus faecium Isolates from the Layer Operation System in Korea.

Linezolid (LNZ) is one of the most important antimicrobial agents against infections caused by gram-positive bacteria, including enterococci. In a layer operation system, antimicrobial resistance can be transferred to commercial layers via the fecal-oral route. This study investigated the presence and distribution of LNZ-resistant Enterococcus faecalis and Enterococcus faecium in a layer operation system. Among 117 E. faecalis and 154 E. faecium, 10 (8.5%) E. faecalis and 5 (3.2%) E. faecium isolates showed resistance to LNZ and chloramphenicol, and they exhibited multidrug resistance against 5 or more classes of antimicrobial agents. Among the resistant isolates, 9 (90.0%) and 2 (20.0%) E. faecalis harbored optrA and cfr genes, respectively. The optrA and fexA genes were not detected in five LNZ-resistant E. faecium. None of the 15 LNZ-resistant isolates harbored the fexA gene, and no mutations were observed in the genes encoding domain V of 23S ribosomal RNA (rRNA) and ribosomal proteins L3 (rplC) and L4 (rplD). Transferability was identified in three of the nine optrA-positive LNZ-resistant isolates. The tetM, tetL, and ermB genes were cotransferred with the optrA gene in all optrA-positive transconjugants. The results indicate that optrA is well-distributed in E. faecalis, implying a greater level of transferability. Thus, enhanced surveillance efforts are needed to monitor the emergence and spread of optrA in enterococci in layer operation system.

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

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

Mutasynthesis of Antibacterial Halogenated Actinomycin Analogues.

Through precursor-directed biosynthesis, feeding halogenated (F-, Cl-, Br-, I-) or methoxy-substituted 4-methyl-3-hydroxyanthranilic acid (4-MHA) analogues to the acnGHLM-deleted mutant strain of Streptomyces costaricanus SCSIO ZS0073 led to the production of ten new actinomycin analogues (4-13). Several of the actinomycin congeners displayed impressive antimicrobial activities, with MIC values spanning 0.06-64 µg/mL to clinically derived antibiotic resistant pathogens, including Staphylococcus aureus, Enterococcus faecium, and Candida albicans, with low cytotoxicity.

Effect of Vancomycin on Cytoplasmic Peptidoglycan Intermediates and van Operon mRNA Levels in VanA-Type Vancomycin-Resistant Enterococcus faecium.

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.