High-level Vancomycin Resistance Research Articles

Identification of pathways to high-level vancomycin resistance in Clostridioides difficile that incur high fitness costs in key pathogenicity traits.

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.

Occurrence of Antimicrobial-Resistant Enterococcus spp. in Healthy Chickens Never Exposed to Antimicrobial Agents in Central Italy.

Enterococci are part of the natural flora of the gastrointestinal tract of mammals, including humans, birds and invertebrates. They can cause infection, mainly among hospitalized patients, as well as acquire and transfer antimicrobial resistance genes. The present study allowed the isolation of 98 Enterococcus (73.47% E. faecium, 23.47% E. faecalis, 3.06% E. avium) strains from 120-day-old healthy chickens that had never been treated with antimicrobials. Their antimicrobial resistance was evaluated by the agar disk diffusion method; high-level aminoglycoside (streptomycin and gentamicin) and vancomycin resistance were established using the microbroth dilution method. The highest percentages of resistant isolates were detected with quinupristin-dalfopristin (88.78%), rifampicin (64.29%), tetracyclines (45.92%), and enrofloxacin (41.84%). High percentages of susceptible strains were found with teicoplanin (100%), amoxicillin-clavulanic acid (97.96%), nitrofurantoin (94.90%), ampicillin (92.86%), chloramphenicol (90.82%), and linezolid (88.78%). About 60% of the strains were classified as MDR (multidrug-resistant). Moreover, PCR was carried out to investigate genes encoding for tetracyclines resistance determinants: tet(M), tet(L), tet(O), tet(K), and Int-Tn. Genes were detected in 68 (69.38%) strains: 36 were shown to be resistant with the agar disk diffusion method, while 28 were intermediate, and 2 were susceptible. The present study showed that chickens never treated with antimicrobials potentially harbor enterococci having phenotypic and genotypic characters of antimicrobial resistance.

Outbreak of vancomycin-resistant Enterococcus faecium ST1133 in paediatric patients with acute lymphoblastic leukaemia from southern Brazil

We aimed to investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) in paediatric patients from Hospital Pequeno Príncipe. The susceptibility profile was determined, and whole-genome sequencing (WGS) was used to analyse the genetic context of the strains. Five VREfm isolates were recovered from sterile sites and surveillance cultures of two paediatric patients with acute lymphoblastic leukaemia. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the minimum inhibitory concentration (MIC) was assessed according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST). WGS was performed to analyse the genetic context of virulence and resistance genes, and in silico multilocus sequence typing was performed to identify the sequence typing of the strains. High-level vancomycin resistance was observed in all isolates (≥256 mg/L). WGS revealed the presence of mobile genetic elements, such as plasmids (rep2, rep11a, repUS15, rep17, and rep18a), insertion sequences, and phages. Multiple resistance genes (aac(6')-aph(2"), dfrG, ermB, and vanA) and virulence genes (acm and efaAfm) were identified. All the isolates were assigned to ST117 (ST1133 - via a novel MLST), an important epidemic lineage associated with nosocomial infections and outbreaks. Our results show that the ST117 (ST1133) VREfm isolates are circulating in paediatric patients, which raises a great concern. The development of new drugs as well as the implementation of an antimicrobial stewardship program are necessary for their correct management, limiting the spread of resistance in oncohematological patients.

Antibiotic resistance and virulence potentials of E. faecalis and E. faecium in hospital wastewater: a case study in Ardabil, Iran.

Hospital wastewater can contaminate the environment with antibiotic-resistant and virulent bacteria. We analyzed wastewater samples from four hospitals in Ardabil province, Iran for Enterococcus faecium and Enterococcus faecalis using culture and molecular methods. We also performed antimicrobial susceptibility testing and polymerase chain reaction testing for resistance and virulence genes. Out of 141 enterococci isolates, 68.8% were E. faecium and 23.4% were E. faecalis. Ciprofloxacin and rifampicin showed the highest level of resistance against E. faecalis and E. faecium isolates at 65%. High-level gentamicin resistance (HLGR), high-level streptomycin resistance (HLSR), ampicillin, and vancomycin resistance were observed in 25, 5, 10, and 5.15% of E. faecium, and 15, 6, 15, and 3.03% of E. faecalis isolates, respectively. The ant(6')-Ia and ant(3')-Ia genes that were responsible for streptomycin resistance were observed in HLSR isolates and aph(3')-IIIa and aac(6') Ie-aph(2″)-Ia genes accounting for gentamicin resistance were detected in HLGR isolates. vanA was the predominant gene detected in vancomycin-resistant isolates. The majority of isolates were positive for gelE, asa1, esp, cylA, and hyl virulence genes. We found that drug-resistant and virulent E. faecalis and E. faecium isolates were prevalent in hospital wastewater. Proper treatment strategies are required to prevent their dissemination into the environment.

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

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

Occurrence, antibiogram, high-level vancomycin and aminoglycosides resistance and potential virulence factors of enterococci in dogs in Nigeria

AbstractThis study was conducted to isolate enterococci from dogs in Nigeria, and to determine the potential virulence, antibiotic susceptibility, phenotypic vancomycin (VAN), high-level ampicillin (AMP) and aminoglycosides susceptibility profile of the isolates. Rectal swabs were collected from 295 randomly-selected, clinically-healthy dogs. The isolation of enterococci was done using Slanetz and Bartley enterococcal selective medium. The resistance of 150 non-repetitive isolates was determined using disc diffusion method. VAN resistance was assessed by high-level disc diffusion and agar-screening methods. High-level AMP and aminoglycosides (gentamicin and streptomycin) resistance was determined by agar-screening method. Potential virulence factors were assayed using phenotypic methods. Out of 295 samples, 234 (80.7%) gave positive growth. From these, 250 enterococcal isolates comprised 229 (91.6%) non-pigmented and 21 (8.4%) pigmented strains, were obtained. Resistance of the isolates was 89% to erythromycin, 92% to rifampicin, 77% to chloramphenicol, 83% to tetracycline, 64% to ciprofloxacin, 32.7% to VAN, 24.7% to high-level streptomycin (HLS) and 6% to high-level gentamicin (HLG). Among 150 non-repetitive resistant isolates, 144 (96%), including all the VAN-, HLS- and HLG-resistant strains, exhibited resistance to at least 3 classes of antibiotics. The mean multiple antibiotic resistance index was 0.54 (range = 0.22 – 0.89). Of these 150 isolates, 94 (62.7%), including all the VAN-, HLS- and HLG-resistant strains, displayed virulence potentials as biofilm (44.7%), surface-layer (13.8%), haemolysin (21.3%), gelatinase (40.4%), caesinase (10.6%) and deoxyribonuclease (12.8%) activities. This study showed that dogs in Nigeria are potential reservoirs and disseminators of potentially-virulent, multidrug-, VAN- and high-level aminoglycosides-resistant enterococci.

High-Level Aminoglycoside and Vancomycin Resistance in Enterococcus spp. Isolated from Hospital Acquired Infections, Ismailia, Egypt.

High-Level Aminoglycoside and Vancomycin Resistance in Enterococcus spp. Isolated from Hospital Acquired Infections, Ismailia, Egypt.

High-Level Aminoglycoside and Vancomycin Resistance in Enterococcus spp. Isolated from Hospital Acquired Infections, Ismailia, Egypt

High-Level Aminoglycoside and Vancomycin Resistance in Enterococcus spp. Isolated from Hospital Acquired Infections, Ismailia, Egypt

Alternative vanHAX promoters and increased vanA-plasmid copy number resurrect silenced glycopeptide resistance in Enterococcus faecium.

BackgroundVancomycin variable enterococci (VVE) are van-positive isolates with a susceptible phenotype that can convert to a resistant phenotype during vancomycin selection.ObjectivesTo describe a vancomycin-susceptible vanA-PCR positive ST203 VVE Enterococcus faecium isolate (VVESwe-S) from a liver transplantation patient in Sweden which reverted to resistant (VVESwe-R) during in vitro vancomycin exposure.MethodsWGS analysis revealed the genetic differences between the isolates. Expression of the van-operon was investigated by qPCR. Fitness and stability of the revertant were investigated by growth measurements, competition and serial transfer.ResultsThe VVESwe-R isolate gained high-level vancomycin (MIC >256 mg/L) and teicoplanin resistance (MIC = 8 mg/L). VVESwe-S has a 5′-truncated vanR activator sequence and the VVESwe-R has in addition acquired a 44 bp deletion upstream of vanHAX in a region containing alternative putative constitutive promoters. In VVESwe-R the vanHAX-operon is constitutively expressed at a level comparable to the non-induced prototype E. faecium BM4147 strain. The vanHAX operon of VVESwe is located on an Inc18-like plasmid, which has a 3–4-fold higher copy number in VVESwe-R compared with VVESwe-S. Resistance has a low fitness cost and the vancomycin MIC of VVESwe-R decreased during in vitro serial culture without selection. The reduction in MIC was associated with a decreased vanA-plasmid copy number.ConclusionsOur data support a mechanism by which vancomycin-susceptible VVE strains may revert to a resistant phenotype through the use of an alternative, constitutive, vanR-activator-independent promoter and a vanA-plasmid copy number increase.

Antibiogram of Urinary Enterococcus Isolates from a Tertiary Care Hospital

Urinary tract infection (UTI) is one of the serious infections caused by the bacteria Enterococci. Vancomycin-Resistant Enterococci (VRE) is a persevering clinical problem globally. This study aims to detect high-level aminoglycoside and vancomycin resistance in uropathogenic Enterococcus spp. A total of 75 clinically relevant Enterococcus spp. grown from urine samples, were collected following convenience non-random sampling method. Identified by standard biochemical tests and susceptibility to antibiotics was studied by Kirby Bauer's disc diffusion method. The MIC of vancomycin was detected by agar dilution test. Van A, and Van B genes in VREs were detected by PCR. Among 75 Enterococcal isolates, 43 (57.3%) were E. faecalis, 12 (16%) were E. faecium, 6 (8%) of each were E. pseudoavium and E. casseliflavus, 5(6.66%) were E. dispar and 3 (4%) were E. durans. E. faecalis (n=19) and E. faecium (n=3) were resistant to High Level Streptomycin (HLS). E. faecalis (n=21) and E. faecium (n=6) were resistant to High Level Gentamicin (HLG). 4 (9.3%) E. faecalis were vancomycin-resistant, out of which 3 were of Van A, and one was both Van A and Van B genotype. Isolation of high level aminoglycoside resistant (HLAR) Enterococci is a challenge for the treating physician because aminoglycoside cannot be used in combination with glycopeptide or ampicillin for such isolates. The occurrence of HLAR, Van A, and Van B VRE genotypes is a cause of concern as they may transfer drug resistance genes to other bacterial isolates, thus leading to limited therapeutic options.

The Extracellular Domain of Two-component System Sensor Kinase VanS from Streptomyces coelicolor Binds Vancomycin at a Newly Identified Binding Site

The glycopeptide antibiotic vancomycin has been widely used to treat infections of Gram-positive bacteria including Clostridium difficile and methicillin-resistant Staphylococcus aureus. However, since its introduction, high level vancomycin resistance has emerged. The genes responsible require the action of the two-component regulatory system VanSR to induce expression of resistance genes. The mechanism of detection of vancomycin by this two-component system has yet to be elucidated. Diverging evidence in the literature supports activation models in which the VanS protein binds either vancomycin, or Lipid II, to induce resistance. Here we investigated the interaction between vancomycin and VanS from Streptomyces coelicolor (VanSSC), a model Actinomycete. We demonstrate a direct interaction between vancomycin and purified VanSSC, and traced these interactions to the extracellular region of the protein, which we reveal adopts a predominantly α-helical conformation. The VanSSC-binding epitope within vancomycin was mapped to the N-terminus of the peptide chain, distinct from the binding site for Lipid II. In targeting a separate site on vancomycin, the effective VanS ligand concentration includes both free and lipid-bound molecules, facilitating VanS activation. This is the first molecular description of the VanS binding site within vancomycin, and could direct engineering of future therapeutics.

634. Incidence of vanC-Mediated Vancomycin-Resistant Enterococcus Bloodstream Infections at Children’s Hospital of Colorado and Implications for Empiric Enterococcal Therapy

BackgroundThe most well-known Enterococcal species, E. faecium and E. faecalis, can harbor high-level vancomycin resistance mediated by acquired vanA and vanB operons. However, other Enterococcal species such as E. gallinarum and E. casseliflavus (VCE), harbor intrinsic low-level vancomycin resistance mediated by an intrinsic vanC operon, and the incidence of these pathogens among pediatric patients is not clear. As the antibiotic resistance pattern of VCE is different than E. faecium and E. faecalis, a high prevalence of VCE may have implications for antibiotic therapy. We describe the incidence and susceptibility of VCE bloodstream infections at a large children’s hospital and compare to E. faecalis and E. faecium.MethodsPositive blood culture results from 2013 to 2018 were obtained from the Children’s Hospital of Colorado data warehouse. All first-time positive cultures for Enterococcus were analyzed for species, susceptibility, and hospital unit location. First-time positive was defined as being at least 2 weeks after any previous positive Enterococcus blood culture. Susceptibilities were categorized by clinical laboratories standards institute (CLSI) guidelines.ResultsOf 240 positive isolates, 7% were ampicillin susceptible and vancomycin nonsusceptible (resistant or intermediate), vs. 6% that were ampicillin resistant and vancomycin susceptible. An additional 3% of isolates were not susceptible to either antibiotic; all of these were E. faecium. VCE accounted for 12% of our isolates while E. faecalis and E. faecium accounted for 66% and 16%, respectively. All VCE were susceptible to ampicillin, but 52% were nonsusceptible to vancomycin. VCE incidence, ampicillin resistance, and vancomycin nonsusceptibility were most prevalent in our hematology, oncology, and bone marrow transplant (BMT) units.ConclusionAt our institution, an as yet unspeciated Enterococcus is equally likely to be ampicillin susceptible and vancomycin nonsusceptible as ampicillin resistant and vancomycin susceptible. This is driven by a significant incidence of VCE, especially on our hematology, oncology, and BMT units. Therefore, vancomycin may not provide adequate empiric Enterococcal coverage on these units, and the addition of ampicillin will be recommended. Disclosures All authors: No reported disclosures.

Faecal carriage of high-level aminoglycoside-resistant and ampicillin-resistant Enterococcus species in healthy Iranian children.

High-level aminoglycoside, ampicillin and vancomycin resistance and virulence genes among enterococcal isolates collected from healthy middle-school children in Ardabil, Iran, during 2016 were investigated. Totally, 305 faecal specimens were collected. Isolates underwent antimicrobial susceptibility testing, virulence gene detection and molecular typing. Totally, 409 enterococcal isolates were collected, comprising Enterococcus faecium (235; 57.5%), Enterococcus faecalis (56; 13.7%) and other Enterococcus spp. (118; 28.9%). Overall, 71 (17.4%), 11 (2.7%) and 10 (2.4%) isolates were identified as high-level streptomycin-resistant (HLSR), high-level gentamicin-resistant (HLGR) and ampicillin-resistant (AR), respectively. Among HLSR isolates, 40 (56.3%), 5 (7.0%) and 26 (36.6%) were E. faecium, E. faecalis and other Enterococcus spp., respectively. Among HLGR isolates 4 (36.4%) and 7 (63.6%) and among AR isolates 7 (70.0%) and 3 (30.0%) were E. faecium and other Enterococcus spp., respectively. Accordingly, 21.6%, 3.6% and 3.3% of subjects were colonised with HLSR, HLGR and AR Enterococcus spp. Carriage of HLGR, HLSR and AR isolates was associated with prior antibiotic consumption (P≤0.05). Additionally, male sex and antacid consumption were associated with AR enterococcal carriage. Moreover, 69 (97.2%), 10 (90.9%) and 9 (90.0%) of HLSR, HLGR and AR isolates were multidrug-resistant, respectively. No vancomycin-resistant enterococci were detected. ERIC-PCR revealed high genetic diversity among isolates. gelE and asa1 were major virulence genes both in E. faecalis and E. faecium. Presence of gelE was associated with HLSR and HLGR phenotypes (P≤0.05). Community intestinal carriage of HLSR enterococci was high; however, carriage of HLGR and AR enterococci was low.

Species, antibiotic susceptibility profiles and van gene frequencies among enterococci isolated from patients at Mulago National Referral Hospital in Kampala, Uganda

BackgroundThe increase in drug resistance to affordable antibiotics used to treat Gram positive bacterial infections has complicated the management of enterococcal infections. Resistance to vancomycin, one of the most powerful antibiotics, is of utmost concern as both intrinsic and acquired forms of resistance do occur in enterococci. This cross-sectional study aimed to determine the species, antibiotic susceptibility profiles and vanA/vanB gene frequencies among enterococci isolated from patients at Mulago Hospital in Kampala, Uganda.MethodsBetween November 2011 and October 2012, stool, urine, sputum and blood samples, as well as vaginal, endocervical, pus, ear and urethra swabs from 3229 patients were processed for isolation of bacteria, yielding 162 enterococci of which 115 were available for analysis (one isolate per specimen/patient). Species-level confirmation and susceptibility testing were determined with the Phoenix™ AST/ID Automated System, while vanA/vanB gene carriage was determined by PCR.ResultsSpecies-level identification revealed 72 isolates of E. faecalis, 20 E. gallinarum/casseliflavus, 5 E. faecium, 4 E. raffinosus and 2 isolates each for E. hirae and E. durans. Ten isolates could not be identified to species level. Antibiotic resistance was generally low especially to ampicillin, quinolones, nitrofurantoin, glycopeptides and linezolid, but high for erythromycin and tetracycline. Equally, vanA and vanB gene frequencies were low (i.e. 15.8 and 7.9%, respectively) and detected only in E. casseliflavus/gallinarum species that are intrinsically resistant to vancomycin. Vancomycin resistant isolates of E. faecalis and E. faecium were not detected.ConclusionsEnterococcus species are frequent in clinical specimens at Mulago Hospital but they are highly susceptible to common antibiotics especially ampicillin. While vancomycin resistant enterococcal (VRE) isolates of E. faecium and E. faecalis are rare in the hospital and frequency of multidrug resistance is low, non-faecium and non-faecalis VRE isolates (i.e. E. gallinarum/casseliflavus) are frequent, some with VanA/VanB (high-level) vancomycin resistance. Therefore, species-level identification of enterococci is necessary in resource limited settings to guide infection control and treatment of enterococcal infections.

Comparison of biochemical and genotypic speciation methods for vancomycin-resistant enterococci isolated from urban wastewater treatment plants

Enterococci species in wastewater including Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus and Enterococcus gallinarum isolates (n = 308) with low or high level vancomycin resistance were determined and compared using a phenotypic method (RapID™ STR system), 16S rRNA sequencing, and multi-locus (atpA, groESL, and pheS) sequence analysis (MLSA). Error rates for the RapID™ STR system were E. faecalis (15.9%), E. faecium (21.5%), and E. casseliflavus/E. gallinarum (56.9%) when referenced to the consensus of all methods tested. Comparison of single nucleotide polymorphism (SNP) distances and phylogenetic trees suggested that the groESL locus delineated species more effectively than other loci. The groESL locus was the most reliable loci for the correct identification of Enterococcus spp., including E. casseliflavus and E. gallinarum, with high congruence compared to the consensus (Adjusted Rand Index = 0.954; Adjusted Wallace Co-efficient = 0.941). All of the methods were compared to whole genome sequencing, which acted as a gold standard, for the isolates from this study and those downloaded from NCBI.

An epidemiological and molecular study regarding the spread of vancomycin-resistant Enterococcus faecium in a teaching hospital in Bogot\xe1, Colombia 2016

BackgroundEnterococcus faecium is ranked worldwide as one of the top ten pathogens identified in healthcare-associated infections (HAI) and is classified as one of the high priority pathogens for research and development of new antibiotics worldwide. Due to molecular biology techniques’ higher costs, the approach for identifying and controlling infectious diseases in developing countries has been based on clinical and epidemiological perspectives. Nevertheless, after an abrupt vancomycin-resistant Enterococcus faecium dissemination in the Méderi teaching hospital, ending up in an outbreak, further measures needed to be taken into consideration. The present study describes the vancomycin-resistant Enterococcus faecium pattern within Colombian’s largest installed-bed capacity hospital in 2016.MethodsThirty-three vancomycin-resistant Enterococcus faecium isolates were recovered during a 5-month period in 2016. Multilocus variable-number tandem-repeat analysis was used for molecular typing to determine clonality amongst strains. A modified time-place-sequence algorithm was used to trace VREfm spread patterns during the outbreak period and estimate transmission routes.ResultsFour clonal profiles were identified. Chronological clonal profile follow-up suggested a transitional spread from profile “A” to profile “B”, returning to a higher prevalence of “A” by the end of the study. Antibiotic susceptibility indicated high-level vancomycin-resistance in most isolates frequently matching vanA gene identification.DiscussionTransmission analysis suggested cross-contamination via healthcare workers. Despite epidemiological control of the outbreak, post-outbreak isolates were still being identified as having outbreak-related clonal profile (A), indicating reduction but not eradication of this clonality. This study supports the use of combined molecular and epidemiological strategies in an approach to controlling infectious diseases. It contributes towards a more accurate evaluation of the effectiveness of the epidemiological measures taken regarding outbreak control and estimates the main cause related to the spread of this microorganism.

Impact on Public Health of the Spread of High-Level Resistance to Gentamicin and Vancomycin in Enterococci.

Antibiotic resistance has turned into a global public health issue. Enterococci are intrinsically resistant to many antimicrobials groups. These bacteria colonize dairy and meat products and integrate the autochthonous microbiota of mammal’s gastrointestinal tract. Over the last decades, detection of vanA genotype in Enterococcus faecium from animals and from food of animal origin has been reported. Vancomycin-resistant E. faecium has become a prevalent nosocomial pathogen. Hospitalized patients are frequently treated with broad-spectrum antimicrobials and this leads to an increase in the presence of VanA or VanB vancomycin-resistant enterococci in patients’ gastrointestinal tract and the risk of invasive infections. In humans, E. faecium is the main reservoir of VanA and VanB phenotypes. Acquisition of high-level aminoglycoside resistance is a significant therapeutic problem for patients with severe infections because it negates the synergistic effect between aminoglycosides and a cell-wall-active agent. The aac(6′)-Ie-aph (2″)-Ia gene is widely spread in E. faecalis and has been detected in strains of human origin and in the food of animal origin. Enzyme AAC(6′)-Ie-APH(2″)-Ia confers resistance to available aminoglycosides, except to streptomycin. Due to the fast dissemination of this genetic determinant, the impact of its horizontal transferability among enterococcal species from different origin has been considered. The extensive use of antibiotics in food-producing animals contributes to an increase in drug-resistant animal bacteria that can be transmitted to humans. Innovation is needed for the development of new antibacterial drugs and for the design of combination therapies with conventional antibiotics. Nowadays, semi-purified bacteriocins and probiotics are becoming an attractive alternative to the antibiotic in animal production. Therefore, a better understanding of a complex and relevant issue for Public Health such as high-level vancomycin and gentamicin resistance in enterococci and their impact is needed. Hence, it is necessary to consider the spread of vanA E. faecium and high-level gentamicin resistant E. faecalis strains of different origin in the environment, and also highlight the potential horizontal transferability of these resistance determinants to other bacteria.

Structural basis for the substrate recognition of peptidoglycan pentapeptides by Enterococcus faecalis VanYB

Vancomycin resistance in Enterococci and its transfer to methicillin-resistant Staphylococcus aureus are challenging problems in health care institutions worldwide. High-level vancomycin resistance is conferred by acquiring either transposable elements of the VanA or VanB type. Enterococcus faecalis VanYB in the VanB-type operon is a d,d-carboxypeptidase that recognizes the peptidyl-d-Ala4-d-Ala5 extremity of peptidoglycan and hydrolyses the terminal d-Ala on the extracellular side of the cell wall, thereby increasing the level of glycopeptide antibiotics resistance. However, at the molecular level, it remains unclear how VanYB manipulates peptidoglycan peptides for vancomycin resistance. In this study, we have determined the crystal structures of E. faecalis VanYB in the d-Ala-d-Ala-bound, d-Ala-bound, and -unbound states. The interactions between VanYB and d-Ala-d-Ala observed in the crystal provide the molecular basis for the recognition of peptidoglycan substrates by VanYB. Moreover, comparisons with the related VanX and VanXY enzymes reveal distinct structural features of E. faecalis VanYB around the active-site cleft, thus shedding light on its unique substrate specificity. Our results could serve as the foundation for unravelling the molecular mechanism of vancomycin resistance and for developing novel antibiotics against the vancomycin-resistant Enterococcus species.

Zn(II) mediates vancomycin polymerization and potentiates its antibiotic activity against resistant bacteria

Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors.

Genomic analysis of vancomycin-resistant Staphylococcus aureus VRS3b and its comparison with other VRSA isolates.

High-level vancomycin resistance among Staphylococcus aureus poses a grave threat to global health as the treatment options for this pathogen are very limited. A detailed evaluation of the genetic background of vancomycin-resistant S. aureus (VRSA) is expected to facilitate the understanding of its origin and pathogenicity. In this study, we performed the genetic analysis of the clinical VRSA isolates and identified the genetic basis of resistance to multiple antibiotics among these strains, based on the available draft genome sequences. In addition, we generated the draft genome of the strain VRS3b, which was considered to be same as VRS3a based on its isolation from the same patient. We found that strain VRS3b did not harbor the genes responsible for tetracycline and gentamicin, which was further confirmed by the sensitivity towards these antibiotics. Our results suggest that the strains VRS3a and VRS3b are different from the view of antibiotic resistance and highlight the possibility of generation of two distinct VRSA strains from the same patient.