Structure of VanS from vancomycin-resistant enterococci: A sensor kinase with weak ATP binding.

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci. VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from vancomycin-resistant enterococci types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with KD values in the low millimolar range. Since these KD values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

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.

The virtue of training: extending phage host spectra against vancomycin-resistant Enterococcus faecium strains using the Appelmans method.

Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra.

Investigation of cross-opsonic effect leads to the discovery of PPIase-domain containing protein vaccine candidate to prevent infections by Gram-positive ESKAPE pathogens.

BACKGROUND: Enterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies. RESULTS: Opsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains. CONCLUSIONS: Given the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy.

Repurposing fusidic acid as an antimicrobial against enterococci with a low probability of resistance development.

Drug repurposing constitutes a strategy to combat antimicrobial resistance, by using agents with known safety, pharmacokinetics, and pharmacodynamics. Previous studies have implemented new fusidic acid (FA) front-loading-dose regimens, allowing higher serum levels than those achievable with ordinary doses. As susceptibility breakpoints are affected by serum level, we evaluated the repurposing of FA as an antimicrobial product against enterococci. FA minimum inhibitory concentrations (MICs) against standard enterococci strains; Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 27270 were 2 and 4 µg/mL, respectively. The MIC against 98 enterococcal clinical isolates was ≤ 8 µg/mL; all would be susceptible if categorized according to recalculated breakpoints (≥ 16 µg/mL), based on the serum level achieved using the front-loading regimen. FA administration in vivo, using the BALB/c mouse infection model, significantly reduced bacterial burden by two to three log10 units in the liver and spleen of mice infected with vancomycin-susceptible and -resistant strains. Exposure of the standard enterococcal strains to increasing, but not fixed, FA concentrations resulted in resistant strains (MIC = 128 µg/mL), with thicker cell walls and slower growth rates. Only one mutation (M651I) was detected in the fusA gene of the resistant strain derived from serial passage of E. faecium ATCC 27270, which was retained in the revertant strain after passage in the FA-free medium. In conclusion, FA can be repurposed as an antimicrobial drug against enterococci with a low probability of mutational resistance development, and can be employed for treatment of infections attributable to vancomycin-resistant enterococci.

Bacteraemia caused by non-faecalis and non-faecium Enterococcus species-a retrospective study of incidence, focus of infection, and prognosis.

BACKGROUND/AIM: Enterococcus faecalis and Enterococcus faecium cause human infections including bacteraemia and infective endocarditis (IE). Only few studies describing non-faecalis and non-faecium Enterococcus (NFE) infections have been conducted. We aimed to describe the incidence, prognosis, and focus of infection of bacteraemia with NFE. METHODS: This retrospective population-based study included all episodes of patients having a blood culture with growth of NFE between 2012 and 2019 in Region Skåne, Sweden. Information was collected from medical records. Episodes of bacteraemia caused by NFE were compared to episodes of bacteraemia caused by E. faecalis and E. faecium. RESULTS: During the study period, 136 episodes with NFE bacteraemia were identified corresponding to an incidence of NFE bacteraemia of 16 cases per 1,000,000 person-years among adults. Enterococcus casseliflavus (n=45), Enterococcus gallinarum (n=34), and Enterococcus avium (n=29) were the most common species. The most common foci of infection were biliary tract infections (n=17) followed by gastrointestinal infections (n=7). Urinary tract infections were not commonly caused by NFE (n=1), and no episodes of IE were caused by NFE. Polymicrobial bacteraemia was more common with NFE (73%) than with E. faecalis (35%) and E. faecium (42%). Community acquired infections were more common in bacteraemia with NFE compared to E. faecium. 30- and 90-day survival rates were 76% and 68%, respectively, and recurrent NFE bacteraemia was seen after 3% of the episodes. CONCLUSION: Bacteraemia caused by NFE is rare and is often polymicrobial. Biliary tract focus is common in NFE bacteraemia whereas IE and urinary tract focus are uncommon.

Genetic analysis of vancomycin-variable Enterococcus faecium clinical isolates in Italy.

PURPOSE: To investigate the occurrence of vancomycin-variable enterococci (VVE) in a hospital in central Italy. METHODS: vanA positive but vancomycin-susceptible Enterococcus faecium isolates (VVE-S) were characterized by antibiotic susceptibility tests, molecular typing (PFGE and MLST), and WGS approach. The reversion of VVE-S to a resistant phenotype was assessed by exposure to increasing vancomycin concentrations, and the revertant isolates were used in filter mating experiments. qPCR was used to analyze the plasmid copy number. RESULTS: Eleven putative VVE-S were selected. WGS revealed two categories of vanA cluster plasmid located: the first type showed the lack of vanR, the deletion of vanS, and an intact vanH/vanA/vanX cluster; the second type was devoid of both vanR and vanS and showed a deletion of 544-bp at the 5'-end of the vanH. Strains (n = 7) carrying the first type of vanA cluster were considered VVE-S and were able to regain a resistance phenotype (VVE-R) in the presence of vancomycin, due to a 44-bp deletion in the promoter region of vanH/vanA/vanX, causing its constitutive expression. VVE-R strains were not able to transfer resistance by conjugation, and the resistance phenotype was unstable: after 11 days of growth without selective pressure, the revertants were still resistant but showed a lower vancomycin MIC. A higher plasmid copy number in the revertant strains was probably related to the resistance phenotype. CONCLUSION: We highlight the importance of VVE transition to VRE under vancomycin therapy resulting in a potential failure treatment. We also report the first-time identification of VVE-S isolates pstS-null belonging to ST1478.

Epidemiological and microbial trends of infective endocarditis in western Norway: a 7-year prospective observational study.

BACKGROUND: In this prospective, observational study, we aimed to investigate epidemiologic and microbial trends of infective endocarditis in western Norway. METHODS: Clinical and microbiological characteristics of 497 cases of infective endocarditis from 2016 through 2022 were investigated. Categorical data were analysed using Chi-squared tests. Survival data were analysed using multiple Cox regression and reported using hazard ratios. RESULTS: The mean age was 67 years, and 74% were men. The annual incidence rates varied from 10.4 to 14.1 per 100,000 inhabitants per year. Infective endocarditis on native valves was observed in 257 (52%) of the cases, whereas infective endocarditis on prosthetic valves and/or cardiac implantable electronic devices was observed in 240 (48%) of the cases: infection on surgically implanted bioprostheses was observed in 124 (25%) of the patients, infection on transcatheter aortic valve implantation was observed in 47 (10%) patients, and infection on mechanical valves was observed in 34 (7%) cases. Infection related to cardiac implantable electronic devices was observed in a total of 50 (10%) cases. Staphylococcus aureus and viridans streptococci were the most common microbial causes, and isolated in 145 (29%) and 130 (26%) of the cases, respectively. Enterococcal endocarditis showed a rising trend during the study period and constituted 90 (18%) of our total cases of infective endocarditis, and 67%, 47%, and 26% of the cases associated with prosthetic material, transcatheter aortic valve implantation and cardiac implantable electronic devices, respectively. There was no significant difference in 90-day mortality rates between the native valve endocarditis group (12%) and the group with infective endocarditis on prosthetic valves or cardiac implants (14%), p = 0.522. In a model with gender, age, people who inject drugs, microbiology and type of valve affected, only advanced age was significantly associated with fatal outcome within 90 days. CONCLUSIONS: The incidence of infective endocarditis, and particularly enterococcal endocarditis, increased during the study period. Enterococci appeared to have a particular affinity for prosthetic cardiac material. Advanced age was the only independent risk factor for death within 90 days.

Antimicrobial resistance patterns, virulence genes, and biofilm formation in enterococci strains collected from different sources.

BACKGROUND: Currently, antibiotic-resistant strains of Enterococcus are considered to be one of the critical health challenges globally. This study aimed to investigate the antibiotic susceptibility pattern, biofilm formation capacity, and virulence genes of enterococci isolated from different sources. METHODS: In this cross-sectional study, environmental and fecal samples were collected from the hospital environment, volunteers, and hospital staff from October 2018 to August 2019. The isolates were identified by morphological and biochemical tests (gram staining, catalase, bile resistance, esculin hydrolysis, carbohydrate fermentation, growth in 6.5% NaCl, Pyrrolidonyl arylamidase, arginine dehydrolase), and PCR for ddl gene. An antimicrobial susceptibility test was performed by the standard disk agar diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Quantitative microplate assays were used to assess biofilm production. The bacterial DNAs were extracted by alkaline lysis method and polymerase chain reaction technique was used detect the esp, ace, and efaA virulence genes. RESULTS: Out of 145 isolates, 84 (57.9%) were identified as E. faecalis and 61 (42.1%) as E. faecium. Resistance to kanamycin and quinupristin-dalfopristin was 82.1% (69/84) and 85.7% (72/84), respectively, in E. faecalis isolates. Out of 61 E. faecalis isolates, 38 (62.4%) were resistant to kanamycin. Among the E. faecalis isolates, esp was the most dominant virulence gene (73.80%), followed by efaA, and ace, which were detected in 60.71%, and 30.95% isolates, respectively. In total, 68.27% of the strains were biofilm producers. Further, esp and efaA genes were more frequently found among E. faecalis strains with moderate and strong biofilm biomass. CONCLUSIONS: According to the findings of our study, enterococci strains isolated from different samples possess distinctive patterns of virulence genes. The esp, ace, and efaA genes were more prevalent among E. faecalis than E. faecium. Besides, the high level antibiotic resistance of normal flora and environmental enterococci strains is alarming the researchers.

Exploring the feasibility of bacteriocins EntK1 and EntEJ97s in treatment of systemic vancomycin resistant enterococci infections in mice.

AIMS: Enterocins K1 and EJ97 have specific antimicrobial activity against Enterococcus faecium and Enterococcus faecalis, respectively. The aim of this study was to investigate the utility of these enterocins for in vivo treatment of systemic enterococcal infections. METHODS AND RESULTS: The antimicrobial effect in blood was analysed and compared against the effect in saline. Colony forming unit counts revealed that the enterocins killed all the bacteria within 1 hour. Additionally, the bactericidal effect against E. faecalis was more rapid in blood, indicating a possible synergy between EntEJ97 and blood. Importantly, no enterocin resistant mutants emerged in these experiments. Injecting the enterocins intraperitoneally in an in vivo mouse model and using fluorescence and minimum inhibitory concentration determination to estimate concentrations of the peptides in plasma, indicate that the enterocins exist in circulation in therapeutic concentrations. Alanine aminotransferase detection, and haemolysis analysis indicates that there is no detectable liver damage or haemolytic effect after injection. CONCLUSIONS: The study revealed that EntK1 and EntEJ97 are able to kill all bacteria ex vivo in the presence of blood. In vivo experiments determine that the enterocins exist in circulation in therapeutic concentrations without causing liver damage or haemolysis. Future experiments should test these peptides for treatment of infection in a relevant in vivo model.

Characteristics and outcomes of urinary tract infections caused by Enterococci: A multicenter retrospective study from two tertiary hospitals in Saudi Arabia.

Enterococci are Gram-positive coccus bacteria that are normally present in the gastrointestinal tract and ordinarily function commensally with humans. Very few studies have investigated the characteristics of enterococcal infections. We aimed to characterize patients with urinary tract infections (UTIs) due to Enterococci and their outcomes. This was a retrospective cohort study between June 2012-November 2022. Patients who had clinically and microbiologically confirmed Enterococcal UTI based on a urine culture positive for E. faecalis or E. faecium with a count of ≥105 CFU/mL and having urinary tract symptoms were included. A total of 396 patients were eligible and included. The patients had a median age of 61 years and were mostly females (56.8 %). The most common characteristics were hospitalization in a non-ICU ward, having a urinary catheter, and recent use of antibiotics within the last 3 months (66.4 %, 59.3 %, and 51.8 %, respectively). Infection with E. faecalis was more common than E. faecium (77.3 % vs. 22.7 %). However, the latter exhibited higher rates of antibiotic resistance (P < 0.001 to several antibiotics) and was associated with significantly higher median C-reactive protein level (26.7 vs. 13 mg/dL; P = 0.025), mortality (23 % vs. 10.1 %; P = 0.002), and median length of stay (25 vs. 11.5 days; P < 0.001). We found that most patients with enterococcal UTIs had a history of having a urinary catheter and recent antibiotic use and were mostly females and hospitalized in non-ICU wards. E. faecium-infected patients experienced more severe episodes and poorer outcomes compared to patients infected with E. faecalis; thus, would need more aggressive therapy.

Improved pharmacokinetics and enhanced efficacy of the vancomycin derivative FU002 using a liposomal nanocarrier.

Antibiotic resistance still represents a global health concern which diminishes the pool of effective antibiotics. With the vancomycin derivative FU002, we recently reported a highly potent substance active against Gram-positive bacteria with the potential to overcome vancomycin resistance. However, the translation of its excellent antimicrobial activity into clinical efficiency could be hampered by its rapid elimination from the blood stream. To improve its pharmacokinetics, we encapsulated FU002 in PEGylated liposomes. For PEG-liposomal FU002, no relevant cytotoxicity on liver, kidney and red blood cells was observed. Studies in Wistar rats revealed a significantly prolonged blood circulation of the liposomal antibiotic. In microdilution assays it could be demonstrated that encapsulation does not diminish the antimicrobial activity against staphylococci and enterococci. Highlighting its great potency, liposomal FU002 exhibited a superior therapeutic efficacy when compared to the free form in a Galleria mellonella larvae infection model.

Vertical stratification and seasonality of fecal indicator bacteria in New York City playground sandboxes.

Sandboxes in public play spaces afford a crucial opportunity for urban children to engage in naturalistic play that fosters development of cognitive, social, and motor skills. As open pits, sandboxes in New York City public playgrounds are potentially exposed to fecal inputs from various sources, including wild and domestic animals. A longitudinal study of thirteen sandboxes located in public playgrounds on the east side of Manhattan reveals ubiquity of the fecal indicator bacteria enterococci and Escherichia coli through all seasons. The highest concentrations of bacteria occur in surface sand (n = 42; mean enterococci 230 MPN/g and E. coli 182 MPN/g dry weight), with significantly lower levels at depths below the surface (n = 35; mean enterococci 21 MPN/g and E. coli 12 MPN/g dry weight), a stratification consistent with fecal loading at the surface. Generalized linear mixed models indicate that sand depth (surface vs. underlayers) is the most influential variable affecting bacterial levels (P <0.001 for both enterococci and E. coli), followed by sampling season (P <0.001 for both). Bacterial concentrations do not vary significantly as a function of playground location or ZIP code within the study area. Children's exposure while playing in sandboxes likely reaches 105 enterococci and 104E. coli in a typical play period. Microbial source tracking to identify fecal hosts reveals dog, bird, and human biomarkers in low concentrations. Open sandbox microcosms installed at ground level in the urban environment of Manhattan are fouled by enterococci and E. coli within two weeks, while adjacent closed microcosms exhibit no fecal contamination over a 33-day sampling period. Collectively, our results indicate that increasing the frequency of sand refills and covering sandboxes during times of disuse would be straightforward management strategies to mitigate fecal contamination in playground sandboxes.

Antibiotic Resistance Characterization and Molecular Characteristics of Enterococcus Species Isolated from Combination Probiotic Preparations in China.

Enterococci can act as reservoirs for antibiotic-resistant genes that are potentially at risk of being transferred to other bacteria that inhabit in the gastrointestinal tract. The aim of this study was to determine the phenotypic and molecular characteristics of antibiotic-resistant enterococci isolated from probiotic preparations. In total, we isolated 15 suspected Enterococcus species from 5 compound probiotics, which were identified by 16S rDNA as 12 Enterococcus faecium and 3 Enterococcus faecalis. Determination of antimicrobial susceptibility by the microdilution broth method showed widespread resistance to sulfamethoxazole (100%), norfloxacin (99.3%), azithromycin (99.3%), gentamicin (86.7%), and chloramphenicol (20%). Whole genome sequencing of five resistant strains revealed that all had circular DNA chromosomes and that E. faecium J-1-A to J-4-A contained a plasmid, while E. faecalis J-5-A did not. The results of the resistance gene analysis revealed that each strain contained approximately 30 resistance genes, with the antibiotic resistance genes and the multidrug resistance efflux pump genes mdtG, lmrC, and lmrD detected in all strains. The chloramphenicol resistance genes ykkC and ykkD were first identified in E. faecalis. And there were 21, 19, 21, 21, and 29 virulence factors involved in strains, respectively. Further analysis of the gene islands (GIs) revealed that each strain contained more than 10 GIs. The above results confirm the existence of hidden dangers in the safety of probiotics and remind us to carefully select probiotic preparations containing enterococcal strains to avoid the potential spread of resistance and pathogenicity.

Evaluation of Antimicrobial Resistancein Clinical Isolates of Enterococcus spp. Obtained from Hospital Patients in Latvia.

Background and Objective: Enterococci are typically found in a healthy human gastrointestinal tract but can cause severe infections in immunocompromised patients. Such infections are treated with antibiotics. This study addresses the rising concern of antimicrobial resistance (AMR) in Enterococci, focusing on the prevalence of vancomycin-resistant enterococcus (VRE) strains. Materials and Methods: The pilot study involved 140 Enterococci isolates collected between 2021 and 2022 from two multidisciplinary hospitals (with and without local therapeutic drug monitoring protocol of vancomycin) in Latvia. Microbiological assays and whole genome sequencing were used. AMR gene prevalence with resistance profiles were determined and the genetic relationship and outbreak evaluation were made by applying core genome multi-locus sequence typing (cgMLST). Results: The acquired genes and mutations were responsible for resistance against 10 antimicrobial classes, including 25.0% of isolates expressing resistance to vancomycin, predominantly of the vanB type. Genetic diversity among E. faecalis and E. faecium isolates was observed and seven potential outbreak clusters were identified, three of them containing sequence types ST6, ST78 and ST80. The prevalence of vancomycin resistance was highest in the hospital without a therapeutic drug-monitoring protocol and in E. faecium. Notably, a case of linezolid resistance due to a mutation was documented. Conclusions: The study illustrates the concerning prevalence of multidrug-resistant Enterococci in Latvian hospitals, showcasing the rather widespread occurrence of vancomycin-resistant strains. This highlights the urgency of implementing efficient infection control mechanisms and the need for continuous VRE surveillance in Latvia to define the scope and pattern of the problem, influencing clinical decision making and planning further preventative measures.

Evaluating the Trend of VRE carriages in Health Facilities: A Retrospective Study from 2019-2022.

Background: Healthcare-associated infections (HAIs) and multidrug resistance (MDR) are a growing public health threat and pose a risk to patient safety in healthcare facilities. Vancomycin-resistant Enterococci (VRE) are responsible for nosocomial infections and have intrinsic and acquired resistance to many antibiotics, including glycopeptides. VRE carriage can remain undetected, increasing the risk of contact transmission. Identifying colonized patients is crucial for the implementation of preventive measures. Aims: The aims of this study were to evaluate the trend of VRE carriage based on rectal swab results between 2019 and February 2022 in a large Italian trust and the percentage of patients with VRE colonization at the time of hospitalization. Methods: This was a retrospective observational study based on results of rectal swabs performed for screening on admission between January 2019 and February 2022 in four hospitals part of a single trust in Turin, North-Western Italy. The study collected data on the date of specimen collection, type of specimen, isolated pathogen and the date of hospital admission. Descriptive analysis of data was performed, and duplicate samples were not considered. Results: From January 2019 to February 2022 we collected 5025 rectal swabs performed in hospitals of the trust, of which 3037 were performed in 2019 (60%), 741 in 2020 (15%), 611 in 2021 (12%) and 636 in the first two months of 2022 (13%). VRE positivity was found in 162 (3%) rectal swabs, of which 2 cases in both 2019 (0.1%) and 2020 (0.3%), 95 in 2021 (15.5%) and 63 in the first two months of 2022 (9.9%). Furthermore, 52% (84/162) of positive rectal swabs were performed at admission, whereas the remaining 48% (78/162) of positive rectal swabs were performed after 48h. Conclusions: This study found an increasing trend of VRE carriage in the study population during the SARS-CoV-2 pandemic, highlighting the importance of screening patients for VRE carriage to prevent worsening clinical conditions, environmental contamination, and prolonged hospitalization.

Priorities and Progress in Gram-positive Bacterial Infection Research by the Antibacterial Resistance Leadership Group: A Narrative Review.

The Antibacterial Resistance Leadership Group (ARLG) has prioritized infections caused by gram-positive bacteria as one of its core areas of emphasis. The ARLG Gram-positive Committee has focused on studies responding to 3 main identified research priorities: (1) investigation of strategies or therapies for infections predominantly caused by gram-positive bacteria, (2) evaluation of the efficacy of novel agents for infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci, and (3) optimization of dosing and duration of antimicrobial agents for gram-positive infections. Herein, we summarize ARLG accomplishments in gram-positive bacterial infection research, including studies aiming to (1) inform optimal vancomycin dosing, (2) determine the role of dalbavancin in MRSA bloodstream infection, (3) characterize enterococcal bloodstream infections, (4) demonstrate the benefits of short-course therapy for pediatric community-acquired pneumonia, (5) develop quality of life measures for use in clinical trials, and (6) advance understanding of the microbiome. Future studies will incorporate innovative methodologies with a focus on interventional clinical trials that have the potential to change clinical practice for difficult-to-treat infections, such as MRSA bloodstream infections.

Web-based prediction of antimicrobial resistance in enterococcal clinical isolates by whole-genome sequencing.

Besides phenotypic antimicrobial susceptibility testing (AST), whole genome sequencing (WGS) is a promising alternative approach for detection of resistance phenotypes. The aim of this study was to investigate the concordance between WGS-based resistance prediction and phenotypic AST results for enterococcal clinical isolates using a user-friendly online tools and databases. A total of 172 clinical isolates (34 E. faecalis, 138 E. faecium) received at the French National Reference Center for enterococci from 2017 to 2020 were included. AST was performed by disc diffusion or MIC determination for 14 antibiotics according to CA-SFM/EUCAST guidelines. The genome of all strains was sequenced using the Illumina technology (MiSeq) with bioinformatic analysis from raw reads using online tools ResFinder 4.1 and LRE-finder 1.0. For both E. faecalis and E. faecium, performances of WGS-based genotype to predict resistant phenotypes were excellent (concordance > 90%), particularly for antibiotics commonly used for treatment of enterococcal infections such as ampicillin, gentamicin, vancomycin, teicoplanin, and linezolid. Note that 100% very major errors were found for quinupristin-dalfopristin, tigecycline, and rifampicin for which resistance mutations are not included in databases. Also, it was not possible to predict phenotype from genotype for daptomycin for the same reason. WGS combined with online tools could be easily used by non-expert clinical microbiologists as a rapid and reliable tool for prediction of phenotypic resistance to first-line antibiotics among enterococci. Nonetheless, some improvements should be made such as the implementation of resistance mutations in the database for some antibiotics.

Nasotracheal enterococcal carriage and resistomes: detection of optrA-, poxtA- and cfrD-carrying strains in migratory birds, livestock, pets, and in-contact humans in Spain.

This study determined the carriage rates and antimicrobial resistance (AMR) genes of enterococci from nasotracheal samples of three healthy animal species and in-contact humans. Nasal samples were collected from 27 dog-owning households (34 dogs, 41 humans) and 4 pig-farms (40 pigs, 10 pig-farmers), and they were processed for enterococci recovery (MALDI-TOF-MS identification). Also, a collection of 144 enterococci previously recovered of tracheal/nasal samples from 87 white stork nestlings were characterized. The AMR phenotypes were determined in all enterococci and AMR genes were studied by PCR/sequencing. MultiLocus-Sequence-Typing was performed for selected isolates. About 72.5% and 60% of the pigs and pig-farmers, and 29.4% and 4.9%, of healthy dogs and owners were enterococci nasal carriers, respectively. In storks, 43.5% of tracheal and 69.2% of nasal samples had enterococci carriages. Enterococci carrying multidrug-resistance phenotype was identified in 72.5%/40.0%/50.0%/23.5%/1.1% of pigs/pig-farmers/dogs/dogs' owners/storks, respectively. Of special relevance was the detection of linezolid-resistant enterococci (LRE) in (a) 33.3% of pigs (E. faecalis-carrying optrA and/or cfrD of ST59, ST330 or ST474 lineages; E. casseliflavus-carrying optrA and cfrD); (b) 10% of pig farmers (E. faecalis-ST330-carrying optrA); (c) 2.9% of dogs (E. faecalis-ST585-carrying optrA); and (d) 1.7% of storks (E. faecium-ST1736-carrying poxtA). The fexA gene was found in all optrA-positive E. faecalis and E. casseliflavus isolates, while fexB was detected in the poxtA-positive E. faecium isolate. The enterococci diversity and AMR rates from the four hosts reflect differences in antimicrobial selection pressure. The detection of LRE carrying acquired and transferable genes in all the hosts emphasizes the need to monitor LRE using a One-Health approach.

Antibiotic choices among healthcare professionals for enterococcal bacteremia with patterns of resistance and risk factors of mortality, in settings of poor antibiotic stewardship program - a five-year retrospective cohort study.

BACKGROUND: Enterococcal bacteremia has become prevalent in the recent decade, especially in hospitalized patients. Moreover, the rise in resistance patterns against antibiotic drugs regarding enterococci infection, such as cephalosporins, ampicillin and vancomycin, is prevailing. The major driving force behind this is the incongruous use of antibiotics with a minor contribution from environmental stressors which calls for vigilant and prudent administration of evidence-based antibiotics. METHODS: A retrospective study was conducted from January 1 2017 until December 31 2021, at the tertiary care center, Dr Ziauddin Hospital in Karachi, Pakistan. RESULTS: Our research revealed ampicillin resistance in 87 (63.5%), with an estimated 25 (18.8%) mortality. Male gender 19 (76%) and vancomycin resistance 13 (52%) were associated with increased mortality. Furthermore, appropriate antibiotic therapy reduced the risk of death compared with inappropriate and excessive use of antibiotics 10 (40%) vs. 15 (60%) vs. 20 (80%) respectively. Targeted therapy with amoxicillin/clavulanic acid was associated with lower mortality 1 (4%) and higher discharge rates 34 (32.1%). On Kaplan-Meier survival, targeted therapy with amoxicillin/clavulanic acid was associated with shorter hospital stays and prolonged survival. UTI was found as the most common source of enterococcal bacteremia 57 (41.6%), followed by respiratory 21 (15.3%) and intra-abdominal 13 (9.5%). In 26 (19%) patients, no identifiable source of infection was found. CONCLUSION: Vancomycin resistance and male gender were found independent risk factors for mortality. The use of inappropriate antibiotics significantly increases mortality in these patients. The appropriate antibiotic therapy reduces the risk of death. Furthermore, overuse of antibiotics didn't reduce mortality; instead increased the financial burden and chances of developing multi-drug resistant strains of other organisms by increasing hospital stays of patients.