Search Results (63)

Background/Objectives: Vancomycin-resistant (VRE) and vancomycin-variable (VVE) Enterococcus spp. represent an increasing clinical challenge due to limited treatment options and the potential for undetected dissemination of such resistance genes. Data on Enterococci genomic epidemiology in healthcare settings remain rather limited. Our study aimed to investigate vancomycin resistance determinants in Enterococcus spp., clonal structure, and occurrence of VVE using whole-genome sequencing (WGS) in Latvia. Methods: Clinical isolates collected from hospitalised patients in two tertiary-level hospitals in Latvia (2021–2024) were analysed using WGS following routine laboratory identification. Vancomycin resistance determinants were identified in silico, along with MLST and cgMLST genotyping. Results: Of 532 sequenced isolates, 482 met the quality and inclusion criteria. E. faecalis (56.64%) and E. faecium (40.25%) predominated. Among 125 isolates carrying vancomycin resistance genes, vanB (54.40%) was the most frequent, followed by vanA (38.20%) and vanC (6.40%); vanC was restricted to E. gallinarum and E. casseliflavus. Vancomycin resistance was more prevalent in E. faecium (51.03%) than in E. faecalis (6.59%). cgMLST identified outbreak clusters among E. faecium ST80 and ST78 with complex type-specific resistance patterns and hospital specificity. E. faecalis showed polyclonal endemicity with the vanB gene present in different clades. Three (0.62%) vancomycin-variable E. faecium (VVE) isolates were identified in one hospital, harbouring vanA-type gene clusters comprising vanHAX but lacking the sensory gene vanS and the regulatory gene vanR. Conclusions: The VanB gene predominated in both hospitals, driven by clonal expansion of hospital-adapted E. faecium ST80/ST78, contrasting with earlier vanA predominance in Europe but aligning with recent regional vanB trends. The detection of VVE highlights clinically relevant genotype–phenotype discordance, underscoring the importance of integrating genomic surveillance with routine phenotypic testing to detect cryptic resistance and guide effective antimicrobial therapy. Full article

Background. Positively charged quaternary phosphonium salts (QPSs) represent new potent weapons to selectively counteract critical superbugs, regardless of their profile of resistance, acting as membrane disruptors. QPSs 1, 3 and 4, and not cationic phosphine 2, recently synthesized, characterized and evaluated for anticancer and cytotoxic effects, were morphologically and microbiologically evaluated. Methods. DLS analysis, minimum inhibitory concentrations (MICs) measurements, time-kill experiments and tests to evaluate biofilm (BF) formation inhibition, on Gram-positive and Gram-negative clinical superbugs, were carried out. Results and Discussion. All compounds demonstrated positive ζ-p = +4.2–+38.1 mV, while 2, 3 and 4 showed nanovesicles of 140, 157, and 605 nm, in water solution. Interesting microbiologic results were obtained for compound 1. Despite not being active against Gram-negative MDR isolates, 1 displayed MICs = 16–32 µg/mL and 16–64 µg/mL against methicillin-susceptible (MSSA) Staphylococcus aureus ATCC 29213, methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE) respectively, while MICs = 32–64 µg/mL were observed against teicoplanin- and vancomycin-resistant (VRE) Enterococcus faecalis and E. faecium, thus overturning MICs previously reported for 1. Novel time-kill experiments established the bactericidal effects of 1 against MRSA within 11 h, without no regrowth in the subsequent 24 h. Further, 1 inhibits up to 100% BF formation by the strongest BF-producers, S. epidermidis and S. aureus isolates, of our collection. Conclusions. All these antibacterial properties and low cytotoxicity on both fibroblasts (3T3) and human keratinocytes (HaCaT) cells make 1 appear as a potential new weapon to treat infections no longer affordable with current antibiotics; it is also thinkable for future use in vivo experiments and clinical development. Full article

Background: Eravaycline is a novel fully synthetic fluorocycline that is currently approved for complicated intra-abdominal infections. However, it is sometimes also used off-label in tertiary care centers for other infection sites as an antibiotic of last resort due to its broad spectrum of activity and efficacy against Enterobacterales, including multidrug-resistant pathogens like extended spectrum β-lactamase (ESBL) producers or carbapenem-resistant Enterobacterales, as well as all Gram-positive organisms including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin- and linezolid-resistant Enterococcus faecium (VRE). Methods: We retrospectively included a total of 78 patients from Austria and Udine who received eravacycline between April 2023 and August 2024 to evaluate the real-world efficacy of eravacycline in various infection sites and pathogens using descriptive statistics. Results: Eravacycline was most commonly used in intra-abdominal infections (44.9%), followed by pneumonia (12.8%) and infections of unknown origin (7.7%). Escherichia coli, including ESBL producers, was the most common pathogen (24.4%), followed by Enterococcus spp. (12.8%) and Klebsiella pneumoniae (12.8%). Clinical cure was achieved in 65% of patients, whereas microbiological cure was documented in 46%; source control was attained in 48.7%, and 16.7% died within 30 days. A total of 48% of patients required intensive care. Conclusions: Eravacycline represents a possible therapeutic option for a wide range of pathogens, but its use must be evaluated in the context of infection site and severity. Full article

Introduction: Bacterial multidrug resistance (MDR) drives treatment with expensive, toxic, or pharmacokinetically suboptimal antibiotics. Objectives: To assess the prevalence of MDR Gram-positive cocci among isolates from cardiac implantable electronic device (CIED) infections at a Polish reference center. Methods: Data come from the “EXTRACT” registry (ClinicalTrials.gov ID NCT05775783), which covers 702 transvenous lead extraction procedures. Blood samples and intraoperative swabs were collected from participants with CIED infection. Results: From 209 cases with isolated pocket infection (PI) (107, 51.2%) or systemic infections (102, 48.8%), 263 Gram-positive cocci were cultured. They were: coagulase-negative staphylococci (CoNS) (177, 67.3%), Staphylococcus aureus (62, 23.6%), enterococci (15, 5.7%), streptococci (8, 3.0%), and others (1, 0.4%). The highest MDR rate was among CoNS (46.9%). CoNS exhibited methicillin resistance (MR-CoNS) in 55.9% with co-resistance to macrolides (73.2%), lincosamides (51.0%), fluoroquinolones (56.1%), aminoglycosides (41.4%), tetracyclines (29.6%), and co-trimoxazole (29.3%). Resistance to daptomycin (5.3%) and linezolid (2.0%) in MR-CoNS was rare. The frequency of MDR S. aureus was 8.1%. Methicillin resistance in S. aureus (MRSA, 6.5%) co-occurred with resistance to macrolides/lincosamides and fluoroquinolones (100% for both) or linezolid (25.0%). All MDR staphylococci were vancomycin-susceptible. High-level aminoglycoside resistance (HLAR) in Enterococcus faecalis (53.8%) was accompanied by levofloxacin co-resistance (66.7%). Conversely, E. faecium HLAR (50.0%) strains showed 100.0% β-lactam resistance. Vancomycin-resistant enterococci (VRE) accounted for 6.7%; the VRE E. faecium strain was tigecycline- and linezolid-susceptible. Among viridans group streptococci, β-lactam and lincosamides resistance was common (40.0% for both), with 50.0% of co-resistance. Conclusions: Epidemiological data may improve the effectiveness of empirical antibiotic therapy for CIED-related infections. Full article

The global rise in antimicrobial resistance among the ESKAPE pathogens represents a major challenge to public health. Here, we report the broad-spectrum antibacterial activity of the synthetic antimicrobial and pore-forming peptide C14R against all six ESKAPE species. Using a radial diffusion assay and resazurin-based viability testing, C14R exhibited a potent bactericidal effect with minimum inhibitory concentrations (MICs), defined as the lowest concentration of an antimicrobial agent that completely inhibits visible growth of planktonic microorganisms, ranging from 3.4 µg/mL (Enterococcus faecium, vancomycin-resistant) to 45.2 µg/mL (Klebsiella quasipneumoniae, ESBL). C14R also inhibited biofilm formation by Gram-positive pathogens, with minimum biofilm inhibitory concentrations (MBICs), referring to the minimal concentration required to prevent the development of biofilms, of 15.0 µg/mL (Staphylococcus aureus, MRSA) and 22.0 µg/mL (E. faecium, VRE), whereas Gram-negative biofilms showed higher tolerance. Together, these findings demonstrate that C14R retains high activity against multidrug-resistant ESKAPE strains, highlighting its potential as a lead compound for the development of next-generation antimicrobial drugs to expand the portfolio of available antibiotics and brace health systems against emerging severe infections. Full article

Vancomycin-resistant Enterococci (VRE) are established nosocomial pathogens; however, vancomycin-dependent Enterococci (VDE) represent a rare and underrecognized phenomenon. These organisms paradoxically require vancomycin for growth due to mutations in cell wall precursor synthesis. Limited awareness and significant diagnostic challenges associated with VDE can lead to delayed recognition and treatment failure. We report a case of vancomycin-dependent Enterococcus faecium isolated from a liver transplant recipient receiving oral vancomycin prophylaxis for recurrent Clostridioides difficile infection. The isolate failed to grow on standard media but exhibited robust growth on vancomycin-supplemented agar, confirmed by vancomycin disc diffusion testing and PCR detection of the vanB gene. Additionally, we reviewed four further VDE cases identified over a two-year period in our tertiary care microbiology laboratory. All patients originated from complex care settings, had significant comorbidities, and had received prolonged glycopeptide therapy. We summarize the clinical features, diagnostic findings, and microbiological challenges encountered across this case series. This series documents the first reported Canadian case of VDE and highlights the critical need for clinical vigilance and diagnostic suspicion in high-risk patients with prior enterococcal colonization and ongoing glycopeptide exposure. Laboratory findings such as failure to grow on blood agar coupled with growth around vancomycin discs should prompt specific evaluation for VDE. Our findings reinforce the necessity for targeted antimicrobial stewardship and infection prevention strategies and underscore the remarkable evolutionary adaptability of Enterococci under sustained antimicrobial pressure. Full article

Blood culture remains the gold standard for identifying bloodstream infections caused by bacteria and fungi. Isolation of the culprit microorganism onto agar plates also facilitates antimicrobial susceptibility testing. The purpose of this study was to determine the contamination rates, pathogen profile, and antimicrobial resistance in a secondary healthcare setting in a two-year timeframe. In this study, data regarding blood cultures of the years 2023 and 2024 were retrospectively analyzed to address the above questions. Blood cultures were incubated for seven days before being discarded as negative. The percentage of positive blood cultures for both years was 14.3%. Most positive cultures contained Gram-positive cocci, with a prevalence of coagulase-negative Staphylococci. In descending order, 72.72% were coagulase-negative Staphylococci, 15.15% were Staphylococcus aureus, and 12.12% were Streptococci. One strain of S. aureus was methicillin-resistant (MRSA), and one strain of Enterococcus faecium was vancomycin-resistant (VRE). Of the Gram-negative rods, 78.3% were Enterobacterales. Of these, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis were the top pathogens. The remainder comprised eight strains of Pseudomonas aeruginosa, four strains of Acinetobacter baumannii (one pandrug-resistant), three strains of Stenotrophomonas maltophilia, one strain of Sphingomonas paucimobilis, and one strain of Campylobacter jejuni. The isolated fungi comprised Candida parapsilosis, Candida glabrata, and Candida tropicalis. Of the isolated Escherichia coli strains, 39.5% were resistant to ciprofloxacin regardless of origin (outpatient or hospitalized patients). Outpatient samples were taken in a Hemodialysis Unit that collaborates with our laboratory, obtained from patients with fever or other signs of infection. Distinguishing true bacteremia from contamination remains challenging. The contamination rate in our study was quite high at 5.3%. Since there is no dedicated phlebotomy team in our healthcare setting, in light of our results, educational courses have been conducted to demonstrate the best practices for sample collection. Full article

Background: To meet the urgent need for novel antibacterial agents that are active also against worrying superbugs, natural pentacyclic triterpenoids, including totally inactive betulin (BET) and betulinic acid (BA), as well as ursolic acid (UA), active on Gram-positive bacteria, have been chemically modified, achieving compounds 1–7. Methods: Triterpenoid derivatives 1–7 and all synthetic intermediates were characterized by chemometric-assisted FTIR and NMR spectroscopy, as well as by other analytical techniques, which confirmed their structure and high purity. Minimum inhibitory concentration values (MICs) of 1–7, BET, BA and UA were determined by the broth dilution method, using a selection of Gram-positive and Gram-negative clinically isolated superbugs. Results: Performed experiments evidenced that compounds 4–7 had potent antibacterial effects against Gram-positive methicillin-resistant Staphylococcus aureus and S. epidermidis (MRSA and MRSE), as well as against vancomycin-resistant Enterococcus faecalis and E. faecium (VRE). The antibacterial effects of 4–7 were due to the insertion of a triphenyl phosphonium (TPP) group and were higher than those reported so far for other BET, BA and UA derivatives, especially considering the complex pattern of resistance of the isolates used here and their clinical source. Conclusions: For the first time, by inserting TPP, a real activity (MICs 2–16 µg/mL) was conferred to inactive BET and BA (MICs > 1024 and 256 µg/mL). Moreover, the antibacterial effects of UA were improved 16- and 32-fold against MRSE and MRSA (MICs = 2 vs. 32 and 64 μg/mL). Future Perspectives: Based on these very promising microbiologic results, new experiments are currently underway with the best-performing compounds 5 and 7 (MICs = 2 μg/mL) on an enlarged number of Gram-positive isolates, to confirm their MICs. Moreover, investigations about their possible antibiofilm activity, time-killing curves and cytotoxicity on eukaryotic cells will be carried out to define their pharmacological behavior and clinical potential. Full article

Background/Objectives: Vancomycin-resistant enterococci (VRE) are significant nosocomial pathogens worldwide, potentially transmitted by food-producing animals and related products. This study investigates the epidemiological role of bovine raw milk in the transmission of VRE to humans. Methods: Bulk milk samples were screened for van gene presence using a multiplex PCR. Mastitogenic enterococci isolated from individual milk samples were tested for antimicrobial susceptibility using the broth microdilution method. Strains not susceptible to vancomycin were whole genome sequenced. Results: Overall, vanC genes were detected in 299/1026 (29.14%) bulk milk samples. Specifically, vanC1 was found in 204 samples (19.88%) and vanC2/3 in 57 samples (5.56%), with both detected simultaneously in 38 samples (3.70%). Clinically significant vanA and vanB genes were not identified. A total of 163 mastitogenic Enterococcus strains were isolated from individual milk samples. Eight different Enterococcus species were detected, with E. faecium (104/163, 63.80%) and E. faecalis (34/163, 20.86%) being the most common. Multidrug resistance was observed in 106/163 (65.03%) isolates. The most common resistance frequencies were to ciprofloxacin and erythromycin (102/163, 62.58% both), followed by quinupristin/dalfopristin (93/163, 57.06%), linezolid (65/163, 39.88%), tetracycline (58/163, 35.58%), daptomycin (46/163, 28.22%), chloramphenicol (33/163, 20.25%), ampicillin, tigecycline, and high-dosage gentamycin (8/163, 4.91% all). Resistance to teicoplanin was not observed. Two vancomycin non-susceptible strains were identified: one vanC2/3 E. casseliflavus and one vanC1 E. gallinarum. Whole genome sequencing confirmed the presence of the complete vanC gene cluster and several virulence genes in both strains. Conclusions: Our findings suggest that while raw milk is unlikely to be a source of vancomycin resistance genes of highest clinical importance (vanA or vanB), it may contribute to the spread of vanC enterococci, which are increasingly associated with human infections. Full article

Background/Objectives: Enterococci, particularly Enterococcus faecalis and Enterococcus faecium, are Gram-positive cocci that can cause severe infections in hospitalized patients. The rise of vancomycin-resistant enterococci (VRE) and vancomycin-variable enterococci (VVE) poses significant challenges in healthcare settings due to their resistance to multiple antibiotics. Methods: We conducted a point prevalence survey (PPS) to assess the prevalence of VRE and VVE colonization in hospitalized patients. Rectal swabs were collected from 160 patients and analyzed using molecular assays (MAs) and culture. Whole-genome sequencing (WGS) and core-genome multilocus sequence typing (cgMLST) were performed to identify the genetic diversity. Results: Of the 160 rectal swabs collected, 54 (33.7%) tested positive for the vanA and/or vanB genes. Culture-based methods identified 47 positive samples (29.3%); of these, 44 isolates were identified as E. faecium and 3 as E. faecalis. Based on the resistance profiles, 35 isolates (74.5%) were classified as VRE, while 12 (25.5%) were classified as VVE. WGS and cgMLST analyses identified seven clusters of E. faecium, with sequence type (ST) 80 being the most prevalent. Various resistance genes and virulence factors were identified, and this study also highlighted intra- and inter-ward transmission of VRE strains. Conclusions: Our findings underscore the potential for virulence and resistance of both the VRE and VVE strains, and they highlight the importance of effective infection control measures to prevent their spread. VVE in particular should be carefully monitored as they often escape detection. Integrating molecular data with clinical information will hopefully enhance our ability to predict and prevent future VRE infections. Full article

Background:Enterococcus spp. is the third leading cause of healthcare-associated infections in the American continent, often because of the virulence factors that protect the bacterium against host defenses and facilitate tissue attachment and genetic material exchange. In addition, vancomycin, considered a last-resort treatment, has shown reduced efficacy in Enterococcus spp. strains. However, the relationship between bacterial resistance and virulence factors remains unclear. This study intends to evaluate the prevalence of glycopeptide-resistant genotypes and virulence factors in Enterococcus spp. strains. Methods: Over six months, 159 Enterococcus spp. strains causing nosocomial infections were analyzed. Multiplex PCR was performed to identify species, glycopeptide-resistant genotypes, and 12 virulence factors. Results: The most abundant species identified were Enterococcus faecalis and E. faecium. Vancomycin resistance was observed in 10.7% of the isolates, and the vanA genotype was present in 47% of resistant samples. The main virulence factors detected were acm (54%), which is related to cell adhesion; gel E (66%), a metalloproteinase linked to tissue damage; and the sex pheromones cpd (64%) and ccf (84%), which are involved in horizontal gene transfer. A significant association was found between the prevalence of acm, ccf, and cpd in VRE isolates, indicating the potential dissemination of genes to emerging strains via horizontal gene transfer. In addition, a new E. faecium, which displayed five virulence factors and harbored the vanA sequence type, was identified and registered as ST2700. Conclusions:Enterococcus faecalis and E. faecium are clinically critical due to multidrug resistance and virulence factors like acm, which aids host colonization. Genes ccf and cpd promote resistance spread via horizontal transfer, while the emerging ST2700 strain requires urgent monitoring to curb its virulent, drug-resistant spread. Full article

Daptomycin (DAP) is a therapeutic option for vancomycin-resistant Enterococcus faecium (VRE) infections, but DAP resistance may occur during treatment. Previously, we identified a mutation within the E. faecium lafB gene that induces hypersusceptibility to DAP. The lafB gene encodes a glycosyltransferase involved in lipoteichoic acid anchor synthesis, which makes it a promising target for enhancing DAP efficacy. In this study, we characterized E. faecium LafB protein (EfLafB) biophysical properties, used AlphaFold3 to predict LafB in silico three-dimensional structure, and determined lafB gene mutation’s role in virulence, comparing E. faecium HBSJRP18 (DAP-hypersusceptible) and a lafB revertant, HBSJRP18_2.7, and analyzing bacterial growth kinetics, biofilm formation ability, and virulence in a Galleria mellonella model. After gene cloning and expressing and purifying EfLafB, circular dichroism and SEC-MALS assays revealed its monomeric nature under in vitro conditions, with approximately a 40 kDa molecular mass and a melting temperature of 50 °C. In silico prediction indicated that LafB is an αβ-type protein with two domains conforming to the GT-4 family glycosyltransferases. These results are further supported by the highly conserved amino acids (E257, D91, R184, and K185), likely involved in UDP-Glc binding. The studied lafB gene mutation resulted in a significant decrease in bacterial growth and virulence in the invertebrate model. Full article

Virulence attributes and putative antibiotic resistance genes from enterococcal isolates from wastewater treatment facilities for sustainable reuse and the areas where they discharge treated water were assessed using phenotypic and molecular methods. This analysis was performed on 269 Enterococci, of which 202 were vancomycin-resistant Enterococcus (VRE). VRE strains show markedly higher resistance across multiple antibiotics, especially glycopeptides and beta-lactams, compared to the more susceptible profile observed in vancomycin-susceptible Enterococcus (VSE) strains. vanC was found in every instance of E. gallinarum among VRE and enterococci susceptible to vancomycin (VSE) isolates but not in VR E. faecium/faecalis. Among VRE, 127 (62.9%) possessed at least one of the tetK, tetL, tetM, or tetO, while 22 (17.3%) had two of these genes. The multidrug efflux pump gene emeA was detected in 27 out of 202 (13.4%) VRE isolates and 8 out of 67 (11.9%) VSE isolates. Exactly 69 (78.4%) possessed at least one of the virulence determinants tested, with 10 (11.4%) and seven (8%) positive for haemolysis and gelatinase activity respectively. The gelatinase gene, gelE, was detected in 16 (18.1%) isolates, while more isolates (n = 23; 26.1%) were positive for gelatinase activity. Cytolytic (cyl) genes (1.1%), Angiotensin-converting-enzyme genes (ace) (13.6%), endocarditis-specific antigen A genes (efaA) (25%), hyaluronidase (hyl) genes (9.1%), enterococcal surface protein (esp) genes (4.5%), among others, were detected. Gelatinase activity and the amplified virulence genes were further validated by sequencing the gel-positive amplicons, which were almost identical (98.97%), and the gelE gene of Enterococcus sp. strain SQ07C was deposited under the GenBank accession number PQ381122. Overall, our results showed that the enterococcal isolates were considered as potential pathogens of notable threat to human health via exposure through reuse, and there is a need for more stringent treatment protocols. Full article

Background: Vancomycin-resistant enterococci (VRE) rectal colonization represents a critical risk factor for subsequent bloodstream infections (BSIs), posing a serious concern in healthcare settings. This study aims to investigate the association between the presence of VRE in rectal swabs and the occurrence of BSIs, highlighting the challenges of rapid detection and patient care implications in an infectious disease hospital setting. Methods: We performed a retrospective analysis of cultural rectal swab screening and molecular assays (MAs) for VRE detection between January 2020 and December 2023. All adult patients admitted with at least one rectal swab screening performed during hospitalization were included. All blood cultures that yielded VRE were identified, and the first Enterococcus-positive blood sample for each patient with at least one prior rectal swab per year was analyzed. Results: The results showed a 15.4% positivity rate for VRE in cultural screening, predominantly Enterococcus faecium. MA showed a higher prevalence of 49.4%, with a significant discordance between MA rectal swab screening and cultural testing. Patients with VRE intestinal colonization by E. faecium were significantly more likely to develop E. faecium BSI, with a risk ratio of 9.78 (p < 0.001). Conclusions: The study identified a strong correlation between VRE rectal colonization and the risk of developing BSI, emphasizing the need for effective screening and infection control measures. The results support the inclusion of molecular testing in VRE detection protocols and highlight the importance of ongoing surveillance for antimicrobial resistance. Full article

Background/Objectives: Vancomycin-resistant enterococci (VRE) are one of the leading causes of antibiotic-resistant infections in the hospital setting worldwide, and this has become a major issue, because most patients infected with this strain are difficult to treat. Multiplex real-time polymerase chain reaction (RT PCR) is an advantageous technique that can amplify multiple targets in a single reaction, and can be used to quickly detect specific targets in VRE within two hours, starting from suspected colonies of bacterial cultures, without sample preparation. Methods: In this study, we selected the glycopeptide/vancomycin resistance genes that are most common in clinical settings, vanA and vanB, in combination with the species markers ddl_faecium and ddl_faecalis for the most common VRE species—Enterococcus faecium and Enterococcus faecalis. Results: DNA from forty clinical VRE strains was prepared using a fast and economic heat lysis method, and a multiplex real-time PCR assay was optimized and carried out subsequently. The results were in concordance with the results from recombinase polymerase amplification (RPA) of the same VRE samples. Conclusions: Multiplex RT PCR and RPA for VRE detection proffers a second method for the confirmation of vancomycin resistance, and it can be developed as a fast screening assay for patients before admission into high-risk settings. Full article