Abstract: ABSTRACT The kākāpō is a critically endangered flightless parrot which suffers from exudative cloacitis, a debilitating disease resulting in inflammation of the vent margin or cloaca. Despite this disease emerging over 20 years ago, the cause of exudative cloacitis remains elusive. We used total RNA sequencing and metatranscriptomic analysis to characterise the infectome of lesions and cloacal swabs from nine kākāpō affected with exudative cloacitis, and compared this to cloacal swabs from 45 non‐diseased kākāpō. We identified three bacterial species— Streptococcus gallolyticus , Enterococcus faecalis and Escherichia coli —as significantly more abundant in diseased kākāpō compared to healthy individuals. The genetic diversity observed in both S. gallolyticus and E. faecalis among diseased kākāpō suggests that these bacteria originate from exogenous sources rather than from kākāpō‐to‐kākāpō transmission. The presence of extraintestinal pathogenic E. coli (ExPEC)‐associated virulence factors in the diseased kākāpō population suggests that E. coli may play a critical role in disease progression by facilitating iron acquisition and causing DNA damage in host cells, possibly in association with E. faecalis . No avian viral, fungal nor other parasitic species were identified. These results, combined with the consistent presence of one E. coli gnd sequence type across multiple diseased birds, suggest that this species may be the primary cause of exudative cloacitis. These findings shed light on possible causative agents of exudative cloacitis, and offer insights into the interplay of microbial factors influencing the disease.

Abstract: Background: The genus Enterococcus includes a diverse group of bacteria that are commonly found in the gastrointestinal tracts of humans and animals, as well as in various environmental habitats. Methods: In this study, Enterococcus lactis RB10, isolated from goat feces, was subjected to comprehensive genomic and functional analysis to assess its safety and potential as a probiotic strain. Results: The genome of E. lactis RB10, with a size of 2,713,772 bp and a GC content of 38.3%, was assembled using Oxford Nanopore Technologies (ONT). Genome annotation revealed 3375 coding sequences (CDSs) and highlighted key metabolic pathways involved in carbohydrate, protein, and amino acid metabolism. The strain was susceptible to important antibiotics, including ampicillin, chloramphenicol, tetracycline, and vancomycin, but exhibited resistance to aminoglycosides, a common trait in Enterococcus species with non-hemolytic activity. Genomic analysis further identified two intrinsic antimicrobial resistance genes (ARGs). The strain also demonstrated antimicrobial activity against Bacillus cereus DMST 11098 and Salmonella Typhi DMST 22842, indicating pathogen-specific effects. Key genes for adhesion, biofilm formation, and stress tolerance were also identified, suggesting that RB10 could potentially colonize the gut and compete with pathogens. Moreover, the presence of bacteriocin and secondary metabolite biosynthetic gene clusters suggests its potential for further evaluation as a biocontrol agent and gut health promoter. Conclusions: However, it is important to note that E. lactis RB10 was isolated from goat feces, a source that may harbor both commensal and opportunistic bacteria, and therefore additional safety assessments are necessary. While further validation is needed, E. lactis RB10 exhibits promising probiotic properties with low pathogenic risk, supporting its potential use in food and health applications.

Abstract: The Enterococcus species originates as non-harmful bacteria indigenous to human intestines but has transformed into severe hospital-acquired pathogens due to antimicrobial resistance (AMR). The clinical species Enterococcus faecalis and Enterococcus faecium create the most relevant infections because they appear in urinary tract infections, bloodstream infections, endocarditis, and wound infections. Enterococcus species demonstrate multiple antibiotic class resistance and resistance determinant acquisition properties that make treatment difficult for medical professionals. Vancomycin-resistant enterococci (VRE) together with high-level aminoglycoside-resistant strains and resistance to both linezolid and daptomycin have exhausted available treatment options. The review investigates the development process of Enterococcus infections by examining virulence characteristics, which involve biofilm production and defense mechanisms against the immune response and transmission of resistance genes. A thorough investigation of medical publications used Google Scholar along with PubMed and ScienceDirect and Medical Subject Headings (MeSH) as appropriate search terms. The traditional classification of Enterococcus species from historical context to modern epidemiology and pathogenesis and available treatment and test approaches are explained in this review. This section examines two categories of resistance together with their mechanisms of action with a specific focus on vancomycin resistance produced by van gene clusters as well as its prevalence trends. An examination of how horizontal gene transfer functions in transferring resistance throughout healthcare facilities is included. The paper investigates the different symptoms of enterococcal infections together with diagnostic obstacles and treatment modalities. Drug-resistant Enterococcus infections continue to increase internationally, so healthcare professionals need new therapeutic methods, better antimicrobial policies, and stronger infection prevention measures. The examination surveys Enterococcus infections through an extensive evaluation of developing resistance patterns combined with emerging intervention requirements.

Abstract: ABSTRACT Screening for carbapenemase-producing Enterobacterales (CPE) and vancomycin-resistant Enterococcus (VRE) colonization among hospitalized patients is a standard infection control procedure that also guides appropriate antibiotic treatment in healthcare settings. Extensive CPE screening in low-prevalence regions imposes a considerable laboratory workload and substantial costs that can be mitigated through the utilization of pool testing. In this study, we evaluated PCR pooling for the detection of CPE and VRE colonization from rectal swabs collected in our hospital, using the Xpert Carba-R and Xpert vanA/vanB assays. CPE pooling demonstrated excellent performance, with 94.44% sensitivity (74.2–99.0 CI 95%), 100% specificity (99.1–100), 100% positive predictive value (81.6–100), and 99.78% (98.7–100) negative predictive value (NPV). In contrast, VRE detection showed lower performance, with 75.86% (63.5–85.0 CI 95%) specificity and 100% (92.0–100) NPV, primarily due to false-positive results for the vanB gene. Overall, the pooling technique showed great potential for CPE screening in low-prevalence settings. Further studies are needed to evaluate VRE pooling and clarify the clinical utility of PCR results for vanB , which currently requires culture confirmation. IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) and vancomycin-resistant enterococci (VRE) pose a major threat to healthcare systems due to their role in hospital-acquired infections and limited treatment options. Routine screening for CPE/VRE colonization is essential for preventing transmission, yet conventional culture-based methods are labor-intensive and time-consuming. This study demonstrates that PCR pooling is a highly effective and resource-efficient approach for CPE screening in low-prevalence settings, maintaining high sensitivity and specificity while reducing laboratory workload. The performance of VRE pooling, particularly for vanB detection, requires further evaluation. These findings support the adoption of pooling strategies for CPE surveillance and highlight the need for improved molecular diagnostics for VRE, offering a promising solution to address growing challenges in clinical microbiology diagnostics.

Abstract: Gastrointestinal colonization by the nosocomial pathogen vancomycin-resistant Enterococcus faecium (VREfm) can lead to bloodstream infections with high mortality rates. Shifts in VREfm lineages found within healthcare settings occur, but reasons underlying these changes are not understood. Here we sequenced 710 VREfm clinical isolates collected between 2017 and 2022 from a large tertiary care centre. Genomic analyses revealed a polyclonal VREfm population, although 46% of isolates formed genetically related clusters, suggesting a high transmission rate. Comparing these data to a global collection of 15,631 publicly available VREfm genomes collected between 2002 and 2022 identified replacement of the sequence type (ST) 17 VREfm lineage by emergent ST80 and ST117 lineages at the local and global level. Comparative genomic and functional analyses revealed that emergent lineages encoded bacteriocin T8, which conferred a competitive advantage over bacteriocin T8-negative strains in vitro and upon colonization of the mouse gut. Bacteriocin T8 carriage was also strongly associated with strain emergence in the global genome collection. These data suggest that bacteriocin T8-mediated competition may have contributed to VREfm lineage replacement. Genomic and functional analyses of healthcare-associated vancomycin-resistant Enterococcus faecium reveal that bacteriocin T8 is enriched in emergent lineages and provides a competitive advantage in vitro and in vivo.

Abstract: Bile salt hydrolase (BSH: EC.3.5.1.24) has been used as a biomarker for probiotics for an extended period. It is mostly present in the gut environment of vertebrates. Additionally, it influences the viability of probiotics. This biomarker is considered a promising nutritional supplement due to its unique ability to effectively address elevated blood cholesterol levels, a common issue in modern society. However, the commercialization of BSH has been limited by an incomplete understanding of the intestinal microbiota and the function of BSH. Hence, in this review, we aim to reveal the current advancements in BSH research and outline the necessary areas of investigation for future studies. The review highlights key findings related to the substrate affinity of BSH in probiotic bacteria and its BSH gene phylogeny that have been researched until today, suggesting further research regarding the differences in multiple BSH genes and corresponding differences in BSH affinity.

Abstract: In microbiota research, whole stool sampling is the conventional approach but can be problematic or infeasible for certain patients. This study aims to validate the use of rectal swabbing as an alternative method for microbiota analysis and determine optimal storage conditions suitable for various clinical settings, including intensive care units. We evaluated different sampling techniques and storage temperatures. Our findings indicated that rectal swabs yield microbiota diversity comparable to whole stool samples. Notably, storage conditions significantly impacted microbiota profiles, with increased E. coli and Enterococcus sp. quantifications observed at room temperature (RT). Consequently, we recommend immediate refrigeration of rectal swabs to reliably assess aerobic and total microbiota, particularly for patients requiring urgent care, such as antibiotic treatment. We developed a pragmatic approach to study total and aerobic gut microbiota, applicable in numerous clinical units, such as intensive care or emergency units, where whole stool sampling is often impractical. This approach employs ESwab devices, which are already commonly used in hospitals.

Abstract: Objective This study analyzes the global prevalence and distribution of vancomycin resistance genes ( van ) in Enterococcus faecium and examines the genetic relationship and epidemiological characteristics of strains carrying these genes. Method A total of 3,256 E. faecium genome sequences were downloaded, and 2,235 high-quality genomes were retained after quality filtering. The blastn tool was used to screen these genomes for van genes, and sequence types (STs) were determined using pubMLST profiles. Result Among the 2,235 genomes, 1,071 (47.9%) harbored van genes, with eight genotypes identified, including vanA , vanB , vanD , and vanM , accounting for 47.6%. There were 83 distinct STs among the strains carrying van genes, with ST17 being the most prevalent. Most strains carrying van genes were isolated from humans, primarily in the United States, and commonly from rectal swabs. In 2015, vanA was the most prevalent van gene, particularly in ST17 strains. Conclusion This study highlights the widespread distribution of van genes and their significant presence in human populations and clinical settings, emphasizing the importance of monitoring and intervening in the spread of ST17 strains.

Abstract: To adapt and persist in the gastrointestinal tract, many enteric pathogens, including Clostridioides difficile, employ strategies such as phase variation to generate phenotypically heterogeneous populations. Notably, the role of the gut microbiota and polymicrobial interactions in shaping population heterogeneity of invading pathogens has not been explored. Here, we show that Enterococcus faecalis , an opportunistic pathogen that thrives in the inflamed gut during C. difficile infection, can impact the phase variable CmrRST signal transduction system in C. difficile . The CmrRST system controls multiple phenotypes including colony morphology, cell elongation, and cell chaining in C. difficile . Here we describe how interactions between E. faecalis and C. difficile on solid media lead to a marked shift in C. difficile phenotypes associated with phase variation of CmrRST. Specifically, E. faecalis drives a switch of the C. difficile population to the cmr- ON state leading to chaining and a rough colony morphology. This phenomenon preferentially occurs with E. faecalis among the enterococci, as other enterococcal species do not show a similar effect, suggesting that the composition of the polymicrobial environment in the gut is likely critical to shaping C. difficile population heterogeneity. Our findings shed light on the complex role that microbial ecology and polymicrobial interactions can have in the phenotypic heterogeneity of invading pathogens. Clostridioides difficile is an enteric pathogen with critical implications for public health. The microbial ecosystem in which C. difficile resides shapes the behavior and fitness of C. difficile ; however, the mechanisms underlying these interactions are not well defined. Here, we demonstrate that Enterococcus faecalis , an opportunistic pathogen known to co-colonize the gut with C. difficile , influences phase variation and downstream growth phenotypes in C. difficile . This phenomenon represents a new paradigm by which co-residing bacteria can modulate phase variation dynamics in C. difficile or other enteric pathogens. Understanding factors that influence C. difficile behavior may elucidate new therapeutic strategies, especially in complex polymicrobial infections.

Abstract: ABSTRACT Background Enterococci are commonly isolated from bile cultures in patients with acute cholangitis (AC) and pose a global concern owing to antibiotic resistance. This study identified predictive factors and clinical characteristics of AC with enterococcal‐positive bile cultures. Methods Consecutive patients with AC and bactobilia who underwent endoscopic retrograde cholangiopancreatography between April 2022 and March 2024 were included. Patients were categorized into enterococcal (E) and non‐E groups based on bile culture results. Predictive factors for enterococcal positivity were analyzed. Empirical antibiotic coverage and clinical outcomes (symptom duration, length of antibiotic therapy, in‐hospital mortality, and short‐term recurrence) were compared within all‐grade and severe AC. Results Among 250 patients, 100 with enterococcal‐positive bile cultures formed the E group. Predictive factors included age > 71 years, immunosuppressed status, prior endoscopic sphincterotomy, and prior biliary stenting. Inappropriate empirical antibiotic coverage was more frequent in the E group across both severity categories. Clinical outcomes did not differ among all‐grade patients except for higher short‐term recurrence in the E group. No significant differences were observed among severe patients for any clinical outcome. Conclusions Enterococcal‐positive bile cultures are predicted by specific factors. Despite frequent inadequate empirical antibiotic coverage, most clinical outcomes were similar to those for non‐enterococcal patients.

Abstract: Abstract Objectives Bloodstream infections (BSIs) due to vancomycin-resistant Enterococcus spp. (VRE) are considered a predictor of mortality among frail patients. The aim of this study was to evaluate the risk factors associated with 30 day mortality and relapse of infection in enterococcal BSI caused by VRE and to evaluate the impact of antibiotic regimens in targeted therapy. Methods We conducted a retrospective study of consecutive hospitalized patients in six teaching hospitals from August 2016 to August 2022 in Italy. All adult patients with a documented VRE BSI were included. Results We enrolled 517 consecutive hospitalized patients with VRE BSI; of these BSIs 496 (96.5%) were caused by Enterococcus faecium and 26 (5.1%) by Enterococcus faecalis. The most frequently used antibiotics as backbone were linezolid (48.1%) and daptomycin (43.7%). Overall, the 30 day mortality was 32.1%. Upon Cox regression analysis, the risk factor independently associated with 30 day mortality was Charlson comorbidity index >3 points (P < 0.001), whereas a Pitt score <4 points (P = 0.031), surgery for source control of infection (P = 0.016) and time to targeted therapy <24 h (P = 0.006) were associated with survival. After propensity score adjustment, a daptomycin-based regimen (P = 0.003) was associated with 30 day survival. Conclusions VRE BSI is an important cause of mortality in frail/critically ill patients. Our data highlighted the role of daptomycin as backbone agent for the treatment of enterococcal BSI caused by vancomycin-resistant strains.

Abstract: The spread of multidrug-resistant (MDR) bacteria and their resistance genes along the food chain and the environment has become a global threat aggravated by incorrect disinfection strategies. This study analysed the effect of induction by sub-inhibitory concentrations of eugenol - a major ingredient in clove essential oil commonly used in disinfectant agents - on the phenotypic and genotypic response of MDR Enterococcus faecalis E9.8 strain, selected based on the phenotypic response of other enterococci. Eugenol treatment irreversibly reduced several antibiotics' minimum inhibitory concentration (MIC), confirmed by kinetic studies for kanamycin, erythromycin, and tetracycline. Furthermore, transcriptomic analysis indicated the reversion of antibiotic resistance through direct and indirect measures, such as down-regulation of genes coding for proteins involved in antibiotic resistance, toxin resistance and virulence factors. Regarding antibiotic resistance genes (ARGs), ten differentially expressed genes (five down-regulated and five up-regulated genes) were related to the main transporter families, which present key targets in antibiotic resistance reversion. Our study thus highlights the importance of considering indirectly related genes as targets for antibiotic resistance reversion besides ARGs sensu stricto. These results allow us to propose using eugenol as an antibiotic resistance reversing agent to be included in disinfectant solutions as an excellent alternative to limit the spread of MDR bacteria and their ARGs in the food chain and the environment.

Abstract: ABSTRACT Difficult-to-treat (DTR) enterococcal infections, particularly those caused by multidrug-resistant Enterococcus faecium and Enterococcus faecalis , pose significant clinical challenges due to limited treatment options and high rates of treatment failure, compounded by a paucity of new antimicrobial agents in the development pipeline. Despite advances in understanding resistance mechanisms and in vitro synergistic antibiotic combinations, robust clinical data to guide therapy for severe or DTR enterococcal infections remain limited. This review synthesizes available evidence to inform optimal management strategies, including drug selection and dosing, while highlighting areas needing further research. Given the ongoing threat posed by multidrug-resistant enterococci, we emphasize the importance of gathering robust clinical data to guide best practices for managing these difficult-to-treat infections.

Abstract: Pathogenic bacteria can inhabit the chicken intestinal tract and potentially cause disease and mortality in chickens. Proteinogenic amino acids are major nutrients available in the intestine that have diverse growth-modulating effects on bacteria. However, limited knowledge exists about the direct association between amino acids and the growth of bacteria in the microbiome of the chicken's intestinal tract. The present study evaluated the growth-modulating effects of the 20 proteinogenic amino acids on chicken pathogens. Seven bacterial species were tested, including avian pathogenic Escherichia coli (APEC), Salmonella enterica., Staphylococcus aureus, Clostridium perfringens, Enterococcus cecorum, Enterococcus faecalis, and Enterococcus faecium. All bacteria were tested in minimal nutrient medium (MNM) in an in vitro model, with or without the addition of each amino acid. Bacterial growth was measured by absorbance (OD₆₀₀) and visualised as growth curves. l-cysteine significantly (p ≤ 0.05) inhibited the growth of APEC, extended the lag phase and increased the maximum density of C. perfringens, Salmonella enterica. and S. aureus. In contrast, l-glutamine and l-glutamic acid significantly shortened the lag phase and increased both the growth rate and maximum density of C. perfringens, Glycine and l-serine significantly (p ≤ 0.05) increased the maximum density of S. aureus. The effect of amino acids was insignificant (p ≥ 0.05) on the total growth of Enterococcus spp. These findings suggested that l-cysteine has inhibiting effects on APEC, C. perfringens, S. enterica and S. aureus. l-glutamine and l-glutamic acid promoted the growth of C. perfringens, while glycine and l-serine promoted the growth of S. aureus.

Abstract: Background: The emergence of linezolid resistance, mediated by genes such as optrA and cfr(D), poses a growing public health threat. While these genes have been detected in clinical and animal-derived Enterococcus species, their presence in underexplored species like Enterococcus saccharolyticus remains undocumented, leaving a significant gap in our understanding of their dissemination and stability. Method: E. saccharolyticus GXN23C125Es was screened for the presence of known linezolid resistance genes via PCR analysis. Conjugation and stability experiments were used to evaluate the transferability and stability of the resistance genes. The complete genome of GXN23C125Es was obtained using both the Illumina and Nanopore platforms. Results: We report the first identification of optrA and cfr(D) in GXN23C125Es from chicken feces in China. Whole-genome sequencing revealed multiple plasmid-borne resistance genes, including optrA, cfr(D), fexA, and erm(A). Stability testing demonstrated that optrA was highly stable, while cfr(D) was rapidly lost without selective pressure. Conclusions: These findings highlight E. saccharolyticus as a potential reservoir for linezolid resistance genes, underscoring the need for enhanced surveillance of resistance determinants in animal-associated bacteria. Understanding the dissemination dynamics of optrA and cfr(D) is crucial for mitigating their impact on public health and guiding antimicrobial resistance management strategies.

Abstract: The continuous growth of global population has promoted the development of new aquaculture techniques for enhancing food supply. Modern approaches, such as Recirculating Aquaculture Systems (RAS), present new challenges concerning the elimination of chemical substances and pathogens that appear because of their operation. In this study, the simultaneous removal of amoxicillin and water disinfection was evaluated using different UV-C photoassisted Advanced Oxidation Processes (AOPs). The processes involved oxidants such as hydrogen peroxide, peracetic acid, sodium hypochlorite, potassium peroxymonosulfate, and sodium persulfate. The optimization of radiation and oxidant dosage was assessed in a batch system photoreactor using simulated aquaculture water. Moreover, the disinfection potential of these treatments, as well of the production of disinfection by-products (DBPs), phytotoxicity generation and matrix interaction are also addressed. Finally, a first approximation to the reaction mechanism is carried out via scavenging studies. Results show that 10 W/m2 of UV-C and optimized dosages in the 0.1-3.2 mM range of oxidant eliminate at least 80 % of amoxicillin in 2 h of operation. Disinfection was achieved in 1 min of treatment and maintained for at least two weeks after storage. While DBPs and matrix parameters variation were minimal. Scavenging study shows that HO⦁, 1O2, and SO4⦁- radicals are the main ones involved in antibiotic degradation. The optimization of operation conditions sets the path towards scale-up of the process in continuous systems, with the objective of application of the process in real aquaculture water.

Abstract: Optimal management of Enterococcus faecium bloodstream infection (BSI) is not fully understood. Multicentre retrospective observational study of all consecutive adult (≥18 years old) patients with E. faecium BSI, between January 2016 and December 2023, at two tertiary teaching hospitals in northern Italy. Patients who died within 48 h from BSI onset were excluded. Primary and secondary endpoints were 30 day mortality and persistent E. faecium BSI, respectively. Cox regression and logistic binary analyses were used. Overall, 391 patients were enrolled: median age was 72 (IQR 61-81) years, 225 were male (57.5%), median Charlson comorbidity index (CCI) was 6 (IQR 4-8) and 94 had immunosuppression (24.0%). BSIs were primary, secondary and device-related in 25.1%, 36.2% and 38.7%, respectively. Vancomycin resistance was found in 30.3%. The appropriate empirical therapy rate was given for 29.1%. All-cause 30 day mortality was 34.3% and the rate of persistent BSI was 18.8%. Variables independently associated with 30 day mortality were immunosuppression (HR 1.638, 95% CI 1.022-2.625, P = 0.040), SOFA (HR 1.205, 95% CI 1.144-1.268, P < 0.001), primary BSI (HR 1.839, 95% CI 1.221-2.770, P = 0.004), source control (HR 0.534, 95% CI 0.260-0.972, P = 0.042) and the performance of follow-up blood cultures (HR 0.403, 95% CI 0.280-0.972, P < 0.001). Factors independently associated with persistent E. faecium BSI were: CCI (OR 1.157, 95% CI 1.030-1.300, P = 0.014), source control not performed (OR 3.275, 95% CI 1.113-9.635, P = 0.031) and teicoplanin (OR 2.023, 95% CI 1.018-4.018, P = 0.044). Among modifiable clinical factors in patients with E. faecium BSI, source control and the execution of follow-up blood cultures demonstrated a protective effect on 30 day mortality. Teicoplanin as targeted antibiotic treatment was independently associated with persistent BSI.