Early identification of the nosocomial spread of vancomycin-resistant Enterococcus faecium by Fourier-transform infrared spectroscopy and performance comparison with PFGE and WGS.

Early detection of disseminating vancomycin-resistant Enterococcus faecium (VREfm) in ICU wards is crucial for outbreak identification and the implementation of prompt infection control measures. Genotypic methods like pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS) are costly and time-consuming, hindering rapid response due to batch dependency. Fourier-transform infrared spectroscopy (FT-IR) offers the potential for real-time outbreak detection and reliable strain typing. We utilized FT-IR to identify clonal VREfm dissemination and compared its performance to PFGE and WGS. Between February through October 2023, an unusually high number of VREfm were recovered at a tertiary hospital in Barcelona. Isolates were examined for antimicrobial susceptibility, carriage of vanA/vanB genes and clonality was also studied using FT-IR, PFGE, and WGS. Routine FT-IR inspections revealed recurring VREfm clustering during the outbreak's initial weeks. In total, 104 isolates were recovered from 75 patients and from multiple wards. However, only one isolate was recovered from an environmental sample, suggesting the absence of environmental reservoirs. An ST80 vancomycin-resistant (vanA) E. faecium strain was the main strain responsible for the outbreak, although a few additional VREfm strains were also identified, all belonging to CC17. PFGE and cgMLST (WGS) yielded identical clustering results to FT-IR, and WGS confirmed vanA/vanB gene carriage in all VREfm isolates. Infection control measures led to a rapid decline in VREfm isolates, with no isolates detected in November. FT-IR spectroscopy offers rapid turnaround times, sensitivity, and reproducibility, comparable to standard typing methods. It proved as an effective tool for monitoring VREfm dissemination and early outbreak detection.

Machine Learning Assisted MALDI Mass Spectrometry for Rapid Antimicrobial Resistance Prediction in Clinicals.

Antimicrobial susceptibility testing (AST) plays a critical role in assessing the resistance of individual microbial isolates and determining appropriate antimicrobial therapeutics in a timely manner. However, conventional AST normally takes up to 72 h for obtaining the results. In healthcare facilities, the global distribution of vancomycin-resistant Enterococcus fecium (VRE) infections underscores the importance of rapidly determining VRE isolates. Here, we developed an integrated antimicrobial resistance (AMR) screening strategy by combining matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) with machine learning to rapidly predict VRE from clinical samples. Over 400 VRE and vancomycin-susceptible E. faecium (VSE) isolates were analyzed using MALDI-MS at different culture times, and a comprehensive dataset comprising 2388 mass spectra was generated. Algorithms including the support vector machine (SVM), SVM with L1-norm, logistic regression, and multilayer perceptron (MLP) were utilized to train the classification model. Validation on a panel of clinical samples (external patients) resulted in a prediction accuracy of 78.07%, 80.26%, 78.95%, and 80.54% for each algorithm, respectively, all with an AUROC above 0.80. Furthermore, a total of 33 mass regions were recognized as influential features and elucidated, contributing to the differences between VRE and VSE through the Shapley value and accuracy, while tandem mass spectrometry was employed to identify the specific peaks among them. Certain ribosomal proteins, such as A0A133N352 and R2Q455, were tentatively identified. Overall, the integration of machine learning with MALDI-MS has enabled the rapid determination of bacterial antibiotic resistance, greatly expediting the usage of appropriate antibiotics.

Antimicrobial Evaluation of Two Polycyclic Polyprenylated Acylphloroglucinol Compounds: PPAP23 and PPAP53.

Polycyclic polyprenylated acylphloroglucinols (PPAPs) comprise a large group of compounds of mostly plant origin. The best-known compound is hyperforin from St. John's wort with its antidepressant, antitumor and antimicrobial properties. The chemical synthesis of PPAP variants allows the generation of compounds with improved activity and compatibility. Here, we studied the antimicrobial activity of two synthetic PPAP-derivatives, the water-insoluble PPAP23 and the water-soluble sodium salt PPAP53. In vitro, both compounds exhibited good activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium. Both compounds had no adverse effects on Galleria mellonella wax moth larvae. However, they were unable to protect the larvae from infection with S. aureus because components of the larval coelom neutralized the antimicrobial activity; a similar effect was also seen with serum albumin. In silico docking studies with PPAP53 revealed that it binds to the F1 pocket of human serum albumin with a binding energy of -7.5 kcal/mol. In an infection model of septic arthritis, PPAP23 decreased the formation of abscesses and S. aureus load in kidneys; in a mouse skin abscess model, topical treatment with PPAP53 reduced S. aureus counts. Both PPAPs were active against anaerobic Gram-positive gut bacteria such as neurotransmitter-producing Clostridium, Enterococcus or Ruminococcus species. Based on these results, we foresee possible applications in the decolonization of pathogens.

Analysis of molecular epidemiological characteristics and antimicrobial susceptibility of vancomycin-resistant and linezolid-resistant Enterococcus in China.

BACKGROUND: This study investigates the distribution and characteristics of linezolid and vancomycin susceptibilities among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and explores the underlying resistance mechanisms. METHODS: A total of 2842 Enterococcus clinical isolates from patients were retrospectively collected, and their clinical data were further analyzed. The minimum inhibitory concentrations (MICs) of vancomycin and linezolid were validated by broth dilution method. The resistance genes optrA, cfr, vanA, vanB and vanM were investigated using polymerase chain reaction (PCR). Housekeeping genes and resistance genes were obtianed through whole-genome sequencing (WGS). RESULTS: Of the 2842 Enterococcus isolates, 88.5% (2516) originated from urine, with E. faecium accounted for 60.1% of these. The vanA gene was identified in 27/28 vancomycin resistant Enterococcus (VRE) isolates, 4 of which carried both vanA and vanM genes. The remaining strain was vanM positive. The optrA gene was identified in all E. faecalis isolates among linezolid resistant Enterococcus (LRE). E. faecium showed a higher multiple antibiotic resistance index (MAR index) compared to E. faecalis. The multi-locus sequence typing (MLST) showed the sequence type of E. faecium mainly belongs to clonal complex (CC) 17, nearly E. faecalis isolates analyzed were differentiated into 7 characteristics of sequence types (STs), among which ST16 of CC16 were the major lineage. CONCLUSION: Urine was the primary source of VRE and LRE isolates in this study. E. faecium showed higher levels of resistance compared to E. faecalis. OptrA gene was detected in 91.6% of LRE, which could explain linezolid resistance, and van genes were detected in all vancomycin resistant Enterococcus strains, while vanA was a key resistance mechanism in VRE identified in this study.

A secondary abdominal aorta-duodenal fistula accompanied with acquired Immune Deficiency Syndrome presented with recurrent sepsis: a case report.

BACKGROUND: Abdominal aorta-duodenal fistulas are rare abnormal communications between the abdominal aorta and duodenum. Secondary abdominal aorta-duodenal fistulas often result from endovascular surgery for aneurysms and can present as severe late complications. CASE PRESENTATION: A 50-year-old male patient underwent endovascular reconstruction for an infrarenal abdominal aortic pseudoaneurysm. Prior to the operation, he was diagnosed with Acquired Immune Deficiency Syndrome and Syphilis. Two years later, he was readmitted with lower extremity pain and fever. Blood cultures grew Enterococcus faecium, Salmonella, and Streptococcus anginosus. Sepsis was successfully treated with comprehensive anti-infective therapy. He was readmitted 6 months later, with blood cultures growing Enterococcus faecium and Escherichia coli. Although computed tomography did not show contrast agent leakage, we suspected an abdominal aorta-duodenal fistula. Esophagogastroduodenoscopy confirmed this suspicion. The patient underwent in situ abdominal aortic repair and received long-term antibiotic therapy. He remained symptom-free during a year and a half of follow-up. CONCLUSIONS: This case suggests that recurrent infections with non-typhoidal Salmonella and gut bacteria may be an initial clue to secondary abdominal aorta-duodenal fistula.

Genome analysis of multidrug resistant Enterococcus faecium and Enterococcus faecalis circulating among hospitalized patients in uMgungundlovu District, KwaZulu-Natal, South Africa.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are important pathogens categorized as high-priority bacteria in the Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics published by the World Health Organization. The aim of this study was to determine the risk factors, resistance, virulence, mobilomes associated with multidrug-resistant and clonal lineages of Enterococcus faecium and faecalis circulating among hospitalized patients following the health system in South Africa, using whole genome sequencing (WGS). METHODS: A cross-sectional study was conducted during a two-month periods among hospitalized patients in 2017. Rectal swabs were collected from patients admitted to medical and surgical wards in an urban tertiary hospital, and a rural district hospital in uMgungundlovu district, South Africa. Enterococci were screened for vancomycin resistance on bile esculin azide agar supplemented with 6 mg/L of vancomycin and confirmation of VRE was done using ROSCO kits. Conventional and real-time PCR methods were used to ascertain the presence of VanA, VanB, VanC-2/3 and VanC-1 genes. All six multidrug-resistant Enterococcus faecalis and faecium selected were identified using multiplexed paired-end libraries (2 × 300 bp) with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and genome sequencing was done using Illumina MiSeq instrument with 100× coverage at the National Institute of Communicable Diseases Sequencing Core Facility, South Africa. Antibiotic resistance genes, virulence factors, plasmids, integrons and CRISPR were characterized using RAST, ResFinder, VirulenceFinder, PlasmidFinder, PHAST and ISFinder respectively. RESULTS: Sequencing analysis revealed that these strains harbouring numerous resistance genes to glycopeptides (vanC[100%], vex3[100%], vex2[83,33%] and vanG[16,66%]), macrolides, lincosamides, sterptogramine B (ermB[33,32%], Isa[16,66%], emeA[16,66%]) and tetracyclines (tetM[33,32%]) in both district and tertiary hospitals. Multidrug efflux pumps including MATE, MFS and pmrA conferring resistance to several classes of antibiotics were also identified. The main transposable elements observed were in the Tn3 family, specifically Tn1546. Four single sequence types (STs) were identified among E. faecium in the district hospital, namely ST822, ST636, ST97 along with a novel ST assigned ST1386, while one lineage, ST29 was detected in the tertiary hospital. CONCLUSION: The study reveals the genetic diversity and high pathogenicity of multidrug-resistant Enterococcus faecalis and faecium circulating among hospitalized patients. It underlines the necessity to implement routine screening of admitted patients coupled with infection control procedures, antimicrobial stewardship and awareness should be strengthened to prevent and/or contain the carriage and spread of multidrug resistant E. faecium and E. faecalis in hospitals and communities in South Africa.

Unraveling host-pathogen dynamics in a murine Model of septic peritonitis induced by vancomycin-resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (E. faecium) infection is associated with higher mortality rates. Previous studies have emphasized the importance of innate immune cells and signalling pathways in clearing E. faecium, but a comprehensive analysis of host-pathogen interactions is lacking. Here, we investigated the interplay of host and E. faecium in a murine model of septic peritonitis. Following injection with a sublethal dose, we observed significantly increased murine sepsis score and histological score, decreased weight and bacterial burden, neutrophils and macrophages infiltration, and comprehensive activation of cytokine-mediated signalling pathway. In mice receiving a lethal dose, hypothermia significantly improved survival, reduced bacterial burden, cytokines, and CD86 expression of MHC-II+ recruited macrophages compared to the normothermia group. A mathematical model constructed by observational data from 80 animals, recapitulated the host-pathogen interplay, and further verified the benefits of hypothermia. These findings indicate that E. faecium triggers a severe activation of cytokine-mediated signalling pathway, and hypothermia can improve outcomes by reducing bacterial burden and inflammation.

Antimicrobial resistance among indicator Enterococcus faecium and Escherichia coli in Swedish pig farms.

Monitoring the use of antimicrobials and the emergence of resistance in animals and people is important for the control of antimicrobial resistance, and for establishing sustainable and effective disease management practices. In this study, we used Enterococcus spp. and Escherichia coli as indicator species to investigate antimicrobial susceptibility patterns and how these change over time, on ten Swedish pig farms. Indoor environmental sock sampling was performed once a month during the entire production cycle of one batch of pigs on each farm, resulting in 60 samples collected in total. Selective culture for E. coli and Enterococcus spp. resulted in 122 isolates of E. coli, 74 isolates of E. faecium, but no isolates of E. faecalis. Microdilution was used to determine minimum inhibitory concentrations for twelve antimicrobial substances in E. coli and fifteen substances in E. faecium. The overall prevalence of resistance was low. Among the E. coli isolates, the proportions non-wild type (resistant, NWT) isolates were as follows: azithromycin and amikacin 1% (n = 1), trimethoprim and sulfamethoxazole 2% (n = 3), ampicillin 6% (n = 7) and tetracycline 9% (n = 11). Among the E. faecium isolates, the NWT proportions were: teicoplanin, linezolid and gentamicin 1% (n = 1), daptomycin 3% (n = 2), erythromycin 26% (n = 19), tetracycline 27% (n = 20), quinupristin/dalfopristin 58% (n = 42). The resistance patterns differed between the farms, likely due to different antimicrobial use, biosecurity measures and source of the animals. The NWT prevalence among E. coli decreased over time, whereas no similar trend could be observed in E. faecium. The results of the current study illustrate the complex factors affecting the antimicrobial resistance patterns observed on each farm, indicating that specific practices and risk factors have an impact on the prevalence and type of antimicrobial resistance. Further studies of the farm environments in combination with antimicrobial use and other risk factor data are needed to elucidate the multifaceted drivers of antimicrobial resistance development on livestock farms.

The mechanism of Enterococcus faecium on the virulence of Listeria monocytogenes during the storage of fermented sausages by whole genome analysis.

This study investigated the safety characteristics and potential probiotic properties of Enterococcus faecium by using whole genome analysis, and then explored the effect of this strain on the virulence of Listeria monocytogenes in vitro and during the storage of fermented sausages. Results showed that E. faecium B1 presented enterocin A, B, and P, enterolysin A, and UviB, and the exotoxin related genes and exoenzyme related genes were not detected in the genome of E. faecium B1. However, the adherence genes including acm and scm were present in this strain, which also positively correlated with characteristics related to probiotic potential. In addition, E. faecium could adapt to the condition of fermented sausages, and decrease the survival of L. monocytogenes in vitro and in vivo. The expression of the virulence genes (prfA, hly, inlA, and inlB) and sigB-related genes (prli42, rsbT, rsbU, rsbV, rsbW, and sigB) were all inhibited by E. faecium B1 to different extents during the storage of fermented sausages at 4 °C. Moreover, compared with the E. faecium B1 group, the expression level of entA, entB, and entP genes of E. faecium B1 in the co-culture of fermented sausages was increased during the storage, which may be the inhibition mechanism of E. faecium B1 on L. monocytogenes. These results demonstrated that E. faecium B1 could potentially be used as bio-protection to control L. monocytogenes in meat products.

Genomic analysis of enterococci carrying optrA, poxtA, and vanA resistance genes from wild boars, Italy.

AIMS: To investigate enterococci carrying linezolid and vancomycin resistance genes from fecal samples recovered from wild boars. METHODS AND RESULTS: Florfenicol- and vancomycin-resistant enterococci, isolated on selective agar plates, were screened by PCR for the presence of linezolid and vancomycin resistance genes. Five isolates carried optrA or poxtA linezolid resistance genes; one strain was resistant to vancomycin for the presence of vanA gene. All isolates were tested for their antibiotic susceptibility and subjected to Whole Genome Sequencing (WGS) analysis. In Enterococcus faecalis (E. faecalis) V1344 and V1676, the optrA was located on the new pV1344-optrA and pV1676-optrA plasmids, respectively, whereas in Enterococcus faecium (E. faecium) V1339 this gene was on a 22 354-bp chromosomal genetic context identical to the one detected in a human E. faecium isolate. In both E. faecium V1682 and E. durans V1343, poxtA was on the p1818-c plasmid previously found in a human E. faecium isolate. In E. faecium V1328, the vanA gene was on the Tn1546 transposon in turn located on a new pV1328-vanA plasmid. Only E. faecium V1682 successfully transferred the poxtA gene to an enterococcal recipient in filter mating assays. CONCLUSIONS: The occurrence of genetic elements carrying linezolid and vancomycin resistance genes in enterococci from wild boars is a matter of concern, moreover, the sharing of plasmids and transposons between isolates from wild animals, human, and environment indicates an exchange of genetic material between these settings.

A bacteriocin expression platform for targeting pathogenic bacterial species.

Bacteriocins are antimicrobial peptides that are naturally produced by many bacteria. They hold great potential in the fight against antibiotic resistant bacteria, including ESKAPE pathogens. Engineered live biotherapeutic products (eLBPs) that secrete bacteriocins can be created to deliver targeted bacteriocin production. Here we develop a modular bacteriocin secretion platform that can be used to express and secrete multiple bacteriocins from non-pathogenic Escherichia coli host strains. As a proof of concept we create Enterocin A (EntA) and Enterocin B (EntB) secreting strains that show strong antimicrobial activity against Enterococcus faecalis and Enterococcus faecium in vitro, and characterise this activity in both solid culture and liquid co-culture. We then develop a Lotka-Volterra model that can be used to capture the interactions of these competitor strains. We show that simultaneous exposure to EntA and EntB can delay Enterococcus growth. Our system has the potential to be used as an eLBP to secrete additional bacteriocins for the targeted killing of pathogenic bacteria.

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.

Metal Nanoparticle-Based Biosensors for the Early Diagnosis of Infectious Diseases Caused by ESKAPE Pathogens in the Fight against the Antimicrobial-Resistance Crisis.

The species included in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and the genus Enterobacter) have a high capacity to develop antimicrobial resistance (AMR), a health problem that is already among the leading causes of death and could kill 10 million people a year by 2050. The generation of new potentially therapeutic molecules has been insufficient to combat the AMR "crisis", and the World Health Organization (WHO) has stated that it will seek to promote the development of rapid diagnostic strategies. The physicochemical properties of metallic nanoparticles (MNPs) have made it possible to design biosensors capable of identifying low concentrations of ESKAPE bacteria in the short term; other systems identify antimicrobial susceptibility, and some have been designed with dual activity in situ (bacterial detection and antimicrobial activity), which suggests that, in the near future, multifunctional biosensors could exist based on MNPs capable of quickly identifying bacterial pathogens in clinical niches might become commercially available. This review focuses on the use of MNP-based systems for the rapid and accurate identification of clinically important bacterial pathogens, exhibiting the necessity for exhaustive research to achieve these objectives. This review focuses on the use of metal nanoparticle-based systems for the rapid and accurate identification of clinically important bacterial pathogens.

A diverse set of Enterococcus-infecting phage provides insight into phage host-range determinants.

Enterococci are robust Gram-positive bacteria that pose a significant threat in healthcare settings due to antibiotic resistance, with vancomycin-resistant enterococci (VRE) most prominent. To tackle this issue, bacteriophages (bacterial viruses) can be exploited as they specifically and efficiently target bacteria. Here, we successfully isolated and characterised a set of novel phages: SHEF10, SHEF11, SHEF13, SHEF14, and SHEF16 which target E. faecalis (SHEF10,11,13), or E. faecium (SHEF13, SHEF14 & SHEF16) strains including a range of clinical and VRE isolates. Genomic analysis shows that all phages are strictly lytic and diverse in terms of genome size and content, quickly and effectively lysing strains at different multiplicity of infections. Detailed analysis of the broad host-range SHEF13 phage revealed the crucial role of the enterococcal polysaccharide antigen (EPA) variable region in its infection of E. faecalis V583. In parallel, the discovery of a carbohydrate-targeting domain (CBM22) found conserved within the three phage genomes indicates a role in cell surface interactions that may be important in phage-bacterial interactons. These findings advance our comprehension of phage-host interactions and pave the way for targeted therapeutic strategies against antibiotic-resistant enterococcal infections.

Fitness costs of Tn1546-type transposons harboring the vanA operon by plasmid type and structural diversity in Enterococcus faecium.

BACKGROUND: This study analyzed the genetic traits and fitness costs of vancomycin-resistant Enterococcus faecium (VREfm) blood isolates carrying Tn1546-type transposons harboring the vanA operon. METHODS: All E. faecium blood isolates were collected from eight general hospitals in South Korea during one-year study period. Antimicrobial susceptibility testing and vanA and vanB PCR were performed. Growth rates of E. faecium isolates were determined. The vanA-positive isolates were subjected to whole genome sequencing and conjugation experiments. RESULTS: Among 308 E. faecium isolates, 132 (42.9%) were positive for vanA. All Tn1546-type transposons harboring the vanA operon located on the plasmids, but on the chromosome in seven isolates. The plasmids harboring the vanA operon were grouped into four types; two types of circular, nonconjugative plasmids (Type A, n = 50; Type B, n = 46), and two types of putative linear, conjugative plasmids (Type C, n = 16; Type D, n = 5). Growth rates of vanA-positive E. faecium isolates were significantly lower than those of vanA-negative isolates (P < 0.001), and reduction in growth rate under vancomycin pressure was significantly larger in isolates harboring putative linear plasmids than in those harboring circular plasmids (P = 0.020). CONCLUSIONS: The possession of vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination. Fitness burden to bacterial hosts was varied by type and size of the vanA operon-harboring plasmid.

Diet-induced changes in the jejunal microbiota of developing broilers reduce the abundance of Enterococcus hirae and Enterococcus faecium.

Modern broiler breeds allow for high feed efficiency and rapid growth, which come at a cost of increased susceptibility to pathogens and disease. Broiler growth rate, feed efficiency, and health are affected by the composition of the gut microbiota, which in turn is influenced by diet. In this study, we therefore assessed how diet composition can affect the broiler jejunal gut microbiota. A total of 96 broiler chickens were divided into four diet groups: control, coated butyrate supplementation, medium-chain fatty acid supplementation, or a high-fibre low-protein content. Diet groups were sub-divided into age groups (4, 12 and 33 days of age) resulting in groups of 8 broilers per diet per age. The jejunum content was used for metagenomic shotgun sequencing to determine the microbiota taxonomic composition at species level. The composed diets resulted in a total of 104 differentially abundant bacterial species. Most notably were the butyrate-induced changes in the jejunal microbiota of broilers 4 days post-hatch, resulting in the reduced relative abundance of mainly Enterococcus faecium (-1.8 l2fc, Padj = 9.9E-05) and the opportunistic pathogen Enterococcus hirae (-2.9 l2fc, Padj = 2.7E-08), when compared to the control diet. This effect takes place during early broiler development, which is critical for broiler health, thus exemplifying the importance of how diet can influence the microbiota composition in relation to broiler health. Future studies should therefore elucidate how diet can be used to promote a beneficial microbiota in the early stages of broiler development.

Navigating ESKAPE Pathogens: Considerations and Caveats for Animal Infection Models Development.

The misuse of antibiotics has led to the global spread of drug-resistant bacteria, especially multi-drug-resistant (MDR) ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). These opportunistic bacteria pose a significant threat, in particular within hospitals, where they cause nosocomial infections, leading to substantial morbidity and mortality. To comprehensively explore ESKAPE pathogenesis, virulence, host immune response, diagnostics, and therapeutics, researchers increasingly rely on necessitate suitable animal infection models. However, no single model can fully replicate all aspects of infectious diseases. Notably when studying opportunistic pathogens in immunocompetent hosts, rapid clearance by the host immune system can limit the expression of characteristic disease symptoms. In this study, we examine the critical role of animal infection models in understanding ESKAPE pathogens, addressing limitations and research gaps. We discuss applications and highlight key considerations for effective models. Thoughtful decisions on disease replication, parameter monitoring, and data collection are crucial for model reliability. By meticulously replicating human diseases and addressing limitations, researchers maximize the potential of animal infection models. This aids in targeted therapeutic development, bridges knowledge gaps, and helps combat MDR ESKAPE pathogens, safeguarding public health.

D-Glucose-Mediated Gold Nanoparticle Fabrication for Colorimetric Detection of Foodborne Pathogens.

Gold nanoparticle (AuNP) fabrication via the oxidation of D-glucose is applied for detecting two foodborne pathogens, Enterococcus faecium (E. faecium) and Staphylococcus aureus (S. aureus). D-glucose is used as a reducing agent due to its oxidation to gluconic acid by sodium hydroxide (NaOH), resulting in the formation of AuNPs. Based on this mechanism, we develop AuNP-based colorimetric detection in conjunction with loop-mediated isothermal amplification (LAMP) for accurately identifying the infectious bacteria. Here, Au+ ions bind to the base of double-stranded DNA. In the presence of D-glucose and NaOH, the LAMP amplicon-Au+ complex maintains its bound state at 65 °C for 10 min while it is reduced to AuNPs in a dispersed form, exhibiting a red color. We aimed to pre-mix D-glucose with LAMP reagents before amplification and induce successful colorimetry without inhibiting amplification to simplify the experimental process and decrease the reaction time. Therefore, the entire process, including LAMP and colorimetric detection, is accomplished in approximately 1 h. The limit of detection of E. faecium and S. aureus is confirmed using the introduced method as 101 CFU/mL and 100 fg/µL, respectively. We expect that colorimetric detection using D-glucose-mediated AuNP synthesis offers an application for simple and immediate molecular diagnosis.

Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy.

Enterococcus faecium is a microbiota species in humans that can modulate host immunity (Griffin and Hang, 2022), but has also acquired antibiotic resistance and is a major cause of hospital-associated infections (Van Tyne and Gilmore, 2014). Notably, diverse strains of E. faecium produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity (Rangan et al., 2016; Pedicord et al., 2016; Kim et al., 2019) and immune checkpoint inhibitor antitumor activity (Griffin et al., 2021). However, the functions of SagA in E. faecium were unknown. Here, we report that deletion of sagA impaired E. faecium growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, ΔsagA showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, the plasmid-based expression of SagA, but not its catalytically inactive mutant, restored ΔsagA growth, production of active muropeptides, and NOD2 activation. SagA is, therefore, essential for E. faecium growth, stress resistance, and activation of host immunity.

Comparative genomic and metabolomic analysis reveals the potential of a newly isolated Enterococcus faecium B6 involved in lipogenic effects.

Evidence has indicated that Enterococcus plays a vital role in non-alcoholic fatty liver disease (NAFLD) development. However, the microbial genetic basis and metabolic potential in the disease are yet unknown. We previously isolated a bacteria Enterococcus faecium B6 (E. faecium B6) from children with NAFLD for the first time. Here, we aim to systematically investigate the potential of strain B6 in lipogenic effects. The lipogenic effects of strain B6 were explored in vitro and in vivo. The genomic and functional characterizations were investigated by whole-genome sequencing and comparative genomic analysis. Moreover, the metabolite profiles were unraveled by an untargeted metabolomic analysis. We demonstrated that strain B6 could effectively induce lipogenic effects in the liver of mice. Strain B6 contained a circular chromosome and two circular plasmids and posed various functions. Compared to the other two probiotic strains of E. faecium, strain B6 exhibited unique functions in pathways of ABC transporters, phosphotransferase system, and amino sugar and nucleotide sugar metabolism. Moreover, strain B6 produced several metabolites, mainly enriched in the protein digestion and absorption pathway. The unique potential of strain B6 in lipogenic effects was probably associated with glycolysis, fatty acid synthesis, and glutamine and choline transport. This study pioneeringly revealed the metabolic characteristics and specific detrimental traits of strain B6. The findings provided new insights into the underlying mechanisms of E. faecium in lipogenic effects, and laid essential foundations for further understanding of E. faecium-related disease.