Integration of MALDI-TOF MS and machine learning to classify enterococci: A comparative analysis of supervised learning algorithms for species prediction.

This research focused on distinguishing distinct matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral signatures of three Enterococcus species. We evaluated and compared the predictive performance of four supervised machine learning algorithms, K-nearest neighbor (KNN), support vector machine (SVM), and random forest (RF), to accurately classify Enterococcus species. This study involved a comprehensive dataset of 410 strains, generating 1640 individual spectra through on-plate and off-plate protein extraction methods. Although the commercial database correctly identified 76.9% of the strains, machine learning classifiers demonstrated superior performance (accuracy 0.991). In the RF model, top informative peaks played a significant role in the classification. Whole-genome sequencing showed that the most informative peaks are biomarkers connected to proteins, which are essential for understanding bacterial classification and evolution. The integration of MALDI-TOF MS and machine learning provides a rapid and accurate method for identifying Enterococcus species, improving healthcare and food safety.

Role of Enterococcus mundtii in gut of the tomato leaf miner (Tuta absoluta) to detoxification of Chlorantraniliprole.

Chlorantraniliprole (CAP) is applied worldwide for the control of caterpillars (Lepidoptera). However, with the overuse of CAP, the resistance problem in pest control is becoming increasingly serious. Recent studies have indicated a central role of the gut symbiont in insect pest resistance to pesticides and these may apply to the tomato leaf miner Tuta absoluta, is one of the most destructive insects worldwide. Here, we successfully isolated seven strains of tolerant CAP bacterium from the CAP-resistant T. absoluta gut, of which Enterococcus mundtii E14 showed the highest CAP tolerance, with a minimum inhibitory concentration (MIC) of 1.6 g/L and CAP degradation rate of 42.4%. Through transcriptomics and metabolism analysis, we studied the detoxification process of CAP by the E. mundtii E14, and found that CAP can be degraded by E. mundtii E14 into non-toxic compounds, such as 3,4-dihydroxy-2-(5-hydroxy-3,7-dimethylocta-2,6-dien-1-yl) benzoic acid and 2-pyridylacetic acid. Additionally, 2-pyridylacetic acid was detected both intracellular and extracellular in E. mundtii E14 treated with CAP. Meanwhile, we identified 52 up-regulated genes, including those associated with CAP degradation, such as RS11670 and RS19130. Transcriptome results annotated using KEGG indicated significant enrichment in up-regulated genes related to the glyoxylate cycle, nitrogen metabolism, and biosynthesis of secondary metabolites. Additionally, we observed that reinfection with E. mundtii E14 may effectively enhance resistance of T. absoluta to CAP. The LC50 values of the antibiotic treatment population of T. absoluta reinfection with E. mundtii E14 is 0.6122 mg/L, which was 18.27 folds higher than before reinfection. These findings offer new insights into T. absoluta resistance to CAP and contribute to a better understanding of the relationship between insecticide resistance and gut symbionts of T. absoluta, which may play a pivotal role in pest management.

Enterococci carbonic anhydrase inhibition.

Carbonic anhydrase metalloenzymes are encoded in genomes throughout all kingdoms of life with a conserved function catalyzing the reversible conversion of CO2 to bicarbonate. Carbonic anhydrases have been well-investigated in humans, but are still relatively understudied in bacterial organisms, including Enterococci. Studies over the past decade have presented bacterial carbonic anhydrases as potential drug targets, with some chemical scaffolds potently inhibiting the Enterococcus carbonic anhydrases in vitro and displaying antimicrobial efficacy against Enterococcus organisms. While carbonic anhydrases in Enterococci still have much to be explored, hypotheses may be drawn from similar Gram-positive organisms for which known information exists about carbonic anhydrase function and relevance. Within this chapter is reported information and rational hypotheses regarding the subcellar locations, potential physiological roles, essentiality, structures, and kinetics of carbonic anhydrases in Enterococci.

Biofilm-specific determinants of enterococci pathogen.

Amongst all Enterococcus spp., E. faecalis and E. faecium are most known notorious pathogen and their biofilm formation has been associated with endocarditis, oral, urinary tract, and wound infections. Biofilm formation involves a pattern of initial adhesion, microcolony formation, and mature biofilms. The initial adhesion and microcolony formation involve numerous surface adhesins e.g. pili Ebp and polysaccharide Epa. The mature biofilms are maintained by eDNA, It's worth noting that phage-mediated dispersal plays a prominent role. Further, the involvement of peptide pheromones in regulating biofilm maintenance sets it apart from other pathogens and facilitating the horizontal transfer of resistance genes. The role of fsr based regulation by regulating gelE expression is also discussed. Thus, we provide a concise overview of the significant determinants at each stage of Enterococcus spp. biofilm formation. These elements could serve as promising targets for antibiofilm strategies.

A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci.

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.

Metal tolerance in enterococci isolated from seabirds in Abrolhos Archipelago, Brazil: Evaluating their role as bioindicators of marine pollution.

Microbiota exposed to pollution provide insights into host physiology and ecosystem disruption. This study evaluated Enterococcus spp. tolerant to arsenic (As), copper (Cu), and mercury (Hg) from red-billed tropicbirds (Phaethon aethereus) and brown boobies (Sula leucogaster), which previously showed these metals in their blood and feathers, and their potential use as bioindicators of metal contamination. Enterococcus casseliflavus (47.9 %), E. faecalis (34.1 %), E. hirae (11.7 %), and E. faecium (5.3 %) were identified. Both seabird species had a high incidence of As-tolerant bacteria (84.0 %), with 40.4 % of these strains containing As efflux system genes (arsA_I and arsA_II). Cu efflux pump gene (tcrB) was detected in 30.9 % of strains, while Hg reductase genes (mer) were not found. As- and Cu-tolerance in enterococci observed in this study underlines their potential as bioindicators in metal-polluted marine environments. Further research may elucidate the role of these metal-tolerant enterococci in seabird gut and their adaptability to polluted environments.

Alternative for HPC22 after repairs in the drinking water distribution system.

There is a risk of contamination by (pathogenic) microorganisms from the outside environment into the drinking water during maintenance or pipe breaches in the drinking water distribution system (DWDS) and, consequently, the drinking water distributed to consumers may result in possible detrimental effects on public health. Traditional time-consuming microbiological testing is, therefore, performed to confirm drinking water is not microbially contaminated. This is done by culturing methods of the faecal indicators Escherichia coli, intestinal enterococci and the technical parameters coliform bacteria and heterotrophic plate counts at 22 °C (HPC22). In this study, fast methods (adenosine triphosphate (ATP), flow cytometry, enzyme activity and qPCR) were compared as an alternative for HPC22. Using dilution series and field samples, ATP (ATPtotal-lab and ATPcell-mob) and enzymatic activity (ALP-2) methods proved to be the more reliable and sensitive than flow cytometry and qPCR methods for detecting microbiological contaminations in drinking water. Significant (p < 0.05) and relatively strong correlations (R2 = 0.61-0.76) were obtained between HPC22 and both ATP methods, enzyme activity and qPCR parameters, but relations with flow cytometry were weak (R2 = 0.24 - 0.52). The samples taken after repairs or a calamity from the DWDS showed in general limited variation in the HPC22 count and were in most cases below the guidance level of 1,000 CFU/mL. We recommend that the best performing alternative methods, i.e. ATPtotal-lab and ATPcell-mob and ALP-2, should be included next to HPC22 in additional field studies to further test and compare these methods to be able to decide which fast method can replace HPC22 analysis after maintenance work in the DWDS.

A case report of empyema caused by Enterococcus gallinarum.

BACKGROUND: Enterococcus gallinarum is an infrequently intestinal symbiotic pathogen associated with nosocomial infection in immunocompromised individuals. To date, rare cases of pulmonary infection attributable to Enterococcus gallinarum were reported. Herein, we presented the first case of empyema resulting from Enterococcus gallinarum infection. CASE PRESENTATION: An 81-year-old male presented with fever and dyspnea upon admission. Chest CT scan and thoracic ultrasonography confirmed the presence of right pleural effusion. Thoracoscopy revealed extensive adhesion, purulent fluid, and necrotic materials within the thoracic cavity. Enterococcus gallinarum was identified through pleural effusion culture. The patient underwent an intrathoracic injection of urokinase along with thoracic drainage. Following surgery, He took oral linezolid for over one month. Undergoing comprehensive treatment, the patient exhibited favorable recovery. CONCLUSIONS: We reported the first case of empyema due to Enterococcus gallinarum infection. It should be suspected in patients with impaired immune function and invasive therapies, without responding to conventional anti-infectious treatment.

Genomic diversity, antibiotic resistance, and virulence in South African Enterococcus faecalis and Enterococcus lactis isolates.

This study presents the empirical findings of an in-depth genomic analysis of Enterococcus faecalis and Enterococcus lactis isolates from South Africa. It offers valuable insights into their genetic characteristics and their significant implications for public health. The study uncovers nuanced variations in the gene content of these isolates, despite their similar GC contents, providing a comprehensive view of the evolutionary diversity within the species. Genomic islands are identified, particularly in E. faecalis, emphasizing its propensity for horizontal gene transfer and genetic diversity, especially in terms of antibiotic resistance genes. Pangenome analysis reveals the existence of a core genome, accounting for a modest proportion of the total genes, with 2157 core genes, 1164 shell genes, and 4638 cloud genes out of 7959 genes in 52 South African E. faecalis genomes (2 from this study, 49 south Africa genomes downloaded from NCBI, and E. faecalis reference genome). Detecting large-scale genomic rearrangements, including chromosomal inversions, underscores the dynamic nature of bacterial genomes and their role in generating genetic diversity. The study uncovers an array of antibiotic resistance genes, with trimethoprim, tetracycline, glycopeptide, and multidrug resistance genes prevalent, raising concerns about the effectiveness of antibiotic treatment. Virulence gene profiling unveils a diverse repertoire of factors contributing to pathogenicity, encompassing adhesion, biofilm formation, stress resistance, and tissue damage. These empirical findings provide indispensable insights into these bacteria's genomic dynamics, antibiotic resistance mechanisms, and virulence potential, underlining the pressing need to address antibiotic resistance and implement robust control measures.

Uninheritable but Widespread Bacterial Symbiont Enterococcus casseliflavus Mediates Detoxification of the Insecticide Chlorantraniliprole in the Agricultural Invasive Pest Spodoptera frugiperda.

Host-symbiont interaction plays a crucial role in determining the host's fitness under toxic stress, as observed in numerous insect species. However, the mechanism of the symbionts involved in the detoxification of insecticides remains poorly known. In this study, through microbiome, proteomic, and genomic analysis, we identified a prevalent symbiont, Enterococcus casseliflavus EMBL-3, in a major invasive insect pest,Spodoptera frugiperda. This symbiont enhances the host's insecticide resistance to chlorantraniliprole by breaking amide bonds and dehalogenating insecticides. Complying with the increase in exposure risk of chlorantraniliprole, the E. casseliflavus isolates of insects' symbionts but not those from mammals or environmental strains showed a significant enrichment of potential chlorantraniliprole degradation genes. EMBL-3 is popular in field population insects with efficient horizontal transmission ability through cross-diet and cannibalism. This study provides a new therapeutic target for agricultural pests based on symbiont-targeted insect control for global crop protection.

Community use of oral antibiotics transiently reprofiles the intestinal microbiome in young Bangladeshi children.

Antibiotics may alter the gut microbiome, and this is one of the mechanisms by which antimicrobial resistance may be promoted. Suboptimal antimicrobial stewardship in Asia has been linked to antimicrobial resistance. We aim to examine the relationship between oral antibiotic use and composition and antimicrobial resistance in the gut microbiome in 1093 Bangladeshi infants. We leverage a trial of 8-month-old infants in rural Bangladesh: 61% of children were cumulatively exposed to antibiotics (most commonly cephalosporins and macrolides) over the 12-month study period, including 47% in the first 3 months of the study, usually for fever or respiratory infection. 16S rRNA amplicon sequencing in 11-month-old infants reveals that alpha diversity of the intestinal microbiome is reduced in children who received antibiotics within the previous 7 days; these samples also exhibit enrichment for Enterococcus and Escherichia/Shigella genera. No effect is seen in children who received antibiotics earlier. Using shotgun metagenomics, overall abundance of antimicrobial resistance genes declines over time. Enrichment for an Enterococcus-related antimicrobial resistance gene is observed in children receiving antibiotics within the previous 7 days, but not earlier. Presence of antimicrobial resistance genes is correlated to microbiome composition. In Bangladeshi children, community use of antibiotics transiently reprofiles the gut microbiome.

Proteolytic bacteria expansion during colitis amplifies inflammation through cleavage of the external domain of PAR2.

Imbalances in proteolytic activity have been linked to the development of inflammatory bowel diseases (IBD) and experimental colitis. Proteases in the intestine play important roles in maintaining homeostasis, but exposure of mucosal tissues to excess proteolytic activity can promote pathology through protease-activated receptors (PARs). Previous research implicates microbial proteases in IBD, but the underlying pathways and specific interactions between microbes and PARs remain unclear. In this study, we investigated the role of microbial proteolytic activation of the external domain of PAR2 in intestinal injury using mice expressing PAR2 with a mutated N-terminal external domain that is resistant to canonical activation by proteolytic cleavage. Our findings demonstrate the key role of proteolytic cleavage of the PAR2 external domain in promoting intestinal permeability and inflammation during colitis. In wild-type mice expressing protease-sensitive PAR2, excessive inflammation leads to the expansion of bacterial taxa that cleave the external domain of PAR2, exacerbating colitis severity. In contrast, mice expressing mutated protease-resistant PAR2 exhibit attenuated colitis severity and do not experience the same proteolytic bacterial expansion. Colonization of wild-type mice with proteolytic PAR2-activating Enterococcus and Staphylococcus worsens colitis severity. Our study identifies a previously unknown interaction between proteolytic bacterial communities, which are shaped by inflammation, and the external domain of PAR2 in colitis. The findings should encourage new therapeutic developments for IBD by targeting excessive PAR2 cleavage by bacterial proteases.

Microbial contamination of spittoons and germicidal effect of irradiation with krypton chloride excimer lamps (Far UV-C 222 nm).

BACKGROUND: In dentistry, instruments, appliances, and body fluids such as saliva or blood are possible sources of infection. Although conventional antiseptic procedures effectively prevent infection, spittoons cannot be sanitized between each treated patient and are usually washed only with running water. However, there is currently no fast and efficient disinfection method that can be implemented between treatments. An optically filtered krypton chloride excimer lamp using ultraviolet light (Far UV-C) in the 200-230 nm wavelength range (innocuous to humans) has been recently used as a virus- and bacteria-inactivating technology. This study aimed to identify the bioburden of a dental spittoon and examine the susceptibility of two oral Streptococcus and two Enterococci to 222-nm Far UV-C by irradiating the spittoon with 222 nm Far UV-C for 5 min before evaluating the disinfection effect. METHODS: Bacterial analysis and real-time polymerase-chain reaction testing was used to confirm the spittoon's biological contamination. Bacterial susceptibility to a 222-nm Far UV-C was determined with a graded dose irradiation test. After each treatment, the spittoon was irradiated with 222-nm Far UV-C for 5 min, and the disinfecting effect was evaluated. Microbial analysis of the spittoon's surface was performed using the Silva database. RESULTS: We found that > 97% of the microbes consisted of six bacterial phyla, whereas no viruses were found. Pseudomonas aeruginosa was frequently detected. The 1-log reduction value of two oral-derived Streptococci and two Enterococci species at 222-nm Far UV-C was 4.5-7.3 mJ/cm2. Exposure of the spittoon to 222-nm Far UV-C at 3.6-13.5 mJ/cm2 significantly decreased bacterial counts (p < 0.001). CONCLUSIONS: Irradiation with 222-nm Far UV-C at 3.6-13.5 mJ/cm2 significantly eliminates bacteria in spittoons, even when they are only rinsed with water. Hence, 222-nm Far UV-C irradiation may inhibit the risk of bacterial transmission from droplets in sink surfaces.

Successful treatment of Enterococcus gallinarum infection in a neonate with vancomycin: a case report.

BACKGROUND: Enterococcus gallinarum (EG) is typically found in the gastrointestinal tracts of birds and mammals. Although its strains are rarely isolated from clinical specimens, EG can lead to septicemia in immunocompromised individuals. EG infections are uncommon in household settings, but their incidence has been rising due to increased antibiotic usage and invasive treatments, particularly in Neonatal Intensive Care Units (NICUs). EG inherently exhibits resistance to vancomycin but is highly sensitive to linezolid. Despite showing in vitro resistance, vancomycin has shown clinical efficacy in treating EG meningitis. CASE PRESENTATION: A neonate born at 30 + 2 weeks gestation was admitted to the Neonatal Intensive Care Unit (NICU) after EG was detected in blood and cerebrospinal fluid cultures. Susceptibility testing indicated that the bacterial strain was resistant to vancomycin and sensitive to linezolid. Initially, vancomycin was selected for treatment. However, due to persistent EG cultures in the blood and cerebrospinal fluid, the treatment was adjusted to linezolid. This led to a rapid decrease in platelet (PLT) count, suspected to be an adverse reaction. Concurrently, the patient experienced recurrent fever and elevated inflammatory marker levels, prompting the discontinuation of linezolid and a return to vancomycin. Subsequent administration of vancomycin stabilized the patient's condition, as evidenced by improved C-reactive protein (CRP), procalcitonin (PCT), and cerebrospinal fluid parameters, ultimately leading to discharge after an eight-week treatment period. CONCLUSION: This retrospective analysis highlights the efficacy of vancomycin in treating EG infections, suggesting that specific genetic phenotypes may influence treatment sensitivity. Monitoring vancomycin blood levels is crucial for determining treatment effectiveness.

Identification of Enterococcus spp. by MALDI-TOF mass spectrometry isolated from clinical mastitis and bulk tank milk samples.

BACKGROUND: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. RESULTS: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%). CONCLUSIONS: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.

The Probiotic Enterococcus Lactis SF68 as a Potential Food Fermentation Microorganism for Safe Food Production.

Due to the reports describing virulent and multidrug resistant enterococci, their use has become a topic of controversy despite most of them being safe and commonly used in traditionally fermented foods worldwide. We have characterized Enterococcus lactis SF68, a probiotic strain approved by the European Food Safety Authority (EFSA) for use in food and feed, and find that it has a remarkable potential in food fermentations. Genome analysis revealed the potential of SF68 to metabolize a multitude of carbohydrates, including lactose and sucrose, which was substantiated experimentally. Bacteriocin biosynthesis clusters were identified and SF68 was found to display a strong inhibitory effect against Listeria monocytogenes. Fermentation-wise, E. lactis SF68 was remarkably like Lactococcus lactis and displayed a clear mixed-acid shift on slowly fermented sugars. SF68 could produce the butter aroma compounds, acetoin and diacetyl, the production of which was enhanced under aerated conditions in a strain deficient in lactate dehydrogenase activity. Overall, E. lactis SF68 was found to be versatile, with a broad carbohydrate utilization capacity, a capacity for producing bacteriocins, and an ability to grow at elevated temperatures. This is key to eliminating pathogenic and spoilage microorganisms that are frequently associated with fermented foods.

Interference-resistant gold nanoparticle assay for detecting Enterococcus in fresh and marine waters.

Enterococci are common indicators of fecal contamination and are used to assess the quality of fresh and marine water, sand, soil, and sediment. However, samples collected from these environments contain various cells and other factors that can interfere with the assays used to detect enterococci. We developed a novel assay for the sensitive and specific detection of enterococci that is resistant to interference from other cells and environmental factors. Our interference-resistant assay used 30-nm gold nanoparticles (AuNPs), streptavidin, and a biotinylated Enterococcus antibody. Enterococci inhibited the interaction between streptavidin and biotin and led to the disaggregation of AuNPs. The absence of enterococci led to the aggregation of AuNPs, and this difference was easily detected by spectrophotometry. This interference-resistant AuNP assay was able to detect whole cells of Enterococcus in the range of 10 to 107 CFU/mL within 3 h, had high specificity for enterococci, and was unaffected by the presence of other intestinal bacteria, such as Escherichia coli. Our examination of fresh and marine water samples demonstrated no interference from other cells or environmental factors. The interference-resistant AuNP assay described here has the potential to be used as a rapid, simple, and effective method for monitoring enterococci in diverse environmental samples.

A novel molecular assay conducted on the BD Max system to facilitate reflex testing for vanA and vanB in clinical isolates of enterococci.

Infections caused by vancomycin-resistant enterococci (VRE) are common. Real-time PCR assays targeting vanA and vanB facilitate screening of patients in healthcare settings to limit the risk of dissemination, especially amongst those at high-risk of infection or with limited treatment options. Such assays are commonly performed as reflex testing procedures where they augment phenotypic techniques and shorten turnaround time to benefit timely clinical management. 'Random access' and 'sample-to-result' real-time PCR platforms are suited for this application as they are of low complexity and less technically demanding. Modelled on these attributes, we configured a real-time PCR assay (VRE BD) for detection of vanA/B in clinical isolates of enterococci, adapted for the BD Max System (Becton Dickinson). We applied an unconventional approach by testing suspensions of microorganisms in water to circumvent the traditional pre-analytical genomic extraction process. Our objective of this study was to assess the performance of this assay for detection of VRE in cultures by validating against a traditional real-time PCR assay based on the LightCycler 2.0 platform (Roche, VRE RO). A high level of analytical sensitivity and specificity (≥99.0%) for both genes was obtained when testing suspensions derived from blood agar. Results for suspensions obtained from chromID VRE (Edwards Group) showed a similar level of performance for vanA detection (100%), but not for the vanB target (≥90.9%) where a lesser number of isolates were available for testing. However, our results for VRE detection in isolates from these media were repeatable and reproducible, and equated to positive and negative predictive values of ≥95.2% and ≥97.8%, respectively. Furthermore, the VRE BD assay was also able to accurately detect VRE in clinical and spiked BacT/ALERT (bioMérieux) blood cultures. Thus, the technical simplicity, short turnaround time and robustness of this high performing assay for VRE is suitable for reflex testing. In addition, the format developed for the BD Max platform has potential application for reflex testing other molecular targets of clinical importance.