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Antimicrobial resistance is a major public health threat, and there is an urgent need for new strategies to address this issue. In a recent study, a library screening strategy was developed in which an FDA-approved drug library was screened against methicillin-resistant Staphylococcus aureus (MRSA) in both its original (unmetabolized [UM]) and its human liver microsome metabolized (postmetabolized [PM]) forms and in the absence and presence of a resistant-to antibiotic. This allows the identification of agents with active metabolites and agents that can act synergistically with the resistant-to antibiotic. In this study, this strategy is applied to VanA-type vancomycin-resistant Enterococcus faecium (VREfm) in the absence and presence of vancomycin. Thirteen drugs with minimum MICs that were ≤12.5 μM under any tested condition (UM/PM vs. -/+vancomycin) were identified. Seven of these appeared to act synergistically with vancomycin, and follow-up checkerboard analyses confirmed synergy (∑FICmin ≤0.5) for six of these. Ultimately four rifamycins, two pleuromutilins, mupirocin, and linezolid were confirmed as synergistic. The most synergistic agent was rifabutin (∑FICmin = 0.19). Linezolid, a protein biosynthesis inhibitor, demonstrated relatively weak synergy (∑FICmin = 0.5). Only mupirocin showed significantly improved activity after microsomal metabolism, indicative of a more active metabolite, but efforts to identify an active metabolite were unsuccessful. Spectra of activity of several hits and related agents were also determined. Gemcitabine showed activity against a number vancomycin-resistant E. faecium and E. faecalis strains, but this activity was substantially weaker than previously observed in MRSA.

Biliary-tract bloodstream infections (BT-BSI) caused by Enterococcus faecalis and E. faecium are associated with inappropriate empirical treatment and worse outcomes compared to other etiologies. The objective of this study was to investigate the risk factors for enterococcal BT-BSI. Patients with BT-BSI from the PROBAC cohort, including consecutive patients with BSI in 26 Spanish hospitals between October 2016 and March 2017, were selected; episodes caused by E. faecalis or E. faecium and other causes were compared. Independent predictors for enterococci were identified by logistic regression, and a predictive score was developed. Eight hundred fifty episodes of BT-BSI were included; 73 (8.5%) were due to target Enterococcus spp. (48 [66%] were E. faecium and 25 [34%] E. faecalis). By multivariate analysis, the variables independently associated with Enterococcus spp. were (OR; 95% confidence interval): cholangiocarcinoma (4.48;1.32 to 15.25), hospital acquisition (3.58;2.11 to 6.07), use of carbapenems in the previous month (3.35;1.45 to 7.78), biliary prosthesis (2.19;1.24 to 3.90), and moderate or severe chronic kidney disease (1.55;1.07 to 2.26). The AUC of the model was 0.74 [95% CI0.67 to 0.80]. A score was developed, with 7, 6, 5, 4, and 2 points for these variables, respectively, with a negative predictive value of 95% for a score ≤ 6. A model, including cholangiocarcinoma, biliary prosthesis, hospital acquisition, previous carbapenems, and chronic kidney disease showed moderate prediction ability for enterococcal BT-BSI. Although the score will need to be validated, this information may be useful for deciding empirical therapy in biliary tract infections when bacteremia is suspected. IMPORTANCE Biliary tract infections are frequent, and a significant cause of morbidity and mortality. Bacteremia is common in these infections, particularly in the elderly and patients with cancer. Inappropriate empirical treatment has been associated with increased risk of mortality in bacteremic cholangitis, and the probability of receiving inactive empirical treatment is higher in episodes caused by enterococci. This is because many of the antimicrobial agents recommended in guidelines for biliary tract infections lack activity against these organisms. To the best of our knowledge, this is the first study analyzing the predictive factors for enterococcal BT-BSI and deriving a predictive score., This work was financed by Plan Nacional de I+D+i 2013‐2016, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades, through grants PI16/01432 and Spanish Network for Research in Infectious Diseases (REIPI) [RD16/0016/0001; RD16/0016/0007; and RD16/0016/0012]; co‐financed by European Development Regional Fund “A Way to Achieve Europe,” Operative program Intelligent Growth 2014–2020.

The purpose of this trial was to evaluate the efficacy of a 4-week supplementation of Lactobacillus rhamnosus GG (LGG) in eliminating the gastrointestinal carrier state of vancomycin-resistant Enterococcus faecium (VREfm) in hospitalized adults. The primary outcome of the study was the number of patients with cleared VREfm colonization after the 4-week intervention. Secondary outcomes were clearance of VREfm colonization at weeks 8, 16, and 24, number of VREfm infections (isolated from nonintestinal foci), and changes in fecal microbiome diversity after the intervention. The trial was a multicenter, randomized, double-blind, placebo-controlled trial in hospitalized adult VREfm carriers. Patients were enrolled and randomized to receive 60 billion CFU of LGG daily or placebo for 4 weeks. For a subgroup of patients, rectal swabs for VREfm were collected also at 8, 16, and 24 weeks and analyzed using shotgun metagenomics. Patients ingesting a minimum of 50% of the probiotic during the 4-week intervention were included in subsequent outcome analyses (48 of 81 patients). Twelve of 21 patients in the LGG group (57%) compared to 15 of 27 patients in the placebo group (56%) cleared their VREfm carriage. Eighteen patients completed the entire 24-week intervention with the same minimum compliancy. Of these, almost 90% in both groups cleared their VREfm carriage. We found a statistically significant difference between VREfm clearers and nonclearers regarding metronidazole and vancomycin usage as well as length of hospitalization after inclusion. The microbiome analyses revealed no significant difference in alpha diversity between the LGG and the placebo group. Beta diversity differed between the groups and the different time points. This study did not show an effect of LGG in eradication of VREfm after a 4-week intervention. IMPORTANCE Whereas other studies exploring the effect of L. rhamnosus in clearing VREfm from the intestine included children and adults, with a wider age range, our study consisted of a geriatric patient cohort. The natural clearance of VREfm in this study was almost 60% after 4 weeks, thus much higher than described previously. Also, this study characterizes the microbiome of VREfm patients in detail. This article showed no effect of the probiotic L. rhamnosus in clearing VREfm from the intestine of patients.

The long-acting lipoglycopeptides (LGPs) dalbavancin and oritavancin are semisynthetic antimicrobials with broad and potent activity against Gram-positive bacterial pathogens. While they are approved by the Food and Drug Administration for acute bacterial skin and soft tissue infections, their pharmacological properties suggest a potential role of these agents for the treatment of deep-seated and severe infections, such as bloodstream and bone and joint infections. The use of these antimicrobials is particularly appealing when prolonged therapy, early discharge, and avoidance of long-term intravascular catheter access are desirable or when multidrug-resistant bacteria are suspected. This review describes the current evidence for the use of oritavancin and dalbavancin in the treatment of invasive infections, as well as the hurdles that are preventing their optimal use. Moreover, this review discusses the current knowledge gaps that need to be filled to understand the potential role of LGPs in highly needed clinical scenarios and the ongoing clinical studies that aim to address these voids in the upcoming years.

Wound infections with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are particularly difficult to treat and present a great challenge to clinicians. Nanoemulsions (NE) are novel oil-in-water emulsions formulated from soybean oil, water, solvent, and surfactants such as benzalkonium chloride (BZK). An optimal ratio of those components produces nanometer-sized particles with the positive-charged surfactant at their oil-water interface. We sought to investigate antimicrobial NE as a novel treatment to address wounds co-infected by MRSA and VRE. Swine split-thickness skin wounds were first infected with MRSA and/or VRE, then treated with the nanoemulsion formulation (X-1735) or placebo controls. Bacterial viability after treatment were determined by nutrient agar plates for total, MRSA-specific, and VRE-specific loads. In addition, inflammation indexes were scored by histopathology. When VRE infected wounds were treated with X-1735, they contained 10

Bacillus cereus group species are widespread, Gram-positive, spore-forming environmental bacteria. B. cereus sensu stricto is one of the major causes of food poisoning worldwide. In high-risk individuals, such as preterm neonates, B. cereus infections can cause fatal infections. It is important to note that the phenotypic identification methods commonly used in clinical microbiology laboratories make no distinction between B. cereus sensu stricto and the other members of the group (Bacillus anthracis excluded). As a result, all the invasive infections attributed to B. cereus are not necessarily due to B. cereus sensu stricto but likely to other closely related species of the B. cereus group. Next-generation sequencing (NGS) should be used to characterize the whole genome of the strains belonging to the B. cereus group. This could confirm whether the strains involved in previously reported B. cereus invasive infections preferentially belong to formerly known or emerging individual species. Moreover, infections related to B. cereus group species have probably been overlooked, since their isolation in human bacteriological samples has for a long time been regarded as an environmental contaminant of the cultures. Recent studies have questioned the emergence or reemergence of B. cereus invasive infections in preterm infants. This review reports our current understanding of B. cereus infections in neonates, including taxonomical updates, microbiological characteristics, bacterial identification, clinical features, host-pathogen interactions, environmental sources of contamination, and antimicrobial resistance.

Healthcare-associated outbreaks of vancomycin-resistant Enterococcus faecium (VREfm) are a worldwide problem with increasing prevalence. The genomic plasticity of this hospital-adapted pathogen contributes to its efficient spread despite infection control measures. Here, we aimed to identify the genomic and phenotypic determinants of health care-associated transmission of VREfm. We assessed the VREfm transmission networks at the tertiary-care University Hospital of Zurich (USZ) between October 2014 and February 2018 and investigated microevolutionary dynamics of this pathogen. We performed whole-genome sequencing for the 69 VREfm isolates collected during this time frame and assessed the population structure and variability of the vancomycin resistance transposon. Phylogenomic analysis allowed us to reconstruct transmission networks and to unveil external or wider transmission networks undetectable by routine surveillance. Notably, it unveiled a persistent clone, sampled 31 times over a 29-month period. Exploring the evolutionary dynamics of this clone and characterizing the phenotypic consequences revealed the spread of a variant with decreased daptomycin susceptibility and the acquired ability to utilize N-acetyl-galactosamine (GalNAc), one of the primary constituents of the human gut mucins. This nutrient utilization advantage was conferred by a novel plasmid, termed pELF_USZ, which exhibited a linear topology. This plasmid, which was harbored by two distinct clones, was transferable by conjugation. Overall, this work highlights the potential of combining epidemiological, functional genomic, and evolutionary perspectives to unveil adaptation strategies of VREfm., mBio, 132 (2), ISSN:2150-7511, ISSN:2161-2129

Oritavancin displayed potent and stable activity (MIC 90 range of 0.06 to 0.5 mg/L) over a 10-year period (2010 to 2019) against Gram-positive pathogens that cause bloodstream infections (BSI), including methicillin-resistant Staphylococcus aureus (MRSA) and resistant subsets of Enterococcus spp. Daptomycin and linezolid were also active against methicillin-resistant S. aureus and vancomycin-resistant Enterococcus (VRE). Only oritavancin and linezolid remained active against Enterococcus faecium isolates displaying an elevated daptomycin MIC (i.e., 2 to 4 mg/L).

Background: Enterococcus faecium is a major cause of clinical infections, often due to multidrug-resistant (MDR) strains. Whole genome sequencing (WGS) is a powerful tool to study MDR bacteria and their antimicrobial resistance (AMR) mechanisms. Here we use WGS to characterize E. faecium clinical isolates and test the feasibility of rules-based genotypic prediction of AMR. Methods: Clinical isolates were divided into derivation and validation sets. Phenotypic susceptibility testing for ampicillin, vancomycin, high-level gentamicin, ciprofloxacin, levofloxacin, doxycycline, tetracycline, and linezolid was performed using the VITEK 2 automated system, with confirmation and discrepancy resolution by broth microdilution, disk diffusion, or gradient diffusion when needed. WGS was performed to identify isolate lineage and AMR genotype. AMR prediction rules were derived by analyzing the genotypic-phenotypic relationship in the derivation set. Results: Phylogenetic analysis demonstrated that 88% of isolates in the collection belonged to hospital-associated clonal complex 17. Additionally, 12% of isolates had novel sequence types. When applied to the validation set, the derived prediction rules demonstrated an overall positive predictive value of 98% and negative predictive value of 99% compared to standard phenotypic methods. Most errors were falsely resistant predictions for tetracycline and doxycycline. Further analysis of genotypic-phenotypic discrepancies revealed potentially novel pbp5 and tet(M) alleles that provide insight into ampicillin and tetracycline class resistance mechanisms. The prediction rules demonstrated generalizability when tested on an external dataset. Conclusions: Known AMR genes and mutations can predict E. faecium phenotypic susceptibility with high accuracy for most routinely tested antibiotics, providing opportunities for advancing molecular diagnostics.

With the increasing reports of community-acquired and nosocomial infection caused by multidrug-resistant Gram-positive pathogens, there is an urgent need to develop new antimicrobial agents with novel antibacterial mechanisms. Here, we investigated the antibacterial activity of the natural product ginkgolic acid (GA) (15:1), derived from Ginkgo biloba, and its potential mode of action against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus. The MIC values of GA (15:1) against clinical E. faecalis and S. aureus isolates from China were ≤4 and ≤8 μg/mL, respectively, from our test results. Moreover, GA (15:1) displayed high efficiency in biofilm formation inhibition and bactericidal activity against E. faecalis and S. aureus. During its inhibition of the planktonic bacteria, the antibacterial activity of GA (15:1) was significantly improved under the condition of abolishing iron homeostasis. When iron homeostasis was abolished, inhibition of planktonic bacteria by GA (15:1) was significantly improved. This phenomenon can be interpreted as showing that iron homeostasis disruption facilitated the disruption of the functions of ribosome and protein synthesis by GA (15:1), resulting in inhibition of bacterial growth and cell death. Genetic mutation of ferric uptake regulator (Fur) led to GA (15:1) tolerance in in vitro-induced resistant derivatives, while overexpression of Fur led to increased GA (15:1) susceptibility. Additionally, GA (15:1) significantly decreased the bacterial loads of S. aureus strain USA300 in the lung tissues of mice in a pneumonic murine model. Conclusively, this study revealed an antimicrobial mechanism of GA (15:1) involving cross talk with iron homeostasis against Gram-positive pathogens. In the future, the natural product GA (15:1) might be applied to combat infections caused by Gram-positive pathogens. IMPORTANCE The increasing emergence of infectious diseases associated with multidrug-resistant Gram-positive pathogens has raised the urgent need to develop novel antibiotics. GA (15:1) is a natural product derived from Ginkgo biloba and possesses a wide range of bioactivities, including antimicrobial activity. However, its antibacterial mechanisms remain unclear. Our current study found that the function of ferric uptake regulator (Fur) was highly correlated with the antimicrobial activity of GA (15:1) against E. faecalis and that the antibacterial activity of GA (15:1) could be strengthened by the disruption of iron homeostasis. This study provided important insight into the mode of action of GA (15:1) against Gram-positive bacteria and suggested that GA (15:1) holds the potential to be an antimicrobial treatment option for infection caused by multidrug-resistant Gram-positive pathogens.

Enterococcus faecium is a clinically important pathogen that can cause significant morbidity and death. In this study, we aimed to develop a machine learning (ML) algorithm-based rapid susceptibility method to distinguish vancomycin-resistant E. faecium (VREfm) and vancomycin-susceptible E. faecium (VSEfm) strains. A predictive model was developed and validated to distinguish VREfm and VSEfm strains by analyzing the matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) spectra of unique E. faecium isolates from different specimen types. The algorithm used 5,717 mass spectra, including 2,795 VREfm and 2,922 VSEfm mass spectra, and was externally validated with 2,280 mass spectra of isolates (1,222 VREfm and 1,058 VSEfm strains). A random forest-based algorithm demonstrated overall good classification performances for the isolates from the specimens, with mean accuracy, sensitivity, and specificity of 0.78, 0.79, and 0.77, respectively, with 10-fold cross-validation, timewise validation, and external validation. Furthermore, the algorithm provided rapid results, which would allow susceptibility prediction prior to the availability of phenotypic susceptibility results. In conclusion, an ML algorithm designed using mass spectra obtained from the routine workflow may be able to rapidly differentiate VREfm strains from VSEfm strains; however, susceptibility results must be confirmed by routine methods, given the demonstrated performance of the assay. IMPORTANCE A modified binning method was incorporated to cluster MS shifting ions into a set of representative peaks based on a large-scale MS data set of clinical VREfm and VSEfm isolates, including 2,795 VREfm and 2,922 VSEfm isolates. Predictions with the algorithm were significantly more accurate than empirical antibiotic use, the accuracy of which was 0.50, based on the local epidemiology. The algorithm improved the accuracy of antibiotic administration, compared to empirical antibiotic prescription. An ML algorithm designed using MALDI-TOF MS spectra obtained from the routine workflow accurately differentiated VREfm strains from VSEfm strains, especially in blood and sterile body fluid samples, and can be applied to facilitate the rapid and accurate clinical testing of pathogens.

Bacterial biofilms are involved in chronic infections and confer 10 to 1,000 times more resistance to antibiotics compared with planktonic growth, leading to complications and treatment failure. When transitioning from a planktonic lifestyle to biofilms, some Gram-positive bacteria are likely to modulate several cellular pathways, including central carbon metabolism, biosynthesis pathways, and production of secondary metabolites. These metabolic adaptations might play a crucial role in biofilm formation by Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis. Here, we performed a transcriptomic approach to identify cellular pathways that might be similarly regulated during biofilm formation in these bacteria. Different strains and biofilm-inducing media were used to identify a set of regulated genes that are common and independent of the environment or accessory genomes analyzed. Our approach highlighted that the de novo purine biosynthesis pathway was upregulated in biofilms of both species when using a tryptone soy broth-based medium but not so when a brain heart infusion-based medium was used. We did not identify other pathways commonly regulated between both pathogens. Gene deletions and usage of a drug targeting a key enzyme showed the importance of this pathway in biofilm formation of S. aureus. The importance of the de novo purine biosynthesis pathway might reflect an important need for purine during biofilm establishment, and thus could constitute a promising drug target. IMPORTANCE Biofilms are often involved in nosocomial infections and can cause serious chronic infections if not treated properly. Current anti-biofilm strategies rely on antibiotic usage, but they have a limited impact because of the biofilm intrinsic tolerance to drugs. Metabolism remodeling likely plays a central role during biofilm formation. Using comparative transcriptomics of different strains of Staphylococcus aureus and Enterococcus faecalis, we determined that almost all cellular adaptations are not shared between strains and species. Interestingly, we observed that the de novo purine biosynthesis pathway was upregulated during biofilm formation by both species in a specific medium. The requirement for purine could constitute an interesting new anti-biofilm target with a wide spectrum that could also prevent resistance evolution. These results are also relevant to a better understanding of the physiology of biofilm formation.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP and ΔdhhPΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of ΔcdaA could be also attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results reveal that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.IMPORTANCEEnterococcus faecalis is an opportunistic pathogen and leading cause of multidrug resistant hospital-acquired infections. During the course of an infection, bacteria encounter multiple adverse (stress) conditions and understanding the adaptive mechanisms used by pathogens to survive these stresses can facilitate the development of new antimicrobial therapies. Here, we used in vitro, ex vivo and in vivo approaches to determine the importance of the second messenger nucleotide c-di-AMP, a global regulator essential for bacterial adaptation to osmotic stress, to E. faecalis pathophysiology. We demonstrated that either accumulation of c-di-AMP or complete loss of c-di-AMP impaired cell fitness and virulence of E. faecalis. Remarkably, the strain that was unable to produce c-di-AMP was avirulent in three animal infection models indicating that c-di-AMP signaling is essential for E. faecalis pathogenesis and a suitable antimicrobial target.

The poxtA gene encodes a protein belonging to the F lineage of the ATP-binding cassette superfamily, which can protect the bacterial ribosome from some anti-ribosomal antibiotics, including oxazolidinones (1).….

Prior research into the bacterium Gardnerella vaginalis has largely focused on its association with bacterial vaginosis (BV). However, G. vaginalis is also frequently found within the urinary microbiota of women with and without lower urinary tract symptoms as well as individuals with chronic kidney disease, interstitial cystitis, and BV., Gardnerella is a frequent member of the urogenital microbiota. Given the association between Gardnerella vaginalis and bacterial vaginosis (BV), significant efforts have been focused on characterizing this species in the vaginal microbiota. However, Gardnerella also is a frequent member of the urinary microbiota. In an effort to characterize the bacterial species of the urinary microbiota, we present here 10 genomes of urinary Gardnerella isolates from women with and without lower urinary tract symptoms. These genomes complement those of 22 urinary Gardnerella strains previously isolated and sequenced by our team. We included these genomes in a comparative genome analysis of all publicly available Gardnerella genomes, which include 33 urinary isolates, 78 vaginal isolates, and 2 other isolates. While once this genus was thought to consist of a single species, recent comparative genome analyses have revealed 3 new species and an additional 9 groups within Gardnerella. Based upon our analysis, we suggest a new group for the species. We also find that distinction between these Gardnerella species/groups is possible only when considering the core or whole-genome sequence, as neither the sialidase nor vaginolysin genes are sufficient for distinguishing between species/groups despite their clinical importance. In contrast to the vaginal microbiota, we found that only five Gardnerella species/groups have been detected within the lower urinary tract. Although we found no association between a particular Gardnerella species/group(s) and urinary symptoms, further sequencing of urinary Gardnerella isolates is needed for both comprehensive taxonomic characterization and etiological classification of Gardnerella in the urinary tract. IMPORTANCE Prior research into the bacterium Gardnerella vaginalis has largely focused on its association with bacterial vaginosis (BV). However, G. vaginalis is also frequently found within the urinary microbiota of women with and without lower urinary tract symptoms as well as individuals with chronic kidney disease, interstitial cystitis, and BV. This prompted our investigation into Gardnerella from the urinary microbiota and all publicly available Gardnerella genomes from the urogenital tract. Our work suggests that while some Gardnerella species can survive in both the urinary tract and vagina, others likely cannot. This study provides the foundation for future studies of Gardnerella within the urinary tract and its possible contribution to lower urinary tract symptoms.

Enterococcus faecalis infections are considered a major public health concern worldwide. The complement system has a crucial role in the protection against different microbial pathogens including E. faecalis Complement can be activated through three different pathways including; the classical, the lectin, and the alternative pathways. There is limited information on the role of the classical pathway (CP) in protection against infections caused by E. faecalis In the present study, we generated Fab fragments that successfully block the CP in mouse via inhibition of a key enzyme C1s-A. Our results showed that anti C1s-A Fab fragments block CP mediated C3b and C4b deposition in vitro We further showed that administration of anti C1s-A Fab fragments significantly impairs the CP functional activity in vitro and in vivo Moreover, treatment of mice infected with E. faecalis using anti C1s-A Fab fragments significantly impairs bacterial clearance as determined from the viable bacterial counts recovered from blood, kidneys, spleens, livers, and lungs of infected mice. Overall, this study highlights the essential role of the CP in host defence against E. faecalis.

“METH mouth” is characterized by severe tooth decay and gum disease, which often causes teeth to break or fall out. METH users are also prone to colonization by cariogenic bacteria such as Streptococcus mutans., “METH mouth” is a common consequence of chronic methamphetamine (METH) use, resulting in tooth decay and painful oral tissue inflammation that can progress to complete tooth loss. METH reduces the amount of saliva in the mouth, promoting bacterial growth, tooth decay, and oral tissue damage. This oral condition is worsened by METH users’ compulsive behavior, including high rates of consumption of sugary drinks, recurrent tooth grinding, and a lack of frequent oral hygiene. Streptococcus mutans is a Gram-positive bacterium found in the oral cavity and associated with caries in humans. Hence, we developed a murine model of METH administration, sugar intake, and S. mutans infection to mimic METH mouth in humans and to investigate the impact of this drug on tooth colonization. We demonstrated that the combination of METH and sucrose stimulates S. mutans tooth adhesion, growth, and biofilm formation in vivo. METH and sucrose increased the expression of S. mutans glycosyltransferases and lactic acid production. Moreover, METH contributes to the low environmental pH and S. mutans sucrose metabolism, providing a plausible mechanism for bacterium-mediated tooth decay. Daily oral rinse treatment with chlorhexidine significantly reduces tooth colonization in METH- and sucrose-treated mice. Furthermore, human saliva inhibits S. mutans colonization and biofilm formation after exposure to either sucrose or the combination of METH and sucrose. These findings suggest that METH might increase the risk of microbial dental disease in users, information that may help in the development of effective public health strategies to deal with this scourge in our society.

Tenascin C (TNC) is an extracellular matrix protein with immunomodulatory properties that plays a major role during tissue injury and repair. TNC levels are increased in patients with pneumonia and pneumosepsis, and they are associated with worse outcomes. Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium that is a major causative pathogen in nosocomial pneumonia and a rising cause of community-acquired pneumonia. To study the role of TNC during MRSA-induced pneumonia, TNC sufficient (TNC+/+) and TNC-deficient (TNC-/-) mice were infected with MRSA via the airways and euthanized after 6, 24, and 48 h for analysis. Pulmonary transcription of TNC peaked at 6 h, while immunohistochemistry revealed higher protein levels at later time points. Although TNC deficiency was not associated with changes in bacterial clearance, TNC-/- mice showed increased levels of TNF-α and IL-6 in bronchoalveolar lavage fluid during the acute phase of infection when compared with TNC+/+ mice. In addition, TNC-/- mice showed more severe pulmonary pathology at 6, but not at 24 or 48 h, after infection. Together, these data suggest that TNC plays a moderate protective role against tissue pathology during the acute inflammatory phase, but not during the bacterial clearance phase, of MRSA-induced pneumonia. These results argue against an important role of TNC on disease outcome during MRSA-induced pneumonia. IMPORTANCE Recently, the immunomodulatory properties of TNC have drawn substantial interest. However, to date most studies made use of sterile models of inflammation. In this study, we examine the pathobiology of MRSA-induced pneumonia in a model of TNC-sufficient and TNC-deficient mice. We have studied the immune response and tissue pathology both during the initial insult and also during the resolution phase. We demonstrate that MRSA-induced pneumonia upregulates pulmonary TNC expression at the mRNA and protein levels. However, the immunomodulatory role of TNC during bacterial pneumonia is distinct from models of sterile inflammation, indicating that the function of TNC is context dependent. Contrary to previous descriptions of TNC as a proinflammatory mediator, TNC-deficient mice seem to suffer from enhanced tissue pathology during the acute phase of infection. Nonetheless, besides its role during the acute phase response, TNC does not seem to play a major role in disease outcome during MRSA-induced pneumonia., peer-reviewed

Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyze the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus species. MRSE (n = 17) and Enterococcus species (Enterococcus faecalis n = 8 and Enterococcus faecium n = 10) strains were used. MIC of the mixture of DTAM, HTAM, and ENDX and that of vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and of vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays. MIC90 of tamoxifen metabolites was 1 mg/liter for MRSE strains and 2 mg/liter for E. faecalis and E. faecium strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 4× MIC for MRSE (SE385 and SE742) and at 2× MIC and 4× MIC for E. faecalis (EVR1 and EVR2) strains, respectively. SE385 and EVR2 strains treated with the tamoxifen metabolites mixture presented higher membrane permeabilization. Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drug treatments against these bacterial pathogens., This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (grants CP15/00132, PI16/01378, and PI19/01453) and by Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009), cofinanced by European Development Regional Fund “A way to achieve Europe,” Operative program Intelligent Growth 2014–2020. Younes Smani is supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/00132).

The enterococci, which are among the leading causes of multidrug-resistant (MDR) hospital infection, are notable for their environmental ruggedness, which extends to intrinsic antibiotic resistance. To identify genes that confer this unique property, we used Tn-seq to comprehensively explore the genome of MDR Enterococcus faecalis strain MMH594 for genes important for growth in nutrient-containing medium and with low-level antibiotic challenge. As expected, a large core of genes for DNA replication, expression, and central metabolism, shared with other bacteria, are intolerant to transposon disruption. However, genes were identified that are important to E. faecalis that are either absent from or unimportant for Staphylococcus aureus and Streptococcus pneumoniae fitness when similarly tested. Further, 217 genes were identified that when challenged by sub-MIC antibiotic levels exhibited reduced tolerance to transposon disruption, including those previously shown to contribute to intrinsic resistance, and others not previously ascribed this role. E. faecalis is one of the few Gram-positive bacteria experimentally shown to possess a functional Entner-Doudoroff pathway for carbon metabolism, a pathway that contributes to stress tolerance in other microbes. Through functional genomics and network analysis we defined the unusual structure of this pathway in E. faecalis and assessed its importance. These approaches also identified toxin-antitoxin and related systems that are unique and active in E. faecalis. Finally, we identified genes that are absent in the closest nonenterococcal relatives, the vagococci, and that contribute importantly to fitness with and without antibiotic selection, advancing an understanding of the unique biology of enterococci. IMPORTANCE Enterococci are leading causes of antibiotic-resistant infection transmitted in hospitals. The intrinsic hardiness of these organisms allows them to survive disinfection practices and then proliferate in the gastrointestinal tracts of antibiotic-treated patients. The objective of this study was to identify the underlying genetic basis for its unusual hardiness. Using a functional genomic approach, we identified traits and pathways of general importance for enterococcal survival and growth that distinguish them from closely related pathogens as well as ancestrally related species. We further identified unique traits that enable them to survive antibiotic challenge, revealing a large set of genes that contribute to intrinsic antibiotic resistance and a smaller set of uniquely important genes that are rare outside enterococci.

Enterococcus faecalis, long implicated in serious systemic infections and failure of root canal treatment, is a persistent inhabitant of oral periapical lesions. Dendritic cells (DCs) and other innate immune cells patrol the oral mucosa for infecting microbes. Dendritic cells are efficient at capturing microbes when immature, whereupon they can transform into potent antigen-presenting cells upon full maturation. Autophagy, a sophisticated intracellular process first described for elimination of damaged organelles, regulates DC maturation and other important immune functions of DCs. The present study examined how E. faecalis influences the differentiation of murine bone marrow-derived stem cells (BMSCs) into functional DCs in the presence of the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Although the viability and differentiation of DCs were not affected by E. faecalis, expression of the autophagy-related proteins ATG7, Beclin1, and LC3bI/II were significantly suppressed in an mTOR-dependent manner. Ultrastructurally, E. faecalis was identified in single-membrane vacuoles, some of which were in the process of binary fission. Bacterium-containing autophagosomes were absent within the cytoplasm. Accessory molecules (major histocompatibility complex class II [MHC-II], CD80, and CD86) and anti-inflammatory cytokine (transforming growth factor β1 [TGF-β1]) were suppressed in E. faecalis-induced DCs, while IL-1β, tumor necrosis factor alpha (TNF-α), and IL-12 levels were upregulated. When pulsed with ovalbumin (OVA), the E. faecalis-induced DCs showed reduction in CD4(+) OVA-specific OT-II T cell proliferation. It is concluded that E. faecalis promotes the differentiation of bone marrow stem cells into CD11c-positive DCs with aberrant immune functions while retaining the capability of proinflammatory cytokine induction.

Concerns regarding increased prevalence of daptomycin (DAP)-resistant strains necessitate novel therapies for Enterococcus faecium infections. Obligately lytic bacteriophages are viruses that target, infect, and kill bacterial cells. Limited studies have evaluated phage-antibiotic combinations against E. faecium . After an initial screen of eight E. faecium strains, three strains with varying DAP/phage susceptibilities were selected for further experiments.

Different linezolid antimicrobial susceptibility testing (AST) methodologies yield various results. In 2018, we transitioned our linezolid AST methodology from the Etest to Vitek 2. We sought to evaluate the impact of this change on antibiotic use among 181 inpatients with vancomycin-resistant enterococcal (VRE) infections. The transition from Etest to Vitek 2 resulted in an increase in linezolid susceptibility (38% versus 96%; P < 0.001) and a reduction in time to active antibiotic therapy (3 versus 2.

Antibiotic resistance is a growing health concern. Resistance mechanisms have evolved that provide bacteria with a growth advantage in their natural habitat such as the soil. We determined that B. subtilis, a Gram-positive soil organism, has a mechanism of resistance to tylosin, a macrolide antibiotic commonly used in the meat industry. Tylosin induces expression of yxjB, which encodes an enzyme that methylates 23S rRNA. YxjB-dependent methylation of 23S rRNA confers tylosin resistance. NusG-dependent RNA polymerase pausing and tylosin-dependent ribosome stalling induce yxjB expression, and hence tylosin resistance, by preventing transcription termination upstream of the yxjB coding sequence and by preventing repression of yxjB translation., Macrolide antibiotics bind to 23S rRNA within the peptide exit tunnel of the ribosome, causing the translating ribosome to stall when an appropriately positioned macrolide arrest motif is encountered in the nascent polypeptide. Tylosin is a macrolide antibiotic produced by Streptomyces fradiae. Resistance to tylosin in S. fradiae is conferred by methylation of 23S rRNA by TlrD and RlmAII. Here, we demonstrate that yxjB encodes RlmAII in Bacillus subtilis and that YxjB-specific methylation of 23S rRNA in the peptide exit tunnel confers tylosin resistance. Growth in the presence of subinhibitory concentrations of tylosin results in increased rRNA methylation and increased resistance. In the absence of tylosin, yxjB expression is repressed by transcription attenuation and translation attenuation mechanisms. Tylosin-dependent induction of yxjB expression relieves these two repression mechanisms. Induction requires tylosin-dependent ribosome stalling at an RYR arrest motif at the C terminus of a leader peptide encoded upstream of yxjB. Furthermore, NusG-dependent RNA polymerase pausing between the leader peptide and yxjB coding sequences is essential for tylosin-dependent induction. Pausing synchronizes the position of RNA polymerase with ribosome position such that the stalled ribosome prevents transcription termination and formation of an RNA structure that sequesters the yxjB ribosome binding site. On the basis of our results, we are renaming yxjB as tlrB.

The anaerobic Gram-positive coccus Finegoldia magna is a rare cause of infections of bone and joints. The aim of this study was to describe the microbiological and clinical characteristics of orthopedic implant-associated infections caused by F. magna . We retrospectively analyzed samples consisting of anaerobic Gram-positive cocci and samples already identified as F. magna from patients with orthopedic infections. The isolates found were determined to the species level using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The antibiotic susceptibility pattern was determined by Etest. Whole-genome sequencing (WGS) was performed. Clinical data were extracted from each patient's journal. In nine patients, orthopedic joint implant-associated infections were identified as being caused by F. magna . The isolates were susceptible to most of the antibiotics tested, with the exception of rifampin and moxifloxacin in a few cases. Five of the nine infections were monomicrobial. The most common antibiotic used to treat the infection was penicillin V, but five of the nine patients received a combination of antibiotics. Eight patients underwent surgical treatment, with extraction of the implant performed in seven cases and reimplantation in only two cases. The WGS showed a relatively small core genome, with 126,647 single nucleotide polymorphisms identified within the core genome. A phylogenomic analysis revealed that the isolates clustered into two distinct clades. Orthopedic implant-associated infections caused by F. magna are rare, but the bacteria are generally susceptible to antibiotics. Despite this, surgical treatment combined with long-term antibiotics is often necessary. The WGS analysis revealed a high heterogeneity and suggested the existence of at least two different Finegoldia species.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) decreases the time to organism identification and improves clinical and financial outcomes. The purpose of this study was to evaluate the impact of MALDI-TOF MS alone versus MALDI-TOF MS combined with real-time, pharmacist-driven, antimicrobial stewardship (AMS) intervention on patient outcomes. This single-center, pre-post, quasiexperimental study evaluated hospitalized patients with positive blood cultures identified via MALDI-TOF MS combined with prospective AMS intervention compared to a control cohort with MALDI-TOF MS identification without AMS intervention. AMS intervention included: real-time MALDI-TOF MS pharmacist notification and prospective AMS provider feedback. The primary outcome was the time to optimal therapy (TTOT). A total of 252 blood cultures, 126 in each group, were included in the final analysis. MALDI-TOF MS plus AMS intervention significantly reduced the overall TTOT (75.17 versus 43.06 h; P < 0.001), the Gram-positive contaminant TTOT (48.21 versus 11.75 h; P < 0.001), the Gram-negative infection (GNI) TTOT (71.83 versus 35.98 h; P < 0.001), and the overall hospital length of stay (LOS; 15.03 versus 9.02 days; P = 0.021). The TTOT for Gram-positive infection (GPI) was improved (64.04 versus 41.61 h; P = 0.082). For GPI, the hospital LOS (14.64 versus 10.31 days; P = 0.002) and length of antimicrobial therapy 24.30 versus 18.97 days; P = 0.018) were reduced. For GNI, the time to microbiologic clearance (51.13 versus 34.51 h; P < 0.001), the hospital LOS (15.40 versus 7.90 days; P = 0.027), and the intensive care unit LOS (5.55 versus 1.19 days; P = 0.035) were reduced. To achieve optimal outcomes, rapid identification with MALDI-TOF MS combined with real-time AMS intervention is more impactful than MALDI-TOF MS alone.

RBx 11760, a bi-aryl oxazolidinone, was investigated for antibacterial activity against Gram-positive bacteria. The MIC 90 s of RBx 11760 and linezolid against Staphylococcus aureus were 2 and 4 mg/liter, against Staphylococcus epidermidis were 0.5 and 2 mg/liter, and against Enterococcus were 1 and 4 mg/liter, respectively. Similarly, against Streptococcus pneumoniae the MIC 90 s of RBx 11760 and linezolid were 0.5 and 2 mg/liter, respectively. In time-kill studies, RBx 11760, tedizolid, and linezolid exhibited bacteriostatic effect against all tested strains except S. pneumoniae . RBx 11760 showed 2-log 10 kill at 4× MIC while tedizolid and linezolid showed 2-log 10 and 1.4-log 10 kill at 16× MIC, respectively, against methicillin-resistant S. aureus (MRSA) H-29. Against S. pneumoniae 5051, RBx 11760 showed bactericidal activity, with 4.6-log 10 kill at 4× MIC compared to 2.42-log 10 and 1.95-log 10 kill for tedizolid and linezolid, respectively, at 16× MIC. RBx 11760 showed postantibiotic effects (PAE) at 3 h at 4 mg/liter against MRSA H-29, and linezolid showed the same effect at 16 mg/liter. RBx 11760 inhibited biofilm production against methicillin-resistant S. epidermidis (MRSE) ATCC 35984 in a concentration-dependent manner. In a foreign-body model, linezolid and rifampin resulted in no advantage over stasis, while the same dose of RBx 11760 demonstrated a significant killing compared to the initial control against S. aureus ( P < 0.05) and MRSE ( P < 0.01). The difference in killing was statistically significant for the lower dose of RBx 11760 ( P < 0.05) versus the higher dose of linezolid ( P > 0.05 [not significant]) in a groin abscess model. In neutropenic mouse thigh infection, RBx 11760 showed stasis at 20 mg/kg of body weight, whereas tedizolid showed the same effect at 40 mg/kg. These data support RBx 11760 as a promising investigational candidate.

Endophthalmitis due to infection with Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Given that the frequency of this condition caused by vancomycin-resistant Enterococcus faecalis has been increasing, the development of novel therapeutics is urgently required. We have demonstrated the therapeutic potential of bacteriophage ΦEF24C-P2 in a mouse model of endophthalmitis caused by vancomycin-sensitive (EF24) or vancomycin-resistant (VRE2) strains of E. faecalis. Phage ΦEF24C-P2 induced rapid and pronounced bacterial lysis in turbidity reduction assays with EF24, VRE2, and clinical isolates derived from patients with E. faecalis-related postoperative endophthalmitis. Endophthalmitis was induced in mice by injection of EF24 or VRE2 (1 × 10(4) cells) into the vitreous. The number of viable bacteria in the eye increased to >1 × 10(7) CFU, and neutrophil infiltration into the eye was detected as an increase in myeloperoxidase activity at 24 h after infection. A clinical score based on loss of visibility of the fundus as well as the number of viable bacteria and the level of myeloperoxidase activity in the eye were all significantly decreased by intravitreous injection of ΦEF24C-P2 6 h after injection of EF24 or VRE2. Whereas histopathologic analysis revealed massive infiltration of inflammatory cells and retinal detachment in vehicle-treated eyes, the number of these cells was greatly reduced and retinal structural integrity was preserved in phage-treated eyes. Our results thus suggest that intravitreous phage therapy is a potential treatment for endophthalmitis caused by vancomycin-sensitive or -resistant strains of E. faecalis.

Industrial farms are unique, human-created ecosystems that provide the perfect setting for the development and dissemination of antibiotic resistance. Agricultural antibiotic use amplifies naturally occurring resistance mechanisms from soil ecologies, promoting their spread and sharing with other bacteria, including those poised to become endemic within hospital environments. To better understand the role of enterococci in the movement of antibiotic resistance from farm to table to clinic, we characterized over 300 isolates of Enterococcus cultured from raw chicken meat purchased at U.S. supermarkets by the Consumers Union in 2013. Enterococcus faecalis and Enterococcus faecium were the predominant species found, and antimicrobial susceptibility testing uncovered striking levels of resistance to medically important antibiotic classes, particularly from classes approved by the FDA for use in animal production. While nearly all isolates were resistant to at least one drug, bacteria from meat labeled as raised without antibiotics had fewer resistances, particularly for E. faecium. Whole-genome sequencing of 92 isolates revealed that both commensal- and clinical-isolate-like enterococcal strains were associated with chicken meat, including isolates bearing important resistance-conferring elements and virulence factors. The ability of enterococci to persist in the food system positions them as vehicles to move resistance genes from the industrial farm ecosystem into more human-proximal ecologies. IMPORTANCE Bacteria that contaminate food can serve as a conduit for moving drug resistance genes from farm to table to clinic. Our results show that chicken meat-associated isolates of Enterococcus are often multidrug resistant, closely related to pathogenic lineages, and harbor worrisome virulence factors. These drug-resistant agricultural isolates could thus represent important stepping stones in the evolution of enterococci into drug-resistant human pathogens. Although significant efforts have been made over the past few years to reduce the agricultural use of antibiotics, continued assessment of agricultural practices, including the roles of processing plants, shared breeding flocks, and probiotics as sources for resistance spread, is needed in order to slow the evolution of antibiotic resistance. Because antibiotic resistance is a global problem, global policies are needed to address this threat. Additional measures must be taken to mitigate the development and spread of antibiotic resistance elements from farms to clinics throughout the world.

Enterococci are unusually well adapted for survival and persistence in a variety of adverse environments, including on inanimate surfaces in the hospital environment and at sites of infection. This intrinsic ruggedness undoubtedly played a role in providing opportunities for enterococci to interact with other overtly drug-resistant microbes and acquire additional resistances on mobile elements. The rapid rise of antimicrobial resistance among hospital-adapted enterococci has rendered hospital-acquired infections a leading therapeutic challenge. With about a quarter of a genome of additional DNA conveyed by mobile elements, there are undoubtedly many more properties that have been acquired that help enterococci persist and spread in the hospital setting and cause diseases that have yet to be defined. Much remains to be learned about these ancient and rugged microbes, particularly in the area of pathogenic mechanisms involved with human diseases.

Weekly oritavancin plus ampicillin continuous infusion combination therapy was used to successfully treat a deep spine vancomycin-resistant Enterococcus faecium infection associated with hardware. Checkerboard and time-kill assays confirmed synergy between these two antibiotics. Further synergies of oritavancin and ampicillin with rifampin or the endogenous human antimicrobial peptide cathelicidin LL-37 were demonstrated.

Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. This is particularly true in immunocompromised patients, where the damage to the microbiota caused by antibiotics can lead to VRE domination of the intestine, increasing a patient’s risk for bloodstream infection., Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. This is particularly true in immunocompromised patients, where the damage to the microbiota caused by antibiotics can lead to VRE domination of the intestine, increasing a patient’s risk for bloodstream infection. In previous studies we observed that the intestinal domination by VRE of patients hospitalized to receive allogeneic bone marrow transplantation can persist for weeks, but little is known about subspecies diversification and evolution during prolonged domination. Here we combined a longitudinal analysis of patient data and in vivo experiments to reveal previously unappreciated subspecies dynamics during VRE domination that appeared to be stable from 16S rRNA microbiota analyses. Whole-genome sequencing of isolates obtained from sequential stool samples provided by VRE-dominated patients revealed an unanticipated level of VRE population complexity that evolved over time. In experiments with ampicillin-treated mice colonized with a single CFU, VRE rapidly diversified and expanded into distinct lineages that competed for dominance. Mathematical modeling shows that in vivo evolution follows mostly a parabolic fitness landscape, where each new mutation provides diminishing returns and, in the setting of continuous ampicillin treatment, reveals a fitness advantage for mutations in penicillin-binding protein 5 (pbp5) that increase resistance to ampicillin. Our results reveal the rapid diversification of host-colonizing VRE populations, with implications for epidemiologic tracking of in-hospital VRE transmission and susceptibility to antibiotic treatment.

Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalisepa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment.

The main strategies used by pathogenic bacteria to infect eukaryotic tissue include their adherence to cells and the extracellular matrix (ECM), the subsequent colonization and invasion as well as the evasion of immune defences. A variety of structurally and functionally characterized adhesins and binding proteins of gram-positive bacteria facilitate these processes by specifically recognizing and interacting with various components of the host ECM, including different collagens, fibronectin and other macromolecules. The ECM affects the cellular physiology of our body and is critical for adhesion, migration, proliferation, and differentiation of many host cell types, but also provides the support for infiltrating pathogens, particularly under conditions of injury and trauma. Moreover, microbial binding to a variety of adhesive components in host tissue fluids leads to structural and/or functional alterations of host proteins and to the activation of cellular mechanisms that influence tissue and cell invasion of pathogens. Since the diverse interactions of gram-positive bacteria with the ECM represent important pathogenicity mechanisms, their characterization not only allows a better understanding of microbial invasion but also provides clues for the design of novel therapeutic strategies to manage infectious diseases.

We would like to focus your attention on ampicillin susceptibility testing for Enterococcus faecium infections. Ampicillin resistance in E. faecium is due to two distinct mechanisms: low-level resistance based on increased quantity of penicillin binding protein 5 (PBP5), which has low affinity for

Numerous studies have demonstrated that adequate hand hygiene among hospital staff is the best measure to prevent hand-to-hand bacterial transmission. The skin microbiome is conditioned by the individual physiological characteristics and anatomical microenvironments. Furthermore, it is important to separate the autochthonous resident microbiota from the transitory microbiota that we can acquire after interactions with contaminated surfaces. Two players participate in the hand-to-hand bacterial transmission process: the bacteria and the person. The particularities of the bacteria have been extensively studied, identifying some genera or species with higher transmission efficiency, particularly those linked to nosocomial infections and outbreaks. However, the human factor remains unstudied, and intrapersonal particularities in bacterial transmission have not been yet explored. Herein we summarize the current knowledge on hand-to-hand bacterial transmission, as well as unpublished results regarding interindividual and interindividual transmission efficiency differences. We designed a simple in vivo test based on four sequential steps of finger-to-finger contact in the same person artificially inoculated with a precise bacterial inoculum. Individuals can be grouped into one of three observed transmission categories: high, medium, and poor finger-to-finger transmitters. Categorization is relevant to predicting the ultimate success of a human transmission chain, particularly for the poor transmitters, who have the ability to cut the transmission chain. Our model allowed us to analyze transmission rate differences among five bacterial species and clones that cause nosocomial infections, from which we detected that Gram-positive microorganisms were more successfully transmitted than Gram-negative.

Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses., Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

The rise in rates of human infection caused by vancomycin-resistant Enterococcus faecium (VREfm) strains between 1988 to the 2000s in Europe was suggested to be associated with acquisition from livestock. As a result, the European Union banned the use of the glycopeptide drug avoparcin as a growth promoter in livestock feed. While some studies reported a decrease in VREfm in livestock, others reported no reduction. Here, we report the first livestock VREfm prevalence survey in the UK since 2003 and the first large-scale study using whole-genome sequencing to investigate the relationship between E. faecium strains in livestock and humans. We found a low prevalence of VREfm in retail meat and limited evidence for recent sharing of strains between livestock and humans with bloodstream infection. There was evidence for limited sharing of genes encoding antibiotic resistance between these reservoirs, a finding which requires further research., Vancomycin-resistant Enterococcus faecium (VREfm) is a major cause of nosocomial infection and is categorized as high priority by the World Health Organization global priority list of antibiotic-resistant bacteria. In the past, livestock have been proposed as a putative reservoir for drug-resistant E. faecium strains that infect humans, and isolates of the same lineage have been found in both reservoirs. We undertook cross-sectional surveys to isolate E. faecium (including VREfm) from livestock farms, retail meat, and wastewater treatment plants in the United Kingdom. More than 600 isolates from these sources were sequenced, and their relatedness and antibiotic resistance genes were compared with genomes of almost 800 E. faecium isolates from patients with bloodstream infection in the United Kingdom and Ireland. E. faecium was isolated from 28/29 farms; none of these isolates were VREfm, suggesting a decrease in VREfm prevalence since the last UK livestock survey in 2003. However, VREfm was isolated from 1% to 2% of retail meat products and was ubiquitous in wastewater treatment plants. Phylogenetic comparison demonstrated that the majority of human and livestock-related isolates were genetically distinct, although pig isolates from three farms were more genetically related to human isolates from 2001 to 2004 (minimum of 50 single-nucleotide polymorphisms [SNPs]). Analysis of accessory (variable) genes added further evidence for distinct niche adaptation. An analysis of acquired antibiotic resistance genes and their variants revealed limited sharing between humans and livestock. Our findings indicate that the majority of E. faecium strains infecting patients are largely distinct from those from livestock in this setting, with limited sharing of strains and resistance genes.

Teicoplanin is a key drug for the treatment of multiresistant staphylococcal bone and joint infections (BJI), yet can only be administered via a parenteral route. The objective of this study was to evaluate the safety and tolerability of subcutaneous (s.c.) teicoplanin for that indication over 42 days. Thirty patients with Gram-positive cocci BJI were included. Once the target of 25 to 40 mg/liter trough serum concentration was achieved, treatment was switched from an intravenous to an s.c. route. No discontinuation of teicoplanin related to injection site reaction and no severe local adverse event were observed. On multivariate analysis, better tolerability was observed at the beginning of treatment, in patients over 70 years old, and for dosages less than 600 mg. In conclusion, we recommend s.c. administration of teicoplanin when needed.

According to studies based on bacterial cultures of middle ear fluids, Streptococcus pneumoniae , Haemophilus influenzae , and Moraxella catarrhalis have been the most common pathogens in acute otitis media. However, bacterial culture can be affected by reduced viability or suboptimal growth of bacteria. PCR detects bacterial DNA from samples with greater sensitivity than culture. In the present study, we analyzed the middle ear pathogens with both conventional culture and semiquantitative real-time PCR in 90 middle ear fluid samples obtained from children aged 5 to 42 months during acute otitis media episodes. Samples were tested for the presence of S. pneumoniae , H. influenzae , M. catarrhalis , Alloiococcus otitidis , Staphylococcus aureus , and Pseudomonas aeruginosa . One or more bacterial pathogens were detected in 42 (47%) samples with culture and in 69 (77%) samples with PCR. According to PCR analysis, M. catarrhalis results were positive in 42 (47%) samples, H. influenzae in 30 (33%), S. pneumoniae in 27 (30%), A. otitidis in 6 (6.7%), S. aureus in 5 (5.6%), and P. aeruginosa in 1 (1.1%). Multibacterial etiology was seen in 34 (38%) samples, and M. catarrhalis was detected in most (85%) of those cases. Fifteen signals for M. catarrhalis were strong, suggesting a highly probable etiological role of the pathogen. In conclusion, even though M. catarrhalis is often a part of mixed flora in acute otitis media, a considerable proportion of cases may be primarily attributable to this pathogen.

Tedizolid and linezolid in vitro activities against 3,032 Gram-positive pathogens collected in Asia-Pacific, Eastern European, and Latin American medical centers during 2014 were assessed. The isolates were tested for susceptibility by the current reference broth microdilution methods. Due to concern over the effect of MIC endpoint criteria on the results of testing the oxazolidinones tedizolid and linezolid, MIC endpoint values were read by two methods: (i) reading the MIC at the first well where the trailing began without regard for pinpoint trailing, according to CLSI M07-A10 and M100-S26 document instructions for reading linezolid (i.e., 80% inhibition of growth; these reads were designated tedizolid 80 and linezolid 80), and (ii) at 100% inhibition of growth (designated tedizolid 100 and linezolid 100). All Staphylococcus aureus , Streptococcus pyogenes , Streptococcus agalactiae , Streptococcus anginosus group, and Enterococcus faecalis isolates were inhibited at tedizolid 80 and 100 MIC values of 0.25 and 0.5, 0.25 and 0.25, 0.25 and 0.5, 0.12 and 0.25, and 0.5 and 1 μg/ml, respectively. Generally, MIC 50 and MIC 90 results for tedizolid 80 and linezolid 80 were one doubling dilution lower than those read at 100% inhibition. Tedizolid was 4- to 8-fold more potent than linezolid against all the isolates tested regardless of the MIC endpoint criterion used. Despite the differences in potency, >99.9% of isolates tested in this survey were susceptible to both linezolid and tedizolid using CLSI and EUCAST interpretive criteria. In conclusion, tedizolid demonstrated greater in vitro potency than linezolid against Gram-positive pathogens isolated from patients in medical centers across the Asia-Pacific region, Eastern Europe, and Latin America.

Enterococcus faecium is an important cause of hospital-associated infections, including urinary tract infections (UTIs), bacteremia, and infective endocarditis. Pili have been shown to play a role in the pathogenesis of Gram-positive bacteria, including E. faecium . We previously demonstrated that a nonpiliated Δ empABC :: cat derivative of E. faecium TX82 was attenuated in biofilm formation and in a UTI model. Here, we studied the contributions of the individual pilus subunits EmpA, EmpB, and EmpC to pilus architecture, biofilm formation, adherence to extracellular matrix (ECM) proteins, and infection. We identified EmpA as the tip of the pili and found that deletion of empA reduced biofilm formation to the same level as deletion of the empABC operon, a phenotype that was restored by reconstituting in situ the empA gene. Deletion of empB also caused a reduction in biofilm, while EmpC was found to be dispensable. Significant reductions in adherence to fibrinogen and collagen type I were observed with deletion of empA and empB , while deletion of empC had no adherence defect. Furthermore, we showed that each deletion mutant was significantly attenuated in comparison to the isogenic parental strain, TX82, in a mixed-inoculum UTI model ( P < 0.001 to 0.048), that reconstitution of empA restored virulence in the UTI model, and that deletion of empA also resulted in attenuation in an infective endocarditis model ( P = 0.0088). Our results indicate that EmpA and EmpB, but not EmpC, contribute to biofilm and adherence to ECM proteins; however, all the Emp pilins are important for E. faecium to cause infection in the urinary tract.