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Previously, we showed that Enterococcus faecium clade B strains outcompeted health care-associated clade A1 strains in murine gastrointestinal colonization. Here, parenterally administered piperacillin-tazobactam and ceftriaxone significantly promoted colonization by clade A1 over clade B strains except that ceftriaxone, at the dose used, did not favor the least β-lactam-resistant A1 strain. The advantage that β-lactam administration gives to more highly ampicillin-resistant E. faecium over ampicillin-susceptible strains mirrors what occurs in hospitalized patients administered these antibiotics.

Serious infections owing to vancomycin-resistant enterococci have historically proven to be difficult clinical cases, requiring combination therapy and management of treatment-related toxicity. Despite the introduction of new antibiotics with activity against vancomycin-resistant enterococci to the therapeutic armamentarium, significant challenges remain. An understanding of the factors driving the emergence of resistance in vancomycin-resistant enterococci, the dynamics of gastrointestinal colonization and microbiota-mediated colonization resistance, and the mechanisms of resistance to the currently available therapeutics will permit clinicians to be better prepared to tackle these challenging hospital-associated pathogens.

In this article, the editors of Clinical Infectious Diseases review some of the most important lessons they have learned about the epidemiology, clinical features, diagnosis, treatment and prevention of SARS-CoV-2 infection and identify essential questions about COVID-19 that remain to be answered., This article reviews important lessons learned to this day, October 15, 2020, about the epidemiology, clinical virology, presentation, complications, diagnosis, treatment and prevention of SARS-CoV-2 infection and identifies essential questions about the COVID-19 pandemic that remain to be answered.

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.

Enterococcus faecalis is a significant cause of hospital-acquired bacteremia. Herein, the discovery is reported that cardiac microlesions form during severe bacteremic E. faecalis infection in mice. The cardiac microlesions were identical in appearance to those formed by Streptococcus pneumoniae during invasive pneumococcal disease. However, E. faecalis does not encode the virulence determinants implicated in pneumococcal microlesion formation. Rather, disulfide bond forming protein A (DsbA) was found to be required for E. faecalis virulence in a Caenorhabditis elegans model and was necessary for efficient cardiac microlesion formation. Furthermore, E. faecalis promoted cardiomyocyte apoptotic and necroptotic cell death at sites of microlesion formation. Additionally, loss of DsbA caused an increase in proinflammatory cytokines, unlike the wild-type strain, which suppressed the immune response. In conclusion, we establish that E. faecalis is capable of forming cardiac microlesions and identify features of both the bacterium and the host response that are mechanistically involved.

Background Infections caused by antibiotic-resistant bacteria, including carbapenem-resistant Enterobacteriaceae, have increased in frequency, resulting in significant patient morbidity and mortality. The Infectious Diseases Society of America continues to propose legislative, regulatory, and funding solutions to address this escalating crisis. This report updates the status of development and approval of systemic antibiotics in the United States as of late 2018. Methods We performed a review of the published literature and on-line clinical trials registry at www.clinicaltrials.gov to identify new systemically acting orally and/or intravenously administered antibiotic drug candidates in the development pipeline, as well as agents approved by the US Food and Drug Administration since 2012. Results Since our 2013 pipeline status report, the number of new antibiotics annually approved for marketing in the United States has reversed its previous decline, likely influenced by new financial incentives and increased regulatory flexibility. Although our survey demonstrates progress in development of new antibacterial drugs that target infections caused by resistant bacterial pathogens, the majority of recently approved agents have been modifications of existing chemical classes of antibiotics, rather than new chemical classes. Furthermore, larger pharmaceutical companies continue to abandon the field, and smaller companies face financial difficulties as a consequence. Conclusions Unfortunately, if 20 × '20 is achieved due to efforts embarked upon in decades past, it could mark the apex of antibiotic drug development for years to come. Without increased regulatory, governmental, industry, and scientific support and collaboration, durable solutions to the clinical, regulatory, and economic problems posed by bacterial multidrug resistance will not be found.

In a mouse urinary tract infection model, omadacycline (OMC) was comparable to gentamicin and better than ciprofloxacin (CIP) against a tetracycline-susceptible (TET-S), CIP-resistant (CIP-R) Escherichia coli strain. Gentamicin showed better efficacy than OMC against a TET-R, CIP-R E. coli strain, and OMC again showed better efficacy than CIP against this strain. OMC may warrant further study as a potential option for urinary tract infection treatment against CIP-R E. coli strains.

Background The cefazolin inoculum effect (CzIE), defined as Cz minimum inhibitory concentration ≥ 16 µg/ml at high inoculum (HI-MIC), has been associated with poor clinical outcomes in patients with MSSA bacteremia or osteomyelitis. The CzIE is correlated with the presence of the blaZ gene, one of the components of the bla operon encoding the BlaZ β-lactamase (type A, B, C or D). Other portions of the bla operon include blaR and blaI (encoding the antibiotic sensor and transcriptional repressor, respectively) and the intergenic region with operator and promoter sequences (Figure 1). In BlaR, residue 293 mediates signal transduction, and the Z and R dyads in the intergenic region are the DNA-binding sites for BlaI (Figure 2). Previous experiments have shown that the regulatory portions of the bla operon play a key role in the CzIE. Here, we investigated the association between the CzIE and specific variations in the regulatory sequences of the bla operon. Figure 1. Functioning of the bla operon and the production of the staphylococcal β-lactamase BlaZ. Figure 2. Structure and key regions of the intergenic region of the bla operon, incluiding the promoter and the BlaI DNA-binding regions (Z and R dyads). Methods A total of 437 MSSA containing blaZ were evaluated for the CzIE using broth microdilution at high inoculum. Using whole genome sequencing, the sequences of the bla operons were classified into cassettes based on unique changes in predicted amino acid sequences of BlaZ, BlaR and BlaI paired with specific nucleotide alterations in the intergenic region. The bla operon sequence of S. aureus ATCC29213 was used as reference (cassette 0). Results Among 437 MSSA isolates, 46% exhibited the CzIE. We identified 55 unique bla cassettes. The bla cassettes were phylogenetically grouped in 7 clusters (Figure 3) which grouped cassettes with different BlaZ types and variations in the Z dyad, the -35 box, residue 293 of BlaR, and the blaI ribosomal binding site. Each cluster had an association to the CzIE and distinct Cz HI-MICs. The combination of: a BlaZ type A, C or D, an adenine in the position -66 of blaZ (-35 box of blaZ), a cytosine in the position -22 of blaZ(Z dyad), and either an arginine or a serine in position 293 of BlaR was a very strong predictor of the CzIE (Figure 4). Figure 3. Phylogenetical organization of bla operon cassettes into clusters, their association with polymorphisms in key regulatory regions and the CzIE. GM Cz-MIC: Geometric mean of the Cefazolin MIC at high Inoculum. Figure 4. Variations of the bla operon, their association with the CzIE, their GM (Geometric Mean) of the Cefazolin MIC and the MIC distribution of the strains with each specific combination of polymorphisms. Conclusion Specific variations in regulatory portions of the bla operon, which are likely to influence BlaZ expression, are highly associated with the CzIE, supporting the notion that regulation of blaZ is the key factor responsible for the CzIE in MSSA. Disclosures Lorena Diaz, PhD , Nothing to disclose William R. Miller, MD , Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD , Entasis (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Grant/Research Support Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

El gen hyl Efm (que codifica una supuesta hialuronidasa) se ha encontrado casi exclusivamente en aislados clínicos de Enterococcus faecium y, recientemente, se ha demostrado que se encuentra en un plásmido que aumenta la capacidad de las cepas de E. faecium para colonizar el tracto gastrointestinal. En este trabajo, los resultados de los experimentos de apareamiento entre cepas de E. faecium que contienen hylz Efm pertenecientes al grupo clonal 17 y aisladas en los Estados Unidos y Colombia indicaron que el hyl EfmEl gen de estas cepas también se transporta en plásmidos grandes (> 145 kb) que mostramos que se transfieren fácilmente de cepas clínicas a huéspedes de E. faecium. También se observó cotransferencia de resistencia a vancomicina y resistencia de alto nivel (HLR) a aminoglucósidos (gentamicina y estreptomicina) y eritromicina. El grupo de genes vanA y los determinantes de resistencia a la gentamicina estaban genéticamente vinculados a hyl Efm , mientras que erm (B) y ant (6) -I, que confieren resistencia a macrólido-lincosamida-estreptogramina B y HLR a estreptomicina, respectivamente, no lo estaban. Un hyl EfmEl transconjugante positivo resultante de un apareamiento entre una cepa de endocarditis bien caracterizada [TX0016 (DO)] y un derivado de una cepa fecal de E. faecium de un voluntario humano sano (TX1330RF) exhibió una mayor virulencia en un modelo de peritonitis de ratón. Estos resultados indican que las cepas de E. faecium utilizan una estrategia que implica el reclutamiento en la misma unidad genética de genes de resistencia a antibióticos y determinantes que aumentan la capacidad de producir enfermedades. Nuestros hallazgos indican que la adquisición de los plásmidos hyl Efm puede explicar, al menos en parte, la reciente aparición exitosa de algunas cepas de E. faecium como patógenos nosocomiales. The hylEfm gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hylzEfm-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hylEfm gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hylEfm, whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hylEfm-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hylEfm plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-β-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin America and the United States. The cefazolin MIC determination at a high inoculum (10(7) CFU/ml) was used as a reference standard (cutoff ≥16 μg/ml). All isolates underwent genome sequencing. A total of 257 (37.3%) of MSSA isolates exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (the most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 μg/ml, the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ. The overall negative predictive value ranged from 81.4% to 95.6% in Latin American countries using published prevalence rates of the CzIE. MSSA isolates from the United States were genetically diverse, with no distinguishing genomic differences from Latin American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high inoculum, the rapid test is inexpensive, feasible, and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

Staphylococcus aureus strain TX0117 is a methicillin-susceptible bacterium with type A beta-lactamase exhibiting a high cefazolin inoculum effect. TX0117 was cured of blaZ, yielding TX0117c with increased antimicrobial peptide resistance. The sequencing and genome assembly of TX0117 elucidate six mutations between TX0117 and TX0117c, including relA truncation and mnA_1 substitution.

Enterococci, such as Enterococcus faecalis, are a significant cause of hospital-acquired bacteremia and mortality among the elderly and patients with comorbid conditions in the healthcare setting. In this study, the novel discovery is made that cardiac microlesions form during severe bacteremic E. faecalis infection in mice. The cardiac microlesions were identical in appearance to those previously observed to form by Streptococcus pneumoniae during invasive pneumococcal disease (IPD) in mice, non-human primates, and humans. However, E. faecalis does not encode the virulence determinants implicated in pneumococcal microlesion formation, suggesting that formation is through an alternative pathway. In previous work, we showed that E. faecalis DsbA is required for the post-translational disulfide bond formation and stability of EntV, a secreted E. faecalis bacteriocin. Here, the role of DsbA in E. faecalis pathogenicity was explored. DsbA was discovered to be required for E. faecalis virulence in a C. elegans model and, importantly, was necessary for efficient cardiac microlesion formation. By investigating the cardiac host response, it was discovered that E. faecalis promotes cardiomyocyte necroptotic cell death at sites of microlesion formation. Additionally, by examining the cytokines induced by exposure of E. faecalis to cardiomyocytes, it was revealed that loss of DsbA caused a pro-inflammatory response unlike the wild type strain, which suppressed the immune response. In conclusion, we establish that E. faecalis is capable of forming cardiac microlesions and identify features of both the bacterial and host responses that are mechanistically involved. Significance Statement E. faecalis is a bacterium capable of causing serious disease in humans, including endocarditis, an infection on the surface of the heart valves with a high mortality rate. In this work, we document for the first time that E. faecalis is capable of also causing an invasive infection of the heart, forming cardiac microlesions in a mouse model of severe bloodstream infection. Additionally, a contributing bacterial factor is identified and the immune response of heart cells to infection with E. faecalis is characterized. Overall, this work makes a significant contribution to the field of infectious diseases by identifying a new enterococcal disease pathology, cardiac microlesion formation, which was previously known to occur only with IPD.

Little is known about the population structure of vancomycin-resistant Enterococcus faecium (VREfm) in Latin America (LATAM). Here, we provide a complete genomic characterization of 55 representative Latin American VREfm recovered from 1998–2015 in 5 countries. The LATAM VREfm population is structured into two main clinical clades without geographical clustering. Using the LATAM genomes, we reconstructed the global population of VREfm by including 285 genomes from 36 countries spanning from 1946 to 2017. In contrast to previous studies, our results show an early branching of animal related isolates and a further split of clinical isolates into two sub-clades within clade A. The overall phylogenomic structure of clade A was highly dependent on recombination (54% of the genome) and the split between clades A and B was estimated to have occurred more than 2,765 years ago. Furthermore, our molecular clock calculations suggest the branching of animal isolates and clinical clades occurred ~502 years ago whereas the split within the clinical clade occurred ~302 years ago (previous studies showed a more recent split between clinical an animal branches around ~74 years ago). By including isolates from Latin America, we present novel insights into the population structure of VREfm and revisit the evolution of these pathogens.

Background Cefazolin (Cz) is commonly used to treat methicillin-sensitive Staphylococcus aureus (MSSA) bacteremia. Yet, some MSSA isolates producing the staphylococcal β-lactamase (BlaZ) exhibit the Cz inoculum effect (CzIE), defined as an increase in the minimum inhibitory concentration (MIC) to ≥ 16 µg/mL at high inoculum (107 CFU/mL, HI-MIC). Retrospective clinical data linked the CzIE to increased 30-day mortality and Cz treatment failure in patients with MSSA bacteremia, yet the mechanistic bases of this phenomenon are unknown. We aimed to explore the contribution of blaZ regulation, via BlaR (antibiotic sensor) and BlaI (transcriptional repressor) (Fig 1) to the CzIE by i) in trans expression assays and ii) analysis of their sequences in a set of isolates Figure 1. Structure of the Staphylococcal bla Operon Methods The blaZ genes (with putative promoters) of strains exhibiting and lacking the CzIE (TX0117 and ATCC29213, respectively) were expressed in trans in RN4220 (blaZ neg) using the promotor-less vector pWM401 (Figure 2). We subsequently cloned the blaR and blaI genes of each TX0117 and ATCC29213 upstream of each blaZ allele (Figure 3). The presence of the CzIE was assessed in transformants using broth microdilution at standard (105 CFU/mL, SI-MIC) and high inoculum. We also performed whole-genome sequencing (WGS) in 104 MSSA isolates exhibiting and lacking the CzIE to compare the sequences of BlaZ, BlaR, and BlaI and classified them by allotypes (unique amino acid sequences) using ATCC29213 as reference. Figure 2. In trans expression of blaZ genes from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220 Figure 3. In trans expression of the bla Operons from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220 Results Expression of blaZTX0117 and blaZATCC29213 with their native promoters in RN4220 resulted in the CzIE with Cz HI-MICs ≥ 64 µg/mL regardless of the origin of the allele (Table 1). Inclusion of the regulatory elements blaR and blaI from TX0117 (CzIE+) did not change the phenotype. In contrast, addition of blaR and blaI from ATCC29213 (CzIE-) led to a marked decrease in the Cz HI-MIC (Table 1). Sequence analyses of 104 MSSA isolates revealed 10, 17 and 6 BlaZ, BlaR and BlaI allotypes, respectively (Table 2). BlaZ-2 and BlaR-4 were linked to the CzIE in 90% of isolates. Table 1. MIC values of transformans after In trans expression of blaZ genes and bla Operons from a S. aureus CzIE+ strain(TX0117) and a CzIE- strain (ATCC29213) in RN4220 Table 2. BlaZ allotypes of 104 Staphylococcus aureus isolates and their association with the CzIE Conclusion Our results suggest that overexpression of blaZ can lead to the CzIE in any MSSA strain. Thus, the regulation of blaZ expression via BlaR and BlaI seem to play a major role in the CzIE. Identification of specific BlaR and BlaI allotypes could predict the presence of the CzIE. Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

Tedizolid (TZD) and daptomycin (DAP) were assessed in a rat endocarditis model against Enterococcus faecalis , Enterococcus faecium (resistant to vancomycin and ampicillin), and Staphylococcus aureus . As a monotherapy, TZD for 5 days was not effective in a comparison with no-treatment controls, while DAP for 5 days was significantly effective against these bacteria. Step-down therapy (DAP for 3 days followed by TZD for 2 days) was as effective as DAP for 5 days and was comparable to 3 days of DAP plus ceftriaxone against all bacteria and to 3 days of DAP plus gentamicin against E. faecalis OG1RF.

The prevalence of vancomycin-resistantEnterococcus faeciumvaries across geographical regions yet little is known about its population structure in Latin America. Here, we provide a complete genomic characterization of 55 representative Latin American VREfmrecovered from 1998-2015 in 5 countries. We found that VREfmpopulation in the region is structured into two main clinical clades without geographical clustering. To place our regional findings in context, we reconstructed the global population structure of VREfmby including 285 genomes from 36 countries from 1946-2017. Our results differ from previous studies showing an early branching of animal related isolates and a further split of clinical isolates into two sub-clades, all within clade A. The overall phylogenomic structure was highly dependent on recombination (54% of the genome) and the split between clades A and B is estimated to have occurred more than 3585 years BP. Furthermore, while the branching of animal isolates and clinical clades was predicted to have occur ∼894 years BP, our molecular clock calculations suggest that the split within the clinical clade occurred around ∼371 years BP. By including isolates from Latin America, we present novel insights into the population structure of VREfmand revisit the evolution of this pathogen.

Enterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge., Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, are common constituents of the microbiome as well as increasingly problematic causes of infections. Interestingly, we previously showed that these two species antagonize each other’s virulence and that E. faecalis inhibition of C. albicans was specifically mediated by EntV. EntV is a bacteriocin encoded by the entV (ef1097) locus that reduces C. albicans virulence and biofilm formation by inhibiting hyphal morphogenesis. In this report, we studied the posttranslational modifications necessary for EntV antifungal activity. First, we show that the E. faecalis secreted enzyme gelatinase (GelE) is responsible for cleaving EntV into its 68-amino-acid, active form and that this process does not require the serine protease SprE. Furthermore, we demonstrate that a disulfide bond that forms within EntV is necessary for antifungal activity. Abrogating this bond by chemical treatment or genetic modification rendered EntV inactive against C. albicans. Moreover, we identified the likely catalyst of this disulfide bond, a previously uncharacterized thioredoxin within the E. faecalis genome called DsbA. Loss of DsbA, or disruption of its redox-active cysteines, resulted in loss of EntV antifungal activity. Finally, we show that disulfide bond formation is not a prerequisite for cleavage; EntV cleavage proceeded normally in the absence of DsbA. In conclusion, we present a model in which following secretion, EntV undergoes disulfide bond formation by DsbA and cleavage by GelE in order to generate a peptide capable of inhibiting C. albicans.

Cefazolin and ertapenem combination therapy was used successfully to salvage 11 cases (6 endocarditis) of persistent methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, including immediate clearance (≤24 hours) in 8 cases. While in vitro synergy was modest, cefazolin plus ertapenem exhibited synergistic action in a rat model of MSSA endocarditis. The combination of cefazolin and ertapenem provides potent in vivo activity against MSSA beyond what is predicted in vitro and warrants further clinical study in the treatment of refractory MSSA bacteremia and endocarditis.

In a mouse peritonitis model, tedizolid was comparable to linezolid and daptomycin against an Enterococcus faecium strain (VAN r , AMP r ), an Enterococcus faecalis strain, and a methicillin-resistant Staphylococcus aureus (MRSA) strain with and without cfr . Against a cfr (B) + E. faecium , tedizolid was inferior in vivo to linezolid and daptomycin, despite an ∼4-fold lower MIC.

Vancomycin-resistant enterococci are serious health threats due in part to their ability to persist in rugged environments and their propensity to acquire antibiotic resistance determinants. Enterococci have now established a home in our hospitals and possess mechanisms to defeat most currently available antimicrobials. This article reviews the history of the struggle with this pathogen, what is known about the traits associated with its rise in the modern medical environment, and the current understanding of therapeutic approaches in severe infections caused by these microorganisms. As the 21st century progresses, vancomycin-resistant enterococci continue to pose a daunting clinical challenge.

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.

Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of β-methyl-D-glucoside. β-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis.

In a mouse peritonitis model, tedizolid was comparable to linezolid and daptomycin against an Enterococcus faecium strain (VAN(r), AMP(r)), an Enterococcus faecalis strain, and a methicillin-resistant Staphylococcus aureus (MRSA) strain with and without cfr. Against a cfr(B)(+) E. faecium, tedizolid was inferior in vivo to linezolid and daptomycin, despite an ∼4-fold lower MIC.

Ace (Adhesin to collagen from Enterococcus faecalis) is a cell-wall anchored protein that is expressed conditionally and is important for virulence in a rat infective endocarditis (IE) model. Previously, we showed that rats immunized with the collagen binding domain of Ace (domain A), or administered anti-Ace domain A polyclonal antibody, were less susceptible to E. faecalis endocarditis than sham-immunized controls. In this work, we demonstrated that a sub nanomolar monoclonal antibody (mAb), anti-Ace mAb70, significantly diminished E. faecalis binding to ECM collagen IV in in vitro adherence assays and that, in the endocarditis model, anti-Ace mAb70 pre-treatment significantly reduced E. faecalis infection of aortic valves. The effectiveness of anti-Ace mAb against IE in the rat model suggests it might serve as a beneficial agent for passive protection against E. faecalis infections.

We tested the ability of clavulanic acid to restore the efficacy of cefazolin against Staphylococcus aureus TX0117, which exhibits the cefazolin inoculum effect (CzIE). In the rat infective endocarditis model, the coadministration of cefazolin plus clavulanic acid resulted in a significant reduction of bacterial counts (7.1 ± 0.5 log 10 CFU/g) compared to that with cefazolin alone (2 ± 0.6 log 10 CFU/g; P < 0.0001).

Enterococcus faecium isolates that harbor LiaFSR substitutions but are phenotypically susceptible to daptomycin (DAP) by current breakpoints are problematic, since predisposition to resistance may lead to therapeutic failure. Using a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model, we investigated DAP regimens (6, 8, and 10 mg/kg of body weight/day) as monotherapy and in combination with ampicillin (AMP), ceftaroline (CPT), or ertapenem (ERT) against E. faecium HOU503, a DAP-susceptible strain that harbors common LiaS and LiaR substitutions found in clinical isolates (T120S and W73C, respectively). Of interest, the efficacy of DAP monotherapy, at any dose regimen, was dependent on the size of the inoculum. At an inoculum of ∼109 CFU/g, DAP doses of 6 to 8 mg/kg/day were not effective and led to significant regrowth with emergence of resistant derivatives. In contrast, at an inoculum of ∼107 CFU/g, marked reductions in bacterial counts were observed with DAP at 6 mg/kg/day, with no resistance. The inoculum effect was confirmed in a rat model using humanized DAP exposures. Combinations of DAP with AMP, CPT, or ERT demonstrated enhanced eradication and reduced potential for resistance, allowing de-escalation of the DAP dose. Persistence of the LiaRS substitutions was identified in DAP-resistant isolates recovered from the SEV model and in DAP-resistant derivatives of an initially DAP-susceptible clinical isolate of E. faecium (HOU668) harboring LiaSR substitutions that was recovered from a patient with a recurrent bloodstream infection. Our results provide novel data for the use of DAP monotherapy and combinations for recalcitrant E. faecium infections and pave the way for testing these approaches in humans.

Enterococcus faecalis is paradoxically a dangerous nosocomial pathogen and a normal constituent of the human gut microbiome, an environment rich in ethanolamine. E. faecalis carries the eut (ethanolamine utilization) genes, which enable the catabolism of ethanolamine (EA) as a valuable source of carbon and/or nitrogen. EA catabolism was previously shown to contribute to the colonization and growth of enteric pathogens, such as Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli (EHEC), in the gut environment. We tested the ability of eut mutants of E. faecalis to colonize the gut using a murine model of gastrointestinal (GI) tract competition and report the surprising observation that these mutants outcompete the wild-type strain., IMPORTANCE Some bacteria that are normal, harmless colonizers of the human body can cause disease in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote or negate these organisms’ ability to colonize. Previously, ethanolamine, found in high concentrations in the GI tract, was shown to promote the colonization and growth of bacteria associated with food poisoning. Here, we report the surprising, opposite effect of ethanolamine utilization on the commensal colonizer E. faecalis, namely, that loss of this metabolic capacity made it a better colonizer.

Background Suboptimal therapy against methicillin-sensitive Staphylococcus aureus (MSSA) may have catastrophic consequences in severe infections such as endocarditis or epidural abscess. High MSSA inocula have been associated with clinical failure in patients receiving cefazolin (CZ), particularly when used at low doses, associated with a CZ inoculum effect. We previously described that adding ertapenem (ETP) to CZ led to synergism against MSSA and sensitized the pathogen to host innate immune factors. Here we expand our experience with CZ plus ETP as salvage therapy for 11 cases of refractory MSSA bacteremia (lacking source control problems) and explore CZ+ETP combination in vitro and in vivo. Methods Six available MSSA strains from patients treated with CZ+ETP for refractory bacteremia were tested in Mueller–Hinton Broth or RPMI media at standard (105 CFU/mL) or high (107 CFU/mL) inocula by MIC, checkerboard, and time-kill assays using ETP, CZ or nafcillin (NAF) alone vs. ETP+NAF or ETP+CZ. Disk diffusion synergy assays between CZ and ETP were also performed. CZ, ETP and CZ+ETP were tested in a rat endocarditis model using well described MSSA, TX0117 and TX0117c. Results 11 consecutive patients with MSSA bacteremia (6 confirmed endocarditis) refractory to standard CZ or NAF rapidly cleared with CZ+ETP. 9 patients had daily positive blood cultures, and 8 cleared in ≤24 hr, including those with ≥2 cm vegetations. All 11 survived hospitalization. In MHB, 3/6 MSSA exhibited a CZ inoculum effect (CZ MIC >3 log2 in 107 vs. 105 CFU/mL), but only 1 showed a significant CZ inoculum effect in RPMI. CZ+ETP was significantly more efficacious than CZ in a rat model of MSSA endocarditis utilizing a strain displaying a CZ inoculum effect, despite only modest benefit observed in vitro for 6 MSSA isolates. Conclusion CZ+ETP combination therapy yielded profound clinical success in severe MSSA infections with high bacterial densities, as demonstrated by rapid bacteremia clearance. Enhanced efficacy was also observed in a rat endocarditis model. The anti-staphylococcal activity of CZ+ETP in vivo exceeded that observed in vitro, consistent with our prior observations of host innate immune cooperativity with the regimen. CZ+ETP warrants further study for the treatment of refractory MSSA bacteremia and endocarditis. Disclosures All authors: No reported disclosures.

Colonization of the gastrointestinal tract (GIT) generally precedes infection with antibiotic-resistant Enterococcus faecium We used a mouse GIT colonization model to test differences in the colonization levels by strains from different E. faecium lineages: clade B, part of the healthy human microbiota; subclade A1, associated with infections; and subclade A2, primarily associated with animals. After mono-inoculation, there was no significant difference in colonization (measured as the geometric mean number of colony-forming units per gram) by the E. faecium clades at any time point (P > .05). However, in competition assays, with 6 of the 7 pairs, clade B strains outcompeted clade A strains in their ability to persist in the GIT; this difference was significant in some pairs by day 2 and in all pairs by day 14 (P < .0008-.0283). This observation may explain the predominance of clade B in the community and why antibiotic-resistant hospital-associated E. faecium are often replaced by clade B strains once patients leave the hospital.

EfbA is a PavA-like fibronectin adhesin of Enterococcus faecalis previously shown to be important in experimental urinary tract infection. Here, we expressed and purified the E. faecalis OG1RF EfbA and confirmed that this protein binds with high affinity to immobilized fibronectin, collagen I, and collagen V. We constructed an efbA deletion mutant and demonstrated that its virulence was significantly attenuated ( P < 0.0006) versus the wild type in a mixed inoculum rat endocarditis model. Furthermore, efbA deletion resulted in diminished ability to bind fibronectin ( P < 0.0001) and reduced biofilm ( P < 0.001). Reintroduction of efbA into the original chromosomal location restored virulence, adherence to fibronectin, and biofilm formation to wild-type levels. Finally, vaccination of rats with purified recombinant EfbA protein protected against OG1RF endocarditis ( P = 0.008 versus control). Taken together, our results demonstrate that EfbA is an important factor involved in E. faecalis endocarditis and that rEfbA immunization is effective in preventing such infection, likely by interfering with bacterial adherence.

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species.

By integrating the microarray expression data and a global E. faecalis transcriptional network we identified a sub-network activated by zinc and copper. Our analyses indicated that the transcriptional response of the bacterium to copper and zinc exposure involved the activation of two modules, module I that contains genes implicated in zinc homeostasis, including the Zur transcriptional repressor, and module II containing a set of genes associated with general stress response and basal metabolism. Bacterial exposure to zinc and copper led to the repression of the zinc uptake systems of module I. Upon deletion of Zur, exposure to different zinc and copper conditions induced complementary homeostatic mechanisms (ATPase efflux proteins) to control the intracellular concentrations of zinc. The transcriptional activation of zinc homeostasis genes by zinc and copper reveals a functional interplay between these two metals, in which exposure to copper also impacts on the zinc homeostasis. Finally, we present a new zinc homeostasis model in E. faecalis, positioning this bacterium as one of the most complete systems biology model in metals described to date.

Expression of ace ( a dhesin to c ollagen of E nterococcus faecalis ), encoding a virulence factor in endocarditis and urinary tract infection models, has been shown to increase under certain conditions, such as in the presence of serum, bile salts, urine, and collagen and at 46°C. However, the mechanism of ace /Ace regulation under different conditions is still unknown. In this study, we identified a two-component regulatory system GrvRS as the main regulator of ace expression under these stress conditions. Using Northern hybridization and β-galactosidase assays of an ace promoter- lacZ fusion, we found transcription of ace to be virtually absent in a grvR deletion mutant under the conditions that increase ace expression in wild-type OG1RF and in the complemented strain. Moreover, a grvR mutant revealed decreased collagen binding and biofilm formation as well as attenuation in a murine urinary tract infection model. Here we show that GrvR plays a major role in control of ace expression and E. faecalis virulence.

Increasing resistance to antimicrobial agents has led to international pledges to increase funding for public health initiatives as well as for research and development to combat this grave threat....

Certain Staphylococcus aureus strains exhibit an inoculum effect (InE) with cefazolin (CFZ) that has been associated with therapeutic failures in high-inoculum infections. We assessed the i n vitro activities of ceftaroline (CPT), CFZ, and nafcillin (NAF) against 17 type A β-lactamase (βla)-producing, methicillin-susceptible S. aureus (MSSA) strains, including the previously reported TX0117, which exhibits the CFZ InE, and its βla-cured derivative, TX0117c. Additionally, we determined the pharmacokinetics of CPT in rats after single intramuscular doses of 20 and 40 mg/kg of body weight and evaluated the activities of CPT (40 mg/kg every 8 h [q8h]), CFZ, and NAF against TX0117 and TX0117c in a rat model of infective endocarditis. No InE was observed for CPT or NAF, whereas a marked InE was detected for CFZ (MIC, 8 to ≥128 μg/ml). CPT and NAF treatment against TX0117 resulted in mean bacterial counts of 2.3 and 2.1 log 10 CFU/g in vegetations, respectively, compared to a mean of 5.9 log 10 CFU/g in the CFZ-treated group (CPT and NAF versus CFZ, P = 0.001; CPT versus NAF, P = 0.9830). Both CFZ and CPT were efficacious against the βla-cured derivative, TX0117c, compared to time zero ( t 0 ) ( P = in vivo consequences of the CFZ InE and show that CPT is not affected by this phenomenon. CPT might be considered for high-inoculum infections caused by MSSA exhibiting the CFZ InE.

Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of β-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of β-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and β-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications., Author summary Colorectal cancer (CRC) is a leading cause of cancer-related death. The recognition that microbial agents can contribute to the development of CRC raises hope for improving CRC diagnosis and treatment by incorporating both microbial and patient characteristics into clinical strategies. S. gallolyticus subsp. gallolyticus (Sg) has been implicated in CRC for decades. Patients with Sg infections display a much higher risk of having CRC compared to the general population. Despite this, the precise role of Sg in the development of CRC—i.e., whether this organism plays an active role in the development of tumor or its presence is merely a consequence of the tumor environment being favorable for its colonization of the colon—was unknown. Here using in vitro cell cultures and mouse models of CRC, we demonstrate that Sg actively promotes colon cancer cell proliferation and tumor growth, suggesting that it is not an innocent “passenger”. These results represent a major step forward in understanding the relationship between Sg and CRC. This combined with the prevalence of Sg in CRC patients highlight Sg as being both clinically relevant and functionally important for CRC.

Despite progress in antimicrobial drug development, a critical need persists for new, feasible pathways to develop antibacterial agents to treat people infected with drug-resistant bacteria. Infections due to resistant gram-negative bacilli continue to cause unacceptable morbidity and mortality rates. Antibacterial agents have been historically studied in noninferiority clinical trials that focus on a single site of infection (eg, complicated urinary tract infections, intra-abdominal infections), yet these designs may not be optimal, and often are not feasible, for study of infections caused by drug-resistant bacteria. Over the past several years, multiple stakeholders have worked to develop consensus regarding paths forward with a goal of facilitating timely conduct of antimicrobial development. Here we advocate for a novel and pragmatic approach and, toward this end, present feasible trial designs for antibacterial agents that could enable conduct of narrow-spectrum, organism-specific clinical trials and ultimately approval of critically needed new antibacterial agents.