Genomic context as well as sequence of both psr and penicillin-binding protein 5 contributes to ß-lactam resistance in Enterococcus faecium.

Penicillin-binding protein 5 (PBP5) of Enterococcus faecium (Efm) is vital for ampicillin resistance (AMP-R). We previously designated three forms of PBP5, namely, PBP5-S in Efm clade B strains [ampicillin susceptible (AMP-S)], PBP5-S/R (AMP-S or R), and PBP5-R (AMP-R) in clade A strains. Here, pbp5 deletion resulted in a marked reduction in AMP minimum inhibitory concentrations (MICs) to 0.01-0.09 µg/mL for clade B and 0.12-0.19 µg/mL for clade A strains; in situ complementation restored parental AMP MICs. Using D344SRF (lacking ftsW/psr/pbp5), constructs with ftsWA/psrA (from a clade A1 strain) cloned upstream of pbp5-S and pbp5-S/R alleles resulted in modest increases in MICs to 3-8 µg/mL, while high MICs (>64 µg/mL) were seen using pbp5 from A1 strains. Next, using ftsW ± psr from clade B and clade A/B and B/A hybrid constructs, the presence of psrB, even alone or in trans, resulted in much lower AMP MICs (3-8 µg/mL) than when psrA was present (MICs >64 µg/mL). qRT PCR showed relatively greater pbp5 expression (P = 0.007) with pbp5 cloned downstream of clade A1 ftsW/psr (MIC >128 µg/mL) vs when cloned downstream of clade B ftsW/psr (MIC 4-16 µg/mL), consistent with results in western blots. In conclusion, we report the effect of clade A vs B psr on AMP MICs as well as the impact of pbp5 alleles from different clades. While previously, Psr was not thought to contribute to AMP MICs in Efm, our results showed that the presence of psrB resulted in a major decrease in Efm AMP MICs. IMPORTANCE: The findings of this study shed light on ampicillin resistance in Enterococcus faecium clade A strains. They underscore the significance of alterations in the amino acid sequence of penicillin-binding protein 5 (PBP5) and the pivotal role of the psr region in PBP5 expression and ampicillin resistance. Notably, the presence of a full-length psrB leads to reduced PBP5 expression and lower minimum inhibitory concentrations (MICs) of ampicillin compared to the presence of a shorter psrA, regardless of the pbp5 allele involved. Additionally, clade B E. faecium strains exhibit lower AMP MICs when both psr alleles from clades A and B are present, although it is important to consider other distinctions between clade A and B strains that may contribute to this effect. It is intriguing to note that the divergence between clade A and clade B E. faecium and the subsequent evolution of heightened AMP MICs in hospital-associated strains appear to coincide with changes in Pbp5 and psr. These changes in psr may have resulted in an inactive Psr, facilitating increased PBP5 expression and greater ampicillin resistance. These results raise the possibility that a mimicker of PsrB, if one could be designed, might be able to lower MICs of ampicillin-resistant E. faecium, thus potentially resorting ampicillin to our therapeutic armamentarium for this species.

Priorities and Progress in Gram-positive Bacterial Infection Research by the Antibacterial Resistance Leadership Group: A Narrative Review.

The Antibacterial Resistance Leadership Group (ARLG) has prioritized infections caused by gram-positive bacteria as one of its core areas of emphasis. The ARLG Gram-positive Committee has focused on studies responding to 3 main identified research priorities: (1) investigation of strategies or therapies for infections predominantly caused by gram-positive bacteria, (2) evaluation of the efficacy of novel agents for infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci, and (3) optimization of dosing and duration of antimicrobial agents for gram-positive infections. Herein, we summarize ARLG accomplishments in gram-positive bacterial infection research, including studies aiming to (1) inform optimal vancomycin dosing, (2) determine the role of dalbavancin in MRSA bloodstream infection, (3) characterize enterococcal bloodstream infections, (4) demonstrate the benefits of short-course therapy for pediatric community-acquired pneumonia, (5) develop quality of life measures for use in clinical trials, and (6) advance understanding of the microbiome. Future studies will incorporate innovative methodologies with a focus on interventional clinical trials that have the potential to change clinical practice for difficult-to-treat infections, such as MRSA bloodstream infections.

Enterococcus faecium Clade Competition in the Presence of ß-Lactam Antibiotics in a Mouse GI Tract Colonization Model.

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 ß-lactam-resistant A1 strain. The advantage that ß-lactam administration gives to more highly ampicillin-resistant E. faecium over ampicillin-susceptible strains mirrors what occurs in hospitalized patients administered these antibiotics.

Efficacy of Omadacycline against Multidrug-Resistant Enterococcus faecium Strains in a Mouse Peritonitis Model.

Omadacycline (OMC) showed better in vitro potency than daptomycin (DAP) or vancomycin (VAN) against Vanr, Ampr, DAP-nonsusceptible, linezolid-resistant, cfr(B)+ Enterococcus faecium strains. In a mouse peritonitis model, OMC also showed significantly better animal survival during the study and at its end than DAP or VAN with these E. faecium strains. However, OMC, DAP, and VAN showed comparable in vitro and in vivo efficacies against a non-vancomycin-resistant, tetracycline-resistant, DAP-susceptible E. faecium strain.

Efficacy of Omadacycline against Escherichia coli in a Mouse Urinary Tract Infection Model.

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.

Development and Characterization of High-Throughput Caenorhabditis elegans - Enterococcus faecium Infection Model.

The genus Enterococcus includes two Gram-positive pathogens of particular clinical relevance: E. faecalis and E. faecium. Infections with each of these pathogens are becoming more frequent, particularly in the case of hospital-acquired infections. Like most other bacterial species of clinical importance, antimicrobial resistance (and, specifically, multi-drug resistance) is an increasing threat, with both species considered to be of particular importance by the World Health Organization and the US Centers for Disease Control. The threat of antimicrobial resistance is exacerbated by the staggering difference in the speeds of development for the discovery and development of the antimicrobials versus resistance mechanisms. In the search for alternative strategies, modulation of host-pathogen interactions in general, and virulence inhibition in particular, have drawn substantial attention. Unfortunately, these approaches require a fairly comprehensive understanding of virulence determinants. This requirement is complicated by the fact that enterococcal infection models generally require vertebrates, making them slow, expensive, and ethically problematic, particularly when considering the thousands of animals that would be needed for the early stages of experimentation. To address this problem, we developed the first high-throughput C. elegans-E. faecium infection model involving host death. Importantly, this model recapitulates many key aspects of murine peritonitis models, including utilizing similar virulence determinants. Additionally, host death is independent of peroxide production, unlike other E. faecium-C. elegans virulence models, which allows the assessment of other virulence factors. Using this system, we analyzed a panel of lab strains with deletions of targeted virulence factors. Although removal of certain virulence factors (e.g., Δfms15) was sufficient to affect virulence, multiple deletions were generally required to affect pathogenesis, suggesting that host-pathogen interactions are multifactorial. These data were corroborated by genomic analysis of selected isolates with high and low levels of virulence. We anticipate that this platform will be useful for identifying new treatments for E. faecium infection.

A Test for the Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus.

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-ß-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 (107 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.

Cardiac Microlesions Form During Severe Bacteremic Enterococcus faecalis Infection.

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.

COVID-19-Lessons Learned and Questions Remaining.

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.

Resistance in Vancomycin-Resistant Enterococci.

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.

Cryo-electron microscopy structure of the 70S ribosome from Enterococcus faecalis.

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.

Mechanistic Insights Into the Differential Efficacy of Daptomycin Plus ß-Lactam Combinations Against Daptomycin-Resistant Enterococcus faecium.

BACKGROUND: The combination of daptomycin (DAP) plus ampicillin (AMP), ertapenem (ERT), or ceftaroline has been demonstrated to be efficacious against a DAP-tolerant Enterococcus faecium strain (HOU503). However, the mechanism for the efficacy of these combinations against DAP-resistant (DAP-R) E. faecium strains is unknown. METHODS: We investigated the efficacy of DAP in combination with AMP, ERT, ceftaroline, ceftriaxone, or amoxicillin against DAP-R E. faecium R497 using established in vitro and in vivo models. We evaluated pbp expression, levels of penicillin-binding protein (PBP) 5 (PBP5) and ß-lactam binding affinity in HOU503 versus R497. RESULTS: DAP plus AMP was the only efficacious regimen against DAP-R R497 and prevented emergence of resistance. DAP at 8, 6, and 4 mg/kg in combination with AMP was efficacious but showed delayed killing compared with 10 mg/kg. PBP5 of HOU503 exhibited amino acid substitutions in the penicillin-binding domain relative to R497. No difference in pbp mRNA or PBP5 levels was detected between HOU503 and R497. labeling of PBPs with Bocillin FL, a fluorescent penicillin derivative, showed increased ß-lactam binding affinity of PBP5 of HOU503 compared with that of R497. CONCLUSIONS: Only DAP (10 mg/kg) plus AMP or amoxicillin was efficacious against a DAP-R E. faecium strain, and pbp5 alleles may be important contributors to efficacy of DAP plus ß-lactam therapy.

Genome Sequence Comparison of Staphylococcus aureus TX0117 and a Beta-Lactamase-Cured Derivative Shows Increased Cationic Peptide Resistance Accompanying Mutations in relA and mnaA.

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.

Tedizolid as Step-Down Therapy following Daptomycin versus Continuation of Daptomycin against Enterococci and Methicillin- and Vancomycin-Resistant Staphylococcus aureus in a Rat Endocarditis Model.

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.

Genomic Epidemiology of Vancomycin-Resistant Enterococcus faecium (VREfm) in Latin America: Revisiting The Global VRE Population Structure.

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.

Cefazolin and Ertapenem Salvage Therapy Rapidly Clears Persistent Methicillin-Susceptible Staphylococcus aureus Bacteremia.

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.

Antifungal Activity of the Enterococcus faecalis Peptide EntV Requires Protease Cleavage and Disulfide Bond Formation.

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. albicansIMPORTANCEEnterococcus 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.

The Infectious Diseases Society of America's 10 × '20 Initiative (10 New Systemic Antibacterial Agents US Food and Drug Administration Approved by 2020): Is 20 × '20 a Possibility?

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.