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Over the past 2 decades, the bacterial species that cause community-acquired pneumonia have remained relatively constant, but their susceptibilities to antimicrobial agents have changed considerably. In particular, important changes in the susceptibility of Streptococcus pneumoniae and Staphylococcus aureus are influencing prescribing practices. Resistance to beta-lactam and macrolide antibiotics has been associated with a shift toward the use of fluoroquinolones, despite insufficient evidence that such resistance affects clinical outcomes. In a rare reversal of resistance trends, the widespread use of the 7-valent conjugate pneumococcal vaccine has dramatically reduced rates of invasive and resistant pneumococcal infections. The rapid dissemination of methicillin-resistant S aureus (MRSA) in the community, along with case reports of severe and necrotizing pneumonia caused by these strains, has in-creased concern that coverage for MRSA should be added to empirical regimens. [ABSTRACT FROM AUTHOR]

Background: Commonly encountered nosocomially acquired gram-negative bacteria, especially Klebsiella pneumoniae, produce extended-spectrum beta-lactamases (ESBLs) as an antibiotic resistance mechanism.Objective: To determine whether microbiology laboratories should report the presence of ESBLs and to establish the infection-control implications of ESBL-producing organisms.Design: Prospective observational study.Setting: 12 hospitals in South Africa, Taiwan, Australia, Argentina, the United States, Belgium, and Turkey.Patients: 440 patients with 455 consecutive episodes of K. pneumoniae bacteremia between 1 January 1996 and 31 December 1997; of these, 253 episodes were nosocomially acquired.Measurements: The K. pneumoniae isolates were examined for the presence of ESBLs. Pulsed-field gel electrophoresis was used to analyze the molecular epidemiology of nosocomial bacteremia with ESBL-producing K. pneumoniae.Results: Overall, 30.8% (78 of 253) episodes of nosocomial bacteremia and 43.5% (30 of 69) episodes acquired in intensive care units were due to ESBL-producing organisms. After adjustment for potentially confounding variables, previous administration of beta-lactam antibiotics containing an oxyimino group (cefuroxime, cefotaxime, ceftriaxone, ceftazidime, or aztreonam) was associated with bacteremia due to ESBL-producing strains (risk ratio, 3.9 [95% CI, 1.1 to 13.8]). In 7 of 10 hospitals with more than 1 ESBL-producing isolate, multiple strains with the same genotypic pattern were observed, indicating patient-to-patient spread of the organism.Conclusions: Production of ESBLs by Klebsiella pneumoniae is a widespread nosocomial problem. Appropriate infection control and antibiotic management strategies are needed to stem the spread of this emerging form of resistance. [ABSTRACT FROM AUTHOR]

Managing patients infected with antibiotic resistant bacteria is becoming one of the major clinical obstacles facing physicians who treat patients in long-term care facilities (LTCFs). Penicillin-resistant pneumococci (PRP), vancomycin-resistant enterococci (VRE), gram-negative bacteria that produce extended-spectrum and ampC-type beta-lactamase enzymes, and quinolone-resistant gram-positive and gram-negative bacteria are the major resistant pathogens that are emerging in these settings. The mechanisms responsible for the evolution of these antibiotic resistant organisms (molecular rearrangement of penicillin binding protein genes, acquisition of a mobile genetic element, and point mutation that alter the active site) are reviewed. Vancomycin intermediate Staphylococcus aureus (VISA) and multidrug efflux pumps in gram-negative bacteria are also threatening our most potent antimicrobials. Aggressive screening, education, antibiotic-control measures, and immunization are advocated as important preventive measures. The combined efforts of the medical directors, infection-control personnel, and administrators are needed to stem this problem. [ABSTRACT FROM AUTHOR]

Background: Colonization and infection with vancomycin-resistant enterococci have been associated with exposure to antibiotics that are active against anaerobes. In mice that have intestinal colonization with vancomycin-resistant enterococci, these agents promote high-density colonization, whereas antibiotics with minimal antianaerobic activity do not.Methods: We conducted a seven-month prospective study of 51 patients who were colonized with vancomycin-resistant enterococci, as evidenced by the presence of the bacteria in stool. We examined the density of vancomycin-resistant enterococci in stool during and after therapy with antibiotic regimens and compared the effect on this density of antianaerobic agents and agents with minimal antianaerobic activity. In a subgroup of 10 patients, cultures of environmental specimens (e.g., from bedding and clothing) were obtained.Results: During treatment with 40 of 42 antianaerobic-antibiotic regimens (95 percent), high-density colonization with vancomycin-resistant enterococci was maintained (mean [+/-SD] number of organisms, 7.8+/-1.5 log per gram of stool). The density of colonization decreased after these regimens were discontinued. Among patients who had not received antianaerobic antibiotics for at least one week, 10 of 13 patients who began such regimens had an increase in the number of organisms of more than 1.0 log per gram (mean increase, 2.2 log per gram), whereas among 10 patients who began regimens of antibiotics with minimal antianaerobic activity, there was a mean decrease in the number of enterococci of 0.6 log per gram (P=0.006 for the difference between groups). When the density of vancomycin-resistant enterococci in stool was at least 4 log per gram, 10 of 12 sets of cultures of environmental specimens had at least one positive sample, as compared with 1 of 9 sets from patients with a mean number of organisms in stool of less than 4 log per gram (P=0.002).Conclusions: For patients with vancomycin-resistant enterococci in stool, treatment with antianaerobic antibiotics promotes high-density colonization. Limiting the use of such agents in these patients may help decrease the spread of vancomycin-resistant enterococci. [ABSTRACT FROM AUTHOR]

Resistance to ampicillin and imipenem in Enterococcus faecalis is infrequent. However, the evolution of resistance can occur through prolonged antibiotic exposure during the treatment of chronic infections. In this study, we conducted a Long-Term Evolution Experiment (LTEE) using four genetically diverse strains of E. faecalis with varying susceptibilities to ampicillin and imipenem. Each strain was subjected to increasing concentrations of either ampicillin or imipenem over 200 days, with three independent replicates for each strain. Selective pressure from imipenem led to the rapid selection of highly resistant lineages across all genetic backgrounds, compared to ampicillin. In addition to high resistance, we describe, for the first time, the evolution of a β-lactam dependent phenotype observed in lineages from all backgrounds. WGS and bioinformatic analysis revealed mutations in three main functional classes: genes involved in cell wall synthesis and degradation, genes in the walK/R two-component system, and genes in the c-di-AMP pathway. Our analysis identified new mutations in genes known to be involved in resistance as well as novel genes potentially associated with resistance. Furthermore, the newly described β-lactam dependent phenotype was correlated with the inactivation of c-di-AMP degradation, resulting in high levels of this second messenger. Together, these data highlight the diverse genetic mechanisms underlying resistance to ampicillin and imipenem in E. faecalis . The emergence of high resistance levels and β-lactam dependency underscores the importance of understanding evolutionary dynamics in the development of antibiotic resistance., Importance: E. faecalis is a major human pathogen, and treatment is frequently compromised by poor response to first-line antibiotics such ampicillin. Understanding the factors that play a role in susceptibility/resistance to these drugs will help guide the development of much needed treatments.

Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. Both authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. Both authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Penicillin-binding proteins (PBPs) are essential for the formation of the bacterial cell wall. They are also the targets of β-lactam antibiotics. In Enterococcus faecium, high levels of resistance to β-lactams are associated with the expression of PBP5, with higher levels of resistance associated with distinct PBP5 variants. To define the molecular mechanism of PBP5-mediated resistance we leveraged biomolecular NMR spectroscopy of PBP5 - due to its size (>70 kDa) a challenging NMR target. Our data show that resistant PBP5 variants show significantly increased dynamics either alone or upon formation of the acyl-enzyme inhibitor complex. Furthermore, these variants also exhibit increased acyl-enzyme hydrolysis. Thus, reducing sidechain bulkiness and expanding surface loops results in increased dynamics that facilitates acyl-enzyme hydrolysis and, via increased β-lactam antibiotic turnover, facilitates β-lactam resistance. Together, these data provide the molecular basis of resistance of clinical E. faecium PBP5 variants, results that are likely applicable to the PBP family., (© 2023. The Author(s).)

Ampicillin-ceftriaxone has become a first-line therapy for Enterococcus faecalis endocarditis. We characterized the penicillin-binding protein (PBP) profiles of various E. faecalis strains and tested for synergy to better inform beta-lactam options for the treatment of E. faecalis infections. We assessed the affinity of PBP2B from elevated-MIC strain E. faecalis LS4828 compared to type strain JH2-2 using the fluorescent beta-lactam Bocillin FL. We also characterized pbp4 and pbpA structures and PBP4 and PBP2B expression and used deletion and complementation studies to assess the impact of PBP2B on the levels of resistance. We tested penicillin-susceptible and -resistant E. faecalis isolates against ceftriaxone or ceftaroline combinations with other beta-lactams in 24-h time-kill studies. Two penicillin-susceptible strains (JH2-2 and L2052) had identical pbp sequences and similar PBP expression levels. One reduced-penicillin-susceptibility strain (L2068) had pbp sequences identical to those of the susceptible strains but expressed more PBP4. The second decreased-penicillin-susceptibility strain (LS4828) had amino acid substitutions in both PBP4 and PBP2B and expressed increased quantities of both proteins. PBP2B did not appear to contribute significantly to the elevated beta-lactam MICs. No synergy was demonstrable against the strains with both mutated PBPs and increased expression (L2068 and LS4828). Meropenem plus ceftriaxone or ertapenem plus ceftriaxone demonstrated the most consistent synergistic activity. PBP2B of strain LS4828 does not contribute significantly to reduced penicillin susceptibility. Neither the MIC nor the level of PBP expression correlated directly with the identified synergistic combinations when tested at static subinhibitory concentrations.

During the past 4 decades, oral vancomycin has been a mainstay of Clostridioides difficile infection (CDI) therapy with no reports of treatment failure due to emergence of vancomycin resistance. However, C. difficile isolates with high-level phenotypic resistance to vancomycin have recently been reported in 3 distinct geographic regions. There is an urgent need for surveillance to determine if strains with reduced vancomycin susceptibility are circulating in other areas. In a Cleveland-area hospital, screening of 176 CDI stool specimens yielded no C. difficile isolates with reduced vancomycin susceptibility and highlighted the potential for false-positive results due to contamination with vancomycin-resistant enterococci. Additional studies are needed to clarify whether reduced vancomycin susceptibility is an emerging problem that will alter clinical practice. Clinicians should alert their health department if they observe a substantial increase in the frequency of vancomycin treatment failure in patients diagnosed with CDI with no alternative explanation for diarrhea., Competing Interests: Potential conflicts of interest. C. J. D. has received research funding from Clorox, PDI, and Pfizer. All other authors report no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2022.)

The standard of care for serious Enterococcus faecalis infections is ampicillin plus ceftriaxone. Ampicillin's inconvenient dosing schedule, drug instability, allergy potential, along with ceftriaxone's high risk for Clostridioides difficile infection and its promotion of vancomycin-resistant enterococci (VRE), led our team to explore alternative options. This work aimed to understand the role of carbapenems in combination with cephalosporins in these infections. We selected two ampicillin and penicillin susceptible E. faecalis strains (AMP-MIC 0.5-2 μg/mL; PCN-MIC 2 μg/mL) and simulated human therapeutic dosing regimens in a 48-h in vitro pharmacodynamic model (IVPD) with ampicillin (2g q4h), ertapenem (1g q24h), meropenem (2g q8h), ceftriaxone (2g q12h), and ceftaroline (600 mg q8h). As expected, ampicillin plus ceftriaxone demonstrated enhanced activity compared with ampicillin monotherapy with no MIC increases in either isolate. Meropenem and ceftaroline demonstrated significant kill against both isolates, with no regrowth or MIC increases occurring. Meropenem plus ceftriaxone also demonstrated significant kill, and while no MIC increases were identified for meropenem, there was minor regrowth and larger standard deviations. Ertapenem combined with either ceftriaxone or ceftaroline enhanced activity at 24 h, but at 48 h, regrowth occurred, and ertapenem MIC increases were noted. Meropenem-based combination therapy against E. faecalis may provide clinicians with another regimen to treat severe E. faecalis infections. Meropenem plus ceftaroline was as active as the standard of care treatment (ampicillin plus ceftriaxone) and may serve as an alternative for serious E. faecalis infections. Further studies are warranted to determine the clinical efficacy.

Enterococcus faecalis differs from many other common human pathogens in its physiology and in its susceptibility to antimicrobial agents. Multiresistant E. faecalis strains owe their phenotypes to a combination of intrinsic and acquired antimicrobial resistance determinants. Acquired resistance is due to E. faecalis frequenting multicultural environments, its capacity to mate with different species, and the nullification of its own defense mechanisms in some lineages. Intrinsic resistance is a complex phenomenon that is intimately tied to the physiology of the species. In their recent study in mBio , Gilmore and colleagues (M. S. Gilmore, R. Salamzade, E. Selleck, N. Bryan, et al., mBio 11:e02962-20, 2020, https://doi.org/10.1128/mBio.02962-20) use functional genomics to explore the genetic underpinnings of E. faecalis physiology and antimicrobial resistance. While they do not come up with many definitive answers, their work points the way toward new and fruitful areas of investigation., (Copyright © 2021 Rice.)

Since its inaugural issue nearly half a century ago, Antimicrobial Agents and Chemotherapy has served as a premier source for reports on scientific and clinical advances in the field of antimicrobial chemotherapy. As a follow-up to the previous "History of Antimicrobial Agents and Chemotherapy from 1972 to 1998" written by George A. Jacoby (Antimicrob Agents Chemother 43:999-1002, 1999, https://doi.org/10.1128/AAC.43.5.999), we herein highlight the further evolution of this comprehensive and authoritative journal in response to changing science, demographics, and information technology., (Copyright © 2021 American Society for Microbiology.)

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., Competing Interests: Disclosure Dr W.R. Miller has received grants from Merck and Entasis Therapeutics, and honoraria from Achaogen and Shionogi. Dr B.E. Murray has received grant support from Theravance Biopharma, Paratek, and Cubist/Merck, and has served as consultant for Paratek. Dr L.B. Rice has served on data safety monitoring boards for Zavante Pharmaceuticals and VenatoRx Pharmaceuticals. Dr C.A. Arias has received grant support from Merck, MeMed Diagnostics, and Entasis Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: ZTI-01 (fosfomycin for injection) is an epoxide antibiotic with a differentiated mechanism of action (MOA) inhibiting an early step in bacterial cell wall synthesis. ZTI-01 has broad in vitro spectrum of activity, including multidrug-resistant Gram-negative pathogens, and is being developed for treatment of complicated urinary tract infection (cUTI) and acute pyelonephritis (AP) in the United States., Methods: Hospitalized adults with suspected or microbiologically confirmed cUTI/AP were randomized 1:1 to 6 g ZTI-01 q8h or 4.5 g intravenous (IV) piperacillin-tazobactam (PIP-TAZ) q8h for a fixed 7-day course (no oral switch); patients with concomitant bacteremia could receive up to 14 days., Results: Of 465 randomized patients, 233 and 231 were treated with ZTI-01 and PIP-TAZ, respectively. In the microbiologic modified intent-to-treat (m-MITT) population, ZTI-01 met the primary objective of noninferiority compared with PIP-TAZ with overall success rates of 64.7% (119/184 patients) vs 54.5% (97/178 patients), respectively; treatment difference was 10.2% (95% confidence interval [CI]: -0.4, 20.8). Clinical cure rates at test of cure (TOC, day 19-21) were high and similar between treatments (90.8% [167/184] vs 91.6% [163/178], respectively). In post hoc analysis using unique pathogens typed by pulsed-field gel electrophoresis, overall success rates at TOC in m-MITT were 69.0% (127/184) for ZTI-01 versus 57.3% (102/178) for PIP-TAZ (difference 11.7% 95% CI: 1.3, 22.1). ZTI-01 was well tolerated. Most treatment-emergent adverse events, including hypokalemia and elevated serum aminotransferases, were mild and transient., Conclusions: ZTI-01 was effective for treatment of cUTI including AP and offers a new IV therapeutic option with a differentiated MOA for patients with serious Gram-negative infections., Clinical Trial Registration: NCT02753946., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

The genus Enterococcus comprises a ubiquitous group of Gram-positive bacteria that are of great relevance to human health for their role as major causative agents of health care-associated infections. The enterococci are resilient and versatile species able to survive under harsh conditions, making them well adapted to the health care environment. Two species cause the majority of enterococcal infections: Enterococcus faecalis and Enterococcus faecium Both species demonstrate intrinsic resistance to common antibiotics, such as virtually all cephalosporins, aminoglycosides, clindamycin, and trimethoprim-sulfamethoxazole. Additionally, a remarkably plastic genome allows these two species to readily acquire resistance to further antibiotics, such as high-level aminoglycoside resistance, high-level ampicillin resistance, and vancomycin resistance, either through mutation or by horizontal transfer of genetic elements conferring resistance determinants., (Copyright © 2019 American Society for Microbiology.)

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of β-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium , respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the β-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three β-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

Antimicrobial stewardship programs aim to reduce costs, optimize therapeutic outcomes, and reduce antimicrobial resistance. Reductions of antimicrobial resistance are the most elusive because emergence and spread of resistant bacteria involves antimicrobial selective pressure and lapses in infection control techniques. The relationship between antimicrobial usage and resistance is not always direct. The understanding of which techniques are most effective is limited because many studies are descriptive or quasiexperimental. Recent meta-analyses or systematic reviews of stewardship programs offer encouragement that some interventions reduce overall antimicrobial selective pressure and, where associated with infection control interventions, impact resistance rates in individual institutions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Enterococci, one of the most common causes of hospital-associated infections, are responsible for substantial morbidity and mortality. Enterococcus faecalis, the more common and virulent species, causes serious high-inoculum infections, namely infective endocarditis, that are associated with cardiac surgery and mortality rates that remained unchanged for the last 30 years. The best cures for these infections are observed with combination antibiotic therapy; however, optimal treatment has not been fully elucidated. It is the purpose of this review to highlight treatment options and their limitations, and provide direction for future investigative efforts to aid in the treatment of these severe infections. While ampicillin plus ceftriaxone has emerged as a preferred treatment option, mortality rates continue to be high, and from a safety standpoint, ceftriaxone, unlike other cephalosporins, promotes colonization with vancomycin resistant-enterococci due to high biliary concentrations. More research is needed to improve patient outcomes from this high-mortality disease.

Enterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 -35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems. IMPORTANCE Enterococcus faecalis is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an E. faecalis strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to "suppress" infections in prosthetic joints and offer novel insights into the interaction of β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains., (Copyright © 2018 Rice et al.)

Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 μg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays.

The target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB., Competing Interests: The authors declare that no competing interests exist.

The transfer of DNA between Enterococcus faecium strains has been characterized both by the movement of well-defined genetic elements and by the large-scale transfer of genomic DNA fragments. In this work, we report on the whole-genome analysis of transconjugants resulting from mating events between the vancomycin-resistant E. faecium C68 strain and the vancomycin-susceptible D344RRF strain to discern the mechanism by which the transferred regions enter the recipient chromosome. Vancomycin-resistant transconjugants from five independent matings were analyzed by whole-genome sequencing. In all cases but one, the penicillin binding protein 5 (pbp5) gene and the Tn5382 vancomycin resistance transposon were transferred together and replaced the corresponding pbp5 region of D344RRF. In one instance, Tn5382 inserted independently downstream of the D344RRF pbp5 gene. Single nucleotide variant (SNV) analysis suggested that entry of donor DNA into the recipient chromosome occurred by recombination across regions of homology between donor and recipient chromosomes, rather than through insertion sequence-mediated transposition. The transfer of genomic DNA was also associated with the transfer of C68 plasmid pLRM23 and another putative plasmid. Our data are consistent with the initiation of transfer by cointegration of a transferable plasmid with the donor chromosome, with subsequent circularization of the plasmid-chromosome cointegrant in the donor prior to transfer. Entry into the recipient chromosome most commonly occurred across regions of homology between donor and recipient chromosomes., (Copyright © 2016 García-Solache et al.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Enterococcus faecium is an important nosocomial pathogen, causing a substantial health burden due to high resistance to antibiotics and its ability to colonize the gastrointestinal tract. Here, we present the draft genome of vancomycin-susceptible, ampicillin-intermediate strain D344RRF, a rifampicin/fusidic acid-resistant and commonly used laboratory strain, which is useful in studying the transfer of antibiotic resistance., (Copyright © 2016 García-Solache and Rice.)

Enterococcus faecium infections are a rising concern in hospital settings. Vancomycin-resistant enterococci colonize the gastrointestinal tract and replace nonresistant strains, complicating the treatment of debilitated patients. Here, we present a polished genome of the multiantibiotic-resistant strain C68, which was obtained as a clinical isolate and is a useful experimental strain., (Copyright © 2016 García-Solache and Rice.)

Ampicillin-ceftriaxone combination therapy has become a predominant treatment for serious Enterococcus faecalis infections, such as endocarditis. Unfortunately, ceftriaxone use is associated with future vancomycin-resistant enterococcus colonization. We evaluated E. faecalis in an in vitro pharmacodynamic model against simulated human concentration-time profiles of ampicillin plus ceftaroline, cefepime, ceftriaxone, or gentamicin. Ampicillin-cefepime and ampicillin-ceftaroline demonstrated activities similar to those of ampicillin-ceftriaxone against E. faecalis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as "cephalosporins") is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system., Importance: β-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to β-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to β-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a resistant phenotype. The present work identifies a novel, previously uncharacterized, protein that interacts with Pbp5, whose expression increases in conjunction with stimuli that increase resistance to cephalosporins, and that confers increased resistance to cephalosporins when overexpressed. P5AP may represent a promising new target, inhibition of which could restore cephalosporin susceptibility to E. faecium., (Copyright © 2016 Desbonnet et al.)

Background: We identified auranofin as an antimicrobial compound utilizing a high-throughput screen using a Caenorhabditis elegans-Staphylococcus aureus infection model. Results/methodology: Treatment of infected nematodes with auranofin resulted in a prolonged survival rate of 95%, reached with 0.78 μg/ml. Further investigation of the antimicrobial activity of auranofin found inhibition against S. aureus, Enterococcus faecium and Enterococcus faecalis. Importantly, the fungal pathogens Cryptococcus neoformans was also effectively inhibited with an MIC at 0.5 μg/ml. Auranofin appears to target the thioredoxin system., Conclusion: This work provides extensive additional data on the antibacterial effects of auranofin that includes both reference and clinical isolates and reports a novel inhibition of fungal pathogens by this compound., Competing Interests: Financial & competing interests disclosure This research was supported by a COBRE-Immune-based intervention against infectious diseases pilot grant and a Brown University Dean's Emerging Areas of New Science award to B Burgwyn Fuchs, and a NIH P01 grant to E Mylonakis. Our work was also supported with funding provided by the Brown-Brazil Initiative awarded to B Burgwyn Fuchs and E Mylonakis. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Beta-lactam antibiotics sensitize Enterococcus faecium to killing by endogenous antimicrobial peptides (AMPs) of the innate immune system and daptomycin through mechanisms yet to be elucidated. It has been speculated that beta-lactam inactivation of select E. faecium penicillin binding proteins (PBPs) may play a pivotal role in this sensitization process. To characterize the specific PBP inactivation that may be responsible for these phenotypes, we utilized a previously characterized set of E. faecium PBP knockout mutants to determine the effects of such mutations on the activity of daptomycin and the AMP human cathelicidin (LL-37). Enhanced susceptibility to daptomycin was dependent more on a cumulative effect of multiple PBP deletions than on inactivation of any single specific PBP. Selective knockout of PBPZ rendered E. faecium more vulnerable to killing by both recombinant LL-37 and human neutrophils, which produce the antimicrobial peptide in high quantities. Pharmacotherapy targeting multiple PBPs may be used as adjunctive therapy with daptomycin to treat difficult E. faecium infections., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Bypass of the d,d-transpeptidase activity of penicillin-binding proteins by an l,d-transpeptidase (Ldtfm) results in resistance to ampicillin and glycopeptides in Enterococcus faecium M9, a mutant obtained by nine consecutive selection steps. Resistance requires activation of a cryptic locus for production of the essential tetrapeptide-containing substrate of Ldtfm and impaired activity of protein phosphatase StpA. Here, whole-genome sequencing revealed a high mutation rate for the entire selection procedure (79 mutations in 900 generations). Acquisition of a mutation in the mismatch repair gene mutL had little impact on the frequency of rifampin-resistant mutants although the mutation spectrum of M9 was typical of impaired MutL with high transversion to transition (40/11) and substitution to deletion (51/28) ratios. M9 did not mainly accumulate neutral mutations since base substitutions occurred more frequently in coding sequences than expected (χ(2) = 5.0; P < 0.05) and silent mutations were underrepresented (χ(2) = 5.72; P < 0.02). None of the mutations directly affected recognition of the tetrapeptide substrate of Ldtfm by peptidoglycan synthesis enzymes. Instead, mutations appear to remodel regulatory circuits involving two-component regulatory systems and sugar metabolism. The high number of mutations required for activation of the l,d-transpeptidase pathway may strongly limit emergence of cross-resistance to ampicillin and glycopeptides by this mechanism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Vancomycin-resistant enterococci (VRE) have become important nosocomial pathogens causing outbreaks worldwide. Patients undergoing dialysis represent a vulnerable population due to their comorbid conditions, frequent use of antibacterial agents, and frequent contact with health care settings., Study Design: Systematic review and meta-analysis of cross-sectional studies of screening for VRE colonization., Setting & Population: Patients receiving long-term dialysis treatment., Selection Criteria for Studies: We performed a systematic literature search of PubMed and EMBASE databases to identify studies performing screening for VRE colonization among dialysis patients., Predictor: Region, recent use of vancomycin or other antibiotics, previous hospitalization., Outcomes: (1) VRE colonization and (2) rate of VRE infection among colonized and noncolonized individuals. Relative effects were expressed as ORs and 95% CIs., Results: We identified 23 studies that fulfilled the inclusion criteria and provided data for 4,842 dialysis patients from 100 dialysis centers. The pooled prevalence of VRE colonization was 6.2% (95% CI, 2.8%-10.8%), with significant variability between centers. The corresponding number for North American centers was 5.2% (95% CI, 2.8%-8.2%). Recent use of any antibiotic (OR, 3.62; 95% CI, 1.22-10.75), particularly vancomycin (OR, 5.15; 95% CI, 1.56-17.02), but also use of antibiotics other than vancomycin (OR, 2.92; 95% CI, 0.99-8.55) and recent hospitalization (OR, 4.55; 95% CI, 1.93-10.74) significantly increased the possibility of a VRE-positive surveillance culture. Colonized patients had a significantly higher risk of VRE infection (OR, 21.62; 95% CI, 5.33-87.69) than their noncolonized counterparts., Limitations: In 19 of 23 studies, a low percentage of dialysis patients (<80%) consented to participate in the screening procedure. 4 of 8 studies in which patients were followed up for more than 1 month reported VRE infections and only 5 of 23 studies provided extractable data for antibiotic consumption prior to screening., Conclusions: VRE colonization is prevalent in dialysis centers. Previous antibiotic use, in particular vancomycin, and recent hospitalization are important predicting factors of colonization, whereas the risk of VRE infection is significantly higher for colonized patients., (Copyright © 2014 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

The burden of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) colonization among the increasing number of solid organ transplant patients has not been systematically explored. We searched PubMed and EMBASE for pertinent articles, performed a meta-analysis of prevalence across eligible studies and estimated the risk of ensuing MRSA or VRE infections relative to colonization status. We stratified effects in the pretransplant and posttransplant period. Twenty-three studies were considered eligible. Seventeen out of 23 (74%) referred to liver transplants. Before transplantation, the pooled prevalence estimate for MRSA and VRE was 8.5% (95% confidence interval [CI] 3.2–15.8) and 11.9% (95% CI 6.8–18.2), respectively. MRSA estimate was influenced by small studies and was lower (4.0%; 95% CI 0.4–10.2) across large studies (>200 patients). After transplantation, the prevalence estimates were 9.4% (95% CI 3.0–18.5) for MRSA and 16.2% (95% CI 10.7–22.6) for VRE. Pretransplant as well as posttransplant MRSA colonization significantly increased the risk for MRSA infections (pooled risk ratio [RR] 5.51; 95% CI 2.36–12.90 and RR 10.56; 95% CI 5.58–19.95, respectively). Pretransplant and posttransplant VRE colonization were also associated with significant risk of VRE infection (RR 6.65; 95% CI 2.54–17.41 and RR 7.93; 95% CI 2.36–26.67, respectively). Solid organ transplantation is a high-risk setting for MRSA and VRE colonization, and carrier state is associated with infection. Upgraded focus in prevention and eradication strategies is warranted.

The last step of peptidoglycan polymerization involves two families of unrelated transpeptidases that are the essential targets of β-lactam antibiotics. D,D-transpeptidases of the penicillin-binding protein (PBP) family are active-site serine enzymes that use pentapeptide precursors and are the main or exclusive cross-linking enzymes in nearly all bacteria. However, peptidoglycan cross-linking is performed mainly by active-site cysteine L,D-transpeptidases that use tetrapeptides in Mycobacterium tuberculosis, Clostridium difficile, and β-lactam-resistant mutants of Enterococcus faecium. We have investigated reprogramming of the E. faecium peptidoglycan assembly pathway by a switch from pentapeptide to tetrapeptide precursors and bypass of PBPs by L,D-transpeptidase Ldtfm. Mutational alterations of two signal transduction systems were necessary and sufficient for activation of the L,D-transpeptidation pathway, which is essentially cryptic in wild-type strains. The first one is a classical two-component regulatory system, DdcRS, that controls the activity of Ldtfm at the substrate level. As previously described, loss of DdcS phosphatase activity leads to production of the D,D-carboxypeptidase DdcY and conversion of the pentapeptide into the tetrapeptide substrate of Ldtfm. Here we show that full bypass of PBPs by Ldtfm also requires increased Ser/Thr protein phosphorylation resulting from impaired activity of phosphoprotein phosphatase StpA. This enzyme negatively controlled the level of protein phosphorylation both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase Stk. In combination with production of DdcY, increased protein phosphorylation by this eukaryotic-enzyme-like Ser/Thr protein kinase was sufficient for activation of the L,D-transpeptidation pathway in the absence of mutational alteration of peptidoglycan synthesis enzymes. Importance: The mechanism of acquisition of high-level ampicillin resistance involving bypass of the penicillin-binding proteins (PBPs) by L,D-transpeptidase Ldtfm was incompletely understood, as production of tetrapeptide precursors following transcriptional activation of the ddc locus by the DdcRS two-component regulatory system was necessary but not sufficient for full activation of the L,D-transpeptidation pathway. Here, we identified the release of a negative control of Ser/Thr protein phosphorylation mediated by phosphatase StpA as the additional factor essential for ampicillin resistance. Thus, bypass of PBPs by Ldtfm requires the modification of signal transduction regulatory systems without any gain of function by mutational alteration of peptidoglycan biosynthetic enzymes. In contrast, previously characterized mechanisms of antibiotic resistance involve horizontal gene transfer and mutational alteration of drug targets. Activation of the L,D-transpeptidation pathway reported in this study is an unprecedented mechanism of emergence of a new metabolic pathway since it involved the recruitment of preexisting functions following modifications of regulatory circuits., (Copyright © 2014 Sacco et al.)

Synthesis of peptidoglycan precursors ending in D-lactate (D-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by L,D-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in D-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of D-Lac into cytoplasmic precursors. This was due to a D,D-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of D-Lac for D-Ala and Gly. The contribution of L,D-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-D,D-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal D-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by D,D-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of L,D-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-D,D-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that L,D-transpeptidases merely act as a tolerance mechanism in this bacterium.

Background: The burden and significance of vancomycin-resistant enterococci (VRE) colonization in the ICU is not clearly understood., Methods: We searched PubMed and EMBASE up to May 2013 for studies reporting the prevalence of VRE upon admission to the ICU and performed a meta-analysis to assess rates and trends of VRE colonization. We calculated the prevalence of VRE on admission and the acquisition (colonization and/or infection) rates to estimate time trends and the impact of colonization on ensuing VRE infections., Findings: Across 37 studies (62,959 patients at risk), the estimated prevalence of VRE on admission to the ICU was 8.8% (7.1-10.6). Estimates were more consistent when cultures were obtained within 24 hours from admission. The VRE acquisition rate was 8.8% (95% CI 6.9-11.0) across 26 evaluable studies (35,364 patients at risk). Across US studies, VRE acquisition rate was 10.2% (95% CI 7.7-13.0) and demonstrated significant decline in annual trends. We used the US estimate of colonization on admission [12.3% (10.5-14.3)] to evaluate the impact of VRE colonization on admission in overall VRE prevalence. We demonstrated that VRE colonization on admission is a major determinant of the overall VRE burden in the ICU. Importantly, among colonized patients (including admitted and/or acquired cases) the VRE infection rates vary widely from 0-45% (with the risk of VRE bacteremia being reported from 0-16%) and <2% among those without a proven colonization., Conclusion: In summary, up to 10.6% of patients admitted in the ICU are colonized with VRE on admission and a similar percentage will acquire VRE during their ICU stay. Importantly, colonization on admission is a major determinant of VRE dynamics in the ICU and the risk of VRE-related infections is close related to colonization.

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldt(fm)) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldt(fm) does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldt(fm) by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldt(fm) inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt(fm) acylation was followed by rupture of the C(5)-C(6) bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldt(fm) blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

The human gastrointestinal tract is a complex environment of mutualistic associations. As bacteria form a major component of fecal content, the natural balance of the colon can be significantly altered by exposure to antimicrobial agents. However, the effects of antimicrobial therapy on fecal content are difficult to predict and can at times be quite surprising. The emergence and spread of vancomycin-resistant enterococci are cases in point. Resistance to the glycopeptide vancomycin emerged in enterococci (primarily in Enterococcus faecium) in the late 1980s in both Europe and the United States. In Europe, this emergence was tied to the use of the glycopeptide antibiotic avoparcin to promote growth in food animals and had little actual impact on hospital infections. In the United States, where avoparcin has never been licensed, vancomycin-resistant enterococci (VRE) emerged as a major hospital pathogen. Paradoxically, while the initial entry of the vancomycin resistance determinants into enterococci was almost certainly driven by high fecal concentrations of vancomycin associated with treatment for Clostridium difficile colitis, clinical infection and outbreaks were more frequently tied to use of extended-spectrum cephalosporins and agents with potent activity against anaerobic bacteria. Animal studies suggest that cephalosporins promote initial VRE colonization because of the frequent concomitant high-level resistance to β-lactam antibiotics expressed by these strains. Anti-anaerobic agents appear to increase the output of VRE in the feces, presumably by reducing the number of competitive flora in the colon. Intravenously administered vancomycin appears to have little impact because it achieves negligible concentrations in the feces after short courses. Thus, the spread of glycopeptide resistance in enterococci is promoted in a large measure by the administration of non-glycopeptide antibiotics.