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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.

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.

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.

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.

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.

The ferric uptake regulator (Fur) plays a major role in controlling the expression of iron homeostasis genes in bacterial organisms. In this work, we fully characterized the capacity of Fur to reconfigure the global transcriptional network and influence iron homeostasis in Enterococcus faecalis. The characterization of the Fur regulon from E. faecalis indicated that this protein (Fur) regulated the expression of genes involved in iron uptake systems, conferring to the system a high level of efficiency and specificity to respond under different iron exposure conditions. An RNAseq assay coupled with a systems biology approach allowed us to identify the first global transcriptional network activated by different iron treatments (excess and limited), with and without the presence of Fur. The results showed that changes in iron availability activated a complex network of transcriptional factors in E. faecalis, among them global regulators such as LysR, ArgR, GalRS, and local regulators, LexA and CopY, which were also stimulated by copper and zinc treatments. The deletion of Fur impacted the expression of genes encoding for ABC transporters, energy production and [Fe-S] proteins, which optimized detoxification and iron uptake under iron excess and limitation, respectively. Finally, considering the close relationship between iron homeostasis and pathogenesis, our data showed that the absence of Fur increased the internal concentration of iron in the bacterium and also affected its ability to produce biofilm. These results open new alternatives in the field of infection mechanisms of E. faecalis.

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.

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.

Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium . The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium . We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5 , including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.

Treatment of severe infections caused by vancomycin-resistant enterococci (VRE) is challenging due to the scarcity of reliable therapeutic alternatives. In this context, daptomycin (DAP), a lipopeptide antibiotic, has emerged as an interesting alternative as it is one of the few compounds that retain in vitro bactericidal activity against VRE isolates, although it has not been approved for this purpose by regulatory agencies. In this review, we will summarise the clinical, animal and in vitro evidence evaluating the efficacy of DAP for the management of deep-seated VRE infections. In addition, we will address important clinical concerns such as the emergence of DAP resistance during therapy and reports of therapeutic failure with DAP monotherapy. Finally, we will discuss possible future strategies (such as the use of higher doses and/or combination therapies) to optimise the use of this antibiotic against VRE.

Daptomycin is used off-label for enterococcal infections; however, dosing targets for resistance prevention remain undefined. Doses of 4 to 6 mg/kg of body weight/day approved for staphylococci are likely inadequate against enterococci due to reduced susceptibility. We modeled daptomycin regimens in vitro to determine the minimum exposure to prevent daptomycin resistance (Dap r ) in enterococci. Daptomycin simulations of 4 to 12 mg/kg/day (maximum concentration of drug in serum [ C max ] of 57.8, 93.9, 123.3, 141.1, and 183.7 mg/liter; half-life [ t 1/2 ] of 8 h) were tested against one Enterococcus faecium strain (S447) and one Enterococcus faecalis strain (S613) in a simulated endocardial vegetation pharmacokinetic/pharmacodynamic model over 14 days. Samples were plated on media containing 3× the MIC of daptomycin to detect Dap r . Mutations in genes encoding proteins associated with cell envelope homeostasis ( yycFG and liaFSR ) and phospholipid metabolism (cardiolipin synthase [ cls ] and cyclopropane fatty acid synthetase [ cfa ]) were investigated in Dap r derivatives. Dap r derivatives were assessed for changes in susceptibility, surface charge, membrane depolarization, cell wall thickness (CWT), and growth rate. Strains S447 and S613 developed Dap r after simulations of 4 to 8 mg/kg/day but not 10 to 12 mg/kg/day. MICs for Dap r strains ranged from 8 to 256 mg/liter. Some S613 derivatives developed mutations in liaF or cls . S447 derivatives lacked mutations in these genes. Dap r derivatives from both strains exhibited lowered growth rates, up to a 72% reduction in daptomycin-induced depolarization and up to 6-nm increases in CWT ( P < 0.01). Peak/MIC and AUC 0–24 /MIC ratios (AUC 0–24 is the area under the concentration-time curve from 0 to 24 h) associated with Dap r prevention were 72.1 and 780 for S447 and 144 and 1561 for S613, respectively. Daptomycin doses of 10 mg/kg/day may be required to prevent Dap r in serious enterococcal infections.

High-dose daptomycin (DAP) therapy failed in a neutropenic patient with bloodstream infection caused by a DAP-susceptible Enterococcus faecium (minimum inhibitory concentration, 3 µg/mL) harboring genetic changes associated with DAP resistance, with persistent bacteremia and selection of additional resistances. Daptomycin monotherapy should be used cautiously against DAP-susceptible E. faecium strains with minimum inhibitory concentrations >2 µg/mL.

Background Daptomycin (DAP) is one of the mainstay treatments for Enterococcus faecium infections. However, development of resistance threatens its continued viability as a treatment option. Although the mechanisms of DAP resistance in enterococci are not fully comprehended, they are associated with alterations in cell envelope phospholipids assembly which leads to repulsion of the drug from cell exterior and diversion from the cell septum. Previous data suggest that combination of DAP with β-lactams has the potential to improve patient outcomes. In this investigation, we sought to evaluate combinations of DAP with ampicillin (AMP), ceftaroline (CPT), and ertapenem (ERT). Methods E. faecium R497 harboring liaSFR mutations (DAP MIC of 16 mg/L) was evaluated in a simulated endocardial vegetation (SEV) pharmacokinetic and pharmacodynamic model over 336 hours at a starting inoculum of 109 log10 CFU/g of SEV. DAP alone at 10 mg/kg/day or DAP (6, 8, 10 mg/kg/day) in combination with AMP (2 g continuous infusion), CPT 600 mg q12h or ERT 1 g q24h were evaluated. The emergence of DAP resistance was determined daily over the course of the 14-day experiment. Results DAP alone was not bactericidal and high-level DAP resistance was observed (MIC increase from 16 to 256 µg/mL) for all DAP alone regimens. Combination of DAP+AMP offered a significant reduction in log10CFU/g amounts (up to 7 log10 CFU/g and to detection limits) in 24 hours in DAP10+AMP model with no further emergence of DAP resistance. Even in DAP 6 mg/kg/day with AMP (2 g), dramatic killing with no further emergence of resistance was observed. Neither CPT nor ERT in combination with DAP was effective against this strain. At higher doses of DAP (14 mg/kg/day) + CPT or ERT, a temporary (0–48 hours) CFU/g reduction was observed followed by regrowth and the further emergence of DAP resistance. Conclusion Combination of DAP + AMP offered the most encouraging results against E. faecium R497. A DAP dose sparring effect was noted with DAP + AMP but not with CPT or ERT. The reason for the discrepancy is unknown and is under further investigation. Further evaluation of DAP plus β-lactam therapy is warranted to discover the most optimized DAP and β-lactam therapy to improve patient outcome and prevent the emergence of resistance. Disclosures B. Murray, Paratek pharmaceuticals: Consultant and Scientific Advisor, Consulting fee and Speaker honorarium; Forest/Actavis: Grant Investigator, Grant recipient; Cubist/Merck: Grant Investigator, Grant recipient. C. Arias, Merck & Co., Inc.: Grant Investigator, Research support; MeMed: Grant Investigator, Research support; Allergan: Grant Investigator, Research support; M. J. Rybak, Allergan: Consultant, Grant Investigator and Speaker’s Bureau, Research grant and Research support; Achaogen: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support; Bayer: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support; Melinta: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support; Merck: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support; Theravance: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support; Sunovian: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support; Zavante: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support; NIAID: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee, Research grant and Research support

Background Enterococci are a major cause of healthcare-associated infections with limited treatment options. We previously identified that mutations in dak (a gene encoding a putative fatty acid kinase), ace (a collagen adhesin) and the YxdJK stress response system are associated with DAP resistance (DAP-R) in E. faecalis (Efs) in the absence of a functional LiaFSR system. Here, we examined the role of DAK in pathogenesis by examining the ability of the mutants to produce biofilm and bind to collagen, an important protein of the extracellular matrix. Methods Previously, the Efs strain OG1RFΔliaR (inactive LiaFSR system, DAP susceptible) was adapted to make a DAP-R derivative (mutations in yxdK, dak, and ace), and the mutant OG1RFΔliaRΔc-dak, lacking the C-terminal domain of dak, and its complement OG1RFΔliaRΔc-dak::c-dak were constructed to study the dak mutation in isolation. Biofilm formation (BF) for the above strains was assayed after growth in tryptic soy broth with glucose in 96-well plates at 37° C for 24 hours. Bacteria were fixed with Bouin’s fixative, stained with crystal violet, and biofilm was quantitated by absorbance at 570 nm. For collagen adherence, 96-well plates were coated with 10 μg/well type I collagen, with 2% bovine serum albumin (BSA) as a control. Bacteria grown at 46° C (to induce ace expression) were added at OD 600 nm of 1.0 and allowed to bind for 2 hours. Non-adherent bacteria were removed by washing, cells were fixed, stained, and quantified as above. Results When compared with OG1RFΔliaR, the DAP-R derivative exhibited a significant increase in BF (4.6 vs. 2.4, P < 0.001). This enhanced biofilm phenotype was also seen in the OG1RFΔliaRΔc-dak mutant (6.1 vs. 2.4, P < 0.001), and reverted on complementation of the full-length dak in its native chromosomal location (2.4 vs. 2.6, p = 0.72). DAK was also found to impact adherence to collagen, with OG1RFΔliaRΔc-dak showing increased binding to collagen when compared with OG1RFΔliaR (7.9 vs. 3.4, P < 0.001), a phenotype which reverted on complementation (7.9 vs. 1.2, P < 0.001). Conclusion Changes in an enzyme involved in DAP adaptation lead to biofilm formation and adherence to extracellular matrix proteins, potentially enhancing virulence in the setting of DAP-R. Disclosures All authors: No reported disclosures.

Background DAP in combination with β-lactams is a viable option to treat recalcitrant DAP-R/tolerant strains of Efm. Ampicillin (AMP), ceftaroline (CPT), and ertapenem (ERT) have the best synergism. Using a DAP tolerant strain (503; DAP MIC 2 µg/mL) of Efm, we previously showed that AMP, CPT, and ERT combined with DAP were effective in reducing bacterial loads and prevented emergence of resistance in a simulated endocardial vegetation model. However, against a DAP-R Efm strain (R497, DAP MIC of 16 µg/mL), CPT, ERT failed to synergize with DAP. Here, we dissect the mechanistic basis of the differing DAP plus β-lactam synergistic effect. Methods We performed comparative transcriptional profiling of pbp genes in Efm 503 vs. R497 using qRT–PCR. PBP5 protein levels were assessed by immunoblotting. The β-lactam-binding affinity of PBPs was quantified with bocillin-FL staining and SDS–PAGE. PBP5 sequences of Efm Com15 (AMP and DAP-susceptible strain) and clinical strains S447, 503 and R497 (all with AMP MIC > 256 µg/mL) were compared in silico to identify amino acid (AA) differences in key protein sites which were verified with sequencing Results Pbp gene transcripts and PBP5 amounts were similar between 503 vs. R497. Interestingly, bocillin SDS–PAGE showed increased β-lactam binding affinity in PBP5 of 503 compared with that of R497 and S447. PBP5 sequences of S447 and R497 were identical. All three clinical strains had classic mutations (M485A and 466’S) important for high-level AMP-R. However, 503 had additional substitutions in the transpeptidase domain (H408Q, A462V, T546N, T558A, S582G, V586L) and penicillin-binding domain (Q632K, L642P) compared with R497 and S447. The latter AA sequences in 503 are common to AMP-susceptible Efm strains Conclusion We uncovered that a “hybrid” pbp5 allele of 503 (DAP-tolerant) correlated with synergism of DAP plus AMP, CPT or ERT and was associated with increased PBP5 β-lactam binding affinity. Lack of synergism of DAP plus CPT or ERT is associated with specific PBP amino acids in the transpeptidase and penicillin-binding domains. Thus, pbp5 alleles are major determinants of the DAP plus β-lactam synergistic effect and could be used as a diagnostic tool to guide therapy in recalcitrant Efm infections Disclosures All authors: No reported disclosures.

Background We have previously demonstrated that daptomycin (DAP) combinations with β-lactams offer enhanced bactericidal activity and prevent the emergence of resistance in Enterococcus faecium infections. Although the mechanisms of DAP resistance in enterococci are not fully comprehended, they are associated with alterations in cell envelope phospholipids assembly which leads to either repulsion of the drug from cell exterior or diversion from the cell septum. In this context, we sought to evaluate combinations of DAP with a panel of β-lactams including ampicillin (AMP), amoxicillin (AMX), ceftaroline (CPT), ceftriaxone (CRO) and ertapenem (ERT). Methods E. faecium R497 harboring liaSFR mutations (DAP MIC of 16 mg/L) was evaluated in a simulated endocardial vegetation (SEV) pharmacokinetic and pharmacodynamic model over 336 h at a starting inoculum of 109 log10 CFU/g. DAP 10 mg/kg/day combinations with AMP, AMX (2 g continuous infusion), CPT 600 mg q 12 h, CRO 2g q 24 h or ERT 1 g q 24 h were evaluated. The emergence of DAP resistance was determined daily over the course of treatment. Results DAP alone was not bactericidal and high-level DAP resistance was observed (MIC increase from 16 to 64 µg/mL). Combination of DAP+AMP offered a significant reduction in log10CFU/g amounts (Up to 7 log10 CFU/g and to detection limits) in 24h in with no emergence of DAP resistance. DAP 10+ AMX caused 6–6.5 log10 CFU/g reduction and counts were maintained around the detection limit while demonstrating no increased resistance. Dose de-escalation with AMP indicated that even DAP 4 mg/kg/d with AMP (2g) combination, reached detection limit at 168 h with no further resistance. None of the CPT, CRO or ERT regimens in combination with DAP was effective against R497 and elevated DAP MICs (>64 µg/mL) was observed during the 14-day model. Conclusion Combination of DAP+AMP offered the most encouraging results against E. faecium R497, while DAP+AMX caused enhanced reduction. The reason for this discrepancy in various β-lactam activity may be related to diverse β-lactam targeting or affinity toward PBP proteins. Further dissection of our observations is warranted to understand the optimized DAP-β-lactam combination and consequently improve patient outcomes and prevention of resistance. Disclosures All authors: No reported disclosures.

Objectives Clinical failures with cefazolin have been described in high-inoculum infections caused by methicillin-susceptible Staphylococcus aureus (MSSA) producing type A β-lactamase. We investigated the prevalence of the cefazolin inoculum effect (InE) in MSSA from South American hospitals, since cefazolin is used routinely against MSSA due to concerns about the in vivo efficacy of isoxazolyl penicillins. Methods MSSA isolates were recovered from bloodstream (n = 296) and osteomyelitis (n = 68) infections in two different multicentre surveillance studies performed in 2001–02 and 2006–08 in South American hospitals. We determined standard-inoculum (105cfu/mL) and high-inoculum (107 cfu/mL) cefazolin MICs. PFGE was performed on all isolates that exhibited a cefazolin InE. Multilocus sequence typing (MLST) and sequencing of part of blaZ were performed on representative isolates. Results The overall prevalence of the cefazolin InE was 36% (131 isolates). A high proportion (50%) of MSSA isolates recovered from osteomyelitis infections exhibited the InE, whereas it was observed in 33% of MSSA recovered from bloodstream infections. Interestingly, Ecuador had the highest prevalence of the InE (45%). Strikingly, 63% of MSSA isolates recovered from osteomyelitis infections in Colombia exhibited the InE. MLST revealed that MSSA isolates exhibiting the InE belonged to diverse genetic backgrounds, including ST5, ST8, ST30 and ST45, which correlated with the prevalent methicillin-resistant S. aureus clones circulating in South America. Types A (66%) and C (31%) were the most prevalent β-lactamases. Conclusions Our results show a high prevalence of the cefazolin InE associated with type A β-lactamase in MSSA isolates from Colombia and Ecuador, suggesting that treatment of deep-seated infections with cefazolin in those countries may be compromised.

We first assessed telavancin (TLV) pharmacokinetics in rats after a single subcutaneous dose of 35 mg/kg of body weight. The pharmacokinetic data were used to predict a TLV dose that simulates human exposure, and the efficacy of TLV was then evaluated using a TLV dose of 21 mg/kg every 12 h against Enterococcus faecalis OG1RF (TLV MIC of 0.06 μg/ml) in a rat endocarditis model with an indwelling catheter. Therapy was given for 3 days with TLV, daptomycin (DAP), or ampicillin (AMP) monotherapy and with combinations of TLV plus AMP, AMP plus gentamicin (GEN), and AMP plus ceftriaxone (CRO); rats were sacrificed 24 h after the last dose. Antibiotics were given to simulate clinically relevant concentrations or as used in other studies. TLV treatment resulted in a significant decrease in bacterial burden (CFU per gram) in vegetations from 6.0 log 10 at time 0 to 3.1 log 10 after 3 days of therapy. Bacterial burdens in vegetations were also significantly lower in the TLV-treated rats than in the AMP ( P = 0.0009)- and AMP-plus-GEN ( P = 0.035)-treated rats but were not significantly different from that of the AMP-plus-CRO-treated rats. Bacterial burdens from vegetations in TLV monotherapy and TLV-plus-AMP-and-DAP groups were similar to each other ( P ≥ 0.05). Our data suggest that further study of TLV as a therapeutic alternative for deep-seated infections caused by vancomycin-susceptible E. faecalis is warranted.

We evaluated ceftobiprole against the well-characterized Enterococcus faecalis strain OG1RF (with and without the β-lactamase [Bla] plasmid pBEM10) in a murine urinary tract infection (UTI) model. Ceftobiprole was equally effective for Bla + and Bla − OG1 strains, while ampicillin was moderately to markedly (depending on the inoculum) less effective against Bla + than Bla − OG1 strains. These data illustrate an in vivo effect on ampicillin of Bla production by E. faecalis and the stability and efficacy of ceftobiprole in experimental UTI.

Daptomycin MICs for enterococci are typically 1- to 2-fold higher than those for Staphylococcus aureus , and there is an imminent need to establish the optimal dose for appropriate treatment of enterococcal infections. We investigated the bactericidal activity of daptomycin at various dose exposures compared to that of linezolid against vancomycin-resistant enterococcus (VRE) in an in vitro pharmacokinetic/pharmacodynamic model utilizing simulated endocardial vegetations over 96 h. Daptomycin at doses of 6, 8, 10, and 12 mg/kg of body weight/day and linezolid at a dose of 600 mg every 12 h were evaluated against two clinical vancomycin-resistant Enterococcus faecium strains (EFm11499 and 09-184D1051), one of which was linezolid resistant (09-184D1051), and one clinical vancomycin-resistant Enterococcus faecalis strain (EFs11496). Daptomycin MICs were 4, 2, and 0.5 μg/ml for EFm11499, 09-184D1051, and EFs11496, respectively. Bactericidal activity, defined as a ≥3 log 10 CFU/g reduction from the initial colony count, was demonstrated against all three isolates with all doses of daptomycin; however, bactericidal activity was not sustained with the daptomycin 6- and 8-mg/kg/day regimens. Linezolid was bacteriostatic against EFm11499 and displayed no appreciable activity against 09-184D1051 or EFs11496. Concentration-dependent killing was displayed with more sustained reduction in colony count (3.58 to 6.46 and 5.89 to 6.56 log 10 CFU/g) at 96 h for the simulated regimen of daptomycin at doses of 10 and 12 mg/kg/day, respectively ( P ≤ 0.012). No E. faecium mutants with reduced susceptibility were recovered at any dosage regimen; however, the E. faecalis strain developed reduced daptomycin susceptibility with daptomycin at 6, 8, and 10 but not at 12 mg/kg/day. Daptomycin displayed a dose-dependent response against three VRE isolates, with high-dose daptomycin producing sustained bactericidal activity. Further research is warranted.

The treatment of infections caused by vancomycin-resistant enterococci (VRE) has become an important clinical challenge and compromises the care of critically ill patients. A striking increase in the frequency of nosocomial isolation of multidrug-resistant Enterococcus faecium has dramatically reduced the therapeutic alternatives because the majority of E. faecium isolates are resistant to ampicillin and vancomycin. Only 2 agents have US Food and Drug Administration approval for the treatment of VRE (E. faecium) infections, namely, linezolid and quinupristin/dalfopristin (Q/D). However, the use of these compounds in severe VRE infections is hampered by the lack of in vivo bactericidal activity, reports of therapeutic failures with monotherapy, a requirement for central venous access for administration (Q/D), and adverse-effect profile. The lipopeptide antimicrobial daptomycin has in vitro bactericidal activity against VRE; however, clinical use of this compound for VRE has not been well studied, and the reports of resistance emerging during therapy at the approved doses are worrisome. Tigecycline has in vitro bacteriostatic activity against VRE, but its clinical use for serious enterococcal infections is unclear due to low serum levels and static effect. Thus, current reliable therapies for VRE appear to be limited, and clinical data that use the above compounds are certainly scant. Oritavancin is an investigational semisynthetic glycopeptide with potent in vitro activity against VRE (both VanA and VanB phenotypes). Although review of the available preclinical data indicates that this compound used as a single agent is likely to have important limitations for the treatment of a severe VRE infection (ie, endocarditis), combination of oritavancin with other agents such as aminoglycosides may offer promise and deserves further investigation, as does use of oritavancin for less serious infections as monotherapy for vancomycin-susceptible and multidrug-resistant enterococci.

Background The inoculum effect (InE) refers to an increase in the MIC of an antibiotic when a large burden of bacteria is present. MSSA producing type A or C β-lactamase (β-lac) that display this effect may be at risk of clinical failure when treated with cefazolin (CFZ) for a deep-seated infection. We have previously shown that CFZ plus clavulanic acid (CL) abolished the InE in vitro. The aim of this study was to evaluate the effectiveness of the combination in vivo at clinically achievable concentrations of both CFZ and CL. Methods S. aureus TX0117, a type A Bla+ clinical isolate from a patient who failed CFZ therapy and TX0117-cured (TX0117c), a derivative of TX0117 which lacks β-lac activity, were used in a rat model of endocarditis. One animal per strain, in addition to historical controls (n = 22), was sacrificed at the start of therapy to assess colony forming units (CFU) per gram of vegetation at T = 0. CFZ 50mg/kg alone (n = 11) or CFZ 50mg/kg plus CL 4mg/kg (n = 7) was given IM every 8 hours for 72 hours. Doses were selected to mimic mean serum concentration of standard doses (given IM (CFZ) or PO (CL)) in humans. Rats were sacrificed 16 hours after the last antibiotic dose, aortic valves were aseptically excised, weighed, homogenized in 1ml of saline and the entire volume was plated in serial 10-fold dilutions on mannitol salt and/or brain-heart infusion agar. Representative recovered colonies were tested for β-lac activity using nitrocefin. Comparisons of CFU between groups were done by the Mann–Whitney, Wilcoxon unpaired test with significance at p Results At baseline, there was no significant difference between the CFU/g of control animals infected with the two strains (TX0117 7.3±1.3 and TX0117c 7.89±0.83, mean log10 ± SD). Compared with untreated controls, the TX0117 group treated with CFZ alone had a reduction of 2±0.6 CFU/g, while the CFZ plus CL arm had a 7.1±0.5 CFU/g reduction, a statistically significant difference between the two arms (P = 0.0002). CFZ treatment of the TX0117c strain lacking blaZ activity was similar to CFZ+CL (6.5±0.6 log10 CFU/g reduction, P Conclusion Against Bla+ TX0117, the addition of CL, at a dose mimicking human PO kinetics, restored the efficacy of CFZ and overcame the InE. This provides a proof-of-concept for the use of oral CL with CFZ when there is a concern for the InE. Disclosures All authors: No reported disclosures.

Daptomycin is a lipopeptide with bactericidal activity that acts on the cell membrane of enterococci and is often used off-label to treat patients infected with vancomycin-resistant enterococci. However, the emergence of resistance to daptomycin during therapy threatens its usefulness.We performed whole-genome sequencing and characterization of the cell envelope of a clinical pair of vancomycin-resistant Enterococcus faecalis isolates from the blood of a patient with fatal bacteremia; one isolate (S613) was from blood drawn before treatment and the other isolate (R712) was from blood drawn after treatment with daptomycin. The minimal inhibitory concentrations (MICs) of these two isolates were 1 and 12 μg per milliliter, respectively. Gene replacements were made to exchange the alleles found in isolate S613 with those in isolate R712.Isolate R712 had in-frame deletions in three genes. Two genes encoded putative enzymes involved in phospholipid metabolism, GdpD (which denotes glycerophosphoryl diester phosphodiesterase) and Cls (which denotes cardiolipin synthetase), and one gene encoded a putative membrane protein, LiaF (which denotes lipid II cycle-interfering antibiotics protein but whose exact function is not known). LiaF is predicted to be a member of a three-component regulatory system (LiaFSR) involved in the stress-sensing response of the cell envelope to antibiotics. Replacement of the liaF allele of isolate S613 with the liaF allele from isolate R712 quadrupled the MIC of daptomycin, whereas replacement of the gdpD allele had no effect on MIC. Replacement of both the liaF and gdpD alleles of isolate S613 with the liaF and gdpD alleles of isolate R712 raised the daptomycin MIC for isolate S613 to 12 μg per milliliter. As compared with isolate S613, isolate R712--the daptomycin-resistant isolate--had changes in the structure of the cell envelope and alterations in membrane permeability and membrane potential.Mutations in genes encoding LiaF and a GdpD-family protein were necessary and sufficient for the development of resistance to daptomycin during the treatment of vancomycin-resistant enterococci. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.).

Evidence-based guidelines for the management of patients with methicillin-resistant Staphylococcus aureus (MRSA) infections were prepared by an Expert Panel of the Infectious Diseases Society of America (IDSA). The guidelines are intended for use by health care providers who care for adult and pediatric patients with MRSA infections. The guidelines discuss the management of a variety of clinical syndromes associated with MRSA disease, including skin and soft tissue infections (SSTI), bacteremia and endocarditis, pneumonia, bone and joint infections, and central nervous system (CNS) infections. Recommendations are provided regarding vancomycin dosing and monitoring, management of infections due to MRSA strains with reduced susceptibility to vancomycin, and vancomycin treatment failures.

Enterococci are organisms with a remarkable ability to adapt to the environment and acquire antibiotic resistance determinants. The evolution of antimicrobial resistance in these organisms poses enormous challenges for clinicians when faced with patients affected with severe infections. The increased prevalence and dissemination of multidrug-resistant Enterococcus faecium worldwide has resulted in a major decrease in therapeutic options because the majority of E. faecium isolates are now resistant to ampicillin and vancomycin, and exhibit high-level resistance to aminoglycosides, which are three of the traditionally most useful anti-enterococcal antibiotics. Newer antibiotics such as linezolid, daptomycin and tigecycline have good in vitro activity against enterococcal isolates, although their clinical use may be limited in certain clinical scenarios as a result of reduced rates of success, possible underdosing for enterococci and low serum levels, respectively, and also by the emergence of resistance. The experimental agent oritavancin may offer some hope for the treatment of vancomycin-resistant enterococci but clinical data are still lacking. Thus, optimal therapies for the treatment of multidrug-resistant enterococcal infections continue to be based on empirical observations and extrapolations from in vitro and animal data. Clinical studies evaluating new strategies, including combination therapies, to treat severe vancomycin-resistant E. faecium infections are urgently needed.

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm-mediated collagen type I adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (20% of the 60 isolates), fibrinogen (17%) and laminin (13%), while only one or two of the isolates adhered to collagen type V, transferrin or lactoferrin and none to the other host components tested. Adherence to fibronectin and laminin was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to fibronectin and laminin, in addition to collagen type I, may have contributed to the emergence and adaptation of E. faecium, in particular CC17, as a nosocomial pathogen.

Antecedentes: el genogrupo de Enterococcus faecium, denominado complejo clonal 17 (CC17), parece poseer múltiples determinantes que aumentan su capacidad para sobrevivir y causar enfermedades en ambientes nosocomiales. Métodos: Utilizando 53 aislados de E. faecium de EE. UU. Clínicamente y geográficamente diversos que datan de 1971 a 1994, se determinó el tipo de secuencia multilocus; la presencia de 16 genes de virulencia putativos (hyl Efm , esp Efmy genes fms); resistencia a ampicilina (AMP) y vancomicina (VAN); y resistencia de alto nivel a gentamicina y estreptomicina. Resultados: En general, se identificaron 16 tipos de secuencia (ST) diferentes, en su mayoría aislados CC17, en 9 regiones diferentes de los Estados Unidos. Los primeros aislamientos de CC17 fueron parte de un brote que ocurrió en 1982 en Richmond, Virginia. Las características de los aislados de CC17 incluyeron aumentos en la resistencia al AMP, la presencia de hyl Efm y esp Efm., aparición de resistencia a VAN y presencia de al menos 13 de 14 genes fms. Sin embargo, ocho de 41 de los primeros aislados con resistencia a AMP no estaban en CC17. Conclusiones: Aunque no todos los primeros aislamientos de AMP de EE. UU. Estaban relacionados clonalmente, los aislamientos de E. faecium CC17 han estado circulando en los Estados Unidos desde al menos 1982 y parecen haber adquirido progresivamente determinantes adicionales de virulencia y resistencia a los antibióticos, lo que quizás explica el éxito reciente de esta especie. en el entorno hospitalario. Background: The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments. Methods: Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994, we determined the multilocus sequence type; the presence of 16 putative virulence genes (hyl Efm, esp Efm, and fms genes); resistance to ampicillin (AMP) and vancomycin (VAN); and high-level resistance to gentamicin and streptomycin. Results: Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the United States. The earliest CC17 isolates were part of an outbreak that occurred in 1982 in Richmond, Virginia. The characteristics of CC17 isolates included increases in resistance to AMP, the presence of hyl Efm and esp Efm, emergence of resistance to VAN, and the presence of at least 13 of 14 fms genes. Eight of 41 of the early isolates with resistance to AMP, however, were not in CC17. Conclusions: Although not all early US AMP isolates were clonally related, E. faecium CC17 isolates have been circulating in the United States since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment.

Methicillin (meticillin)-susceptible Staphylococcus aureus (MSSA) strains producing large amounts of type A β-lactamase (Bla) have been associated with cefazolin failures, but the frequency and impact of these strains have not been well studied. Here we examined 98 MSSA clinical isolates and found that 26% produced type A Bla, 15% type B, 46% type C, and none type D and that 13% lacked blaZ . The cefazolin MIC 90 was 2 μg/ml for a standard inoculum and 32 μg/ml for a high inoculum, with 19% of isolates displaying a pronounced inoculum effect (MICs of ≥16 μg/ml with 10 7 CFU/ml) (9 type A and 10 type C Bla producers). At the high inoculum, type A producers displayed higher cefazolin MICs than type B or C producers, while type B and C producers displayed higher cefamandole MICs. Among isolates from hemodialysis patients with MSSA bacteremia, three from the six patients who experienced cefazolin failure showed a cefazolin inoculum effect, while none from the six patients successfully treated with cefazolin showed an inoculum effect, suggesting an association between these strains and cefazolin failure ( P = 0.09 by Fisher's exact test). In summary, 19% of MSSA clinical isolates showed a pronounced inoculum effect with cefazolin, a phenomenon that could explain the cases of cefazolin failure previously reported for hemodialysis patients with MSSA bacteremia. These results suggest that for serious MSSA infections, the presence of a significant inoculum effect with cefazolin could be associated with clinical failure in patients treated with this cephalosporin, particularly when it is used at low doses.

Since the discovery, development, and US Food and Drug Administration approval of vancomycin in the 1950s, this agent has remained a mainstay for the treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, because of the development of new antistaphylococcal antibiotics and reports of vancomycin failures, the utility of vancomycin has recently been questioned. Although vancomycin did not undergo the strict US Food and Drug Administration approval process that is in place today to demonstrate efficacy, there is considerable information available that sheds light on the role vancomycin has in infectious diseases pharmacotherapy today. In addition, although we look to in vitro susceptibility testing to assess vancomycin activity against S. aureus, we have come to appreciate that resistance of S. aureus to vancomycin can be a continuous--rather than a categorical--phenomenon. This has resulted in clinical microbiology laboratories having difficulty identifying S. aureus that may not respond to conventional doses of vancomycin. A better understanding is needed of the pharmacodynamic relationship between vancomycin and MRSA as relates to optimal dosing strategies, including consideration for loading doses, and development of rational categorical breakpoints for susceptibility based on clinical outcomes. By better understanding these critical issues, it may be possible to optimize the use of vancomycin, resulting in a cost-effective treatment option for many patients infected with MRSA.

Infective endocarditis due to vancomycin-resistant (VR) Enterococcus faecalis has only rarely been reported. We report a case of VR E. faecalis endocarditis that failed to respond to linezolid therapy, outline the virulence traits of the isolate, and review previously published cases of VR E. faecalis endocarditis.

International audience; We report characterization of a methicillin-susceptible, vancomycin-resistant bloodstream isolate of Staphylococcus aureus recovered from a patient in Brazil. Emergence of vancomycin resistance in methicillin-susceptible S. aureus would indicate that this resistance trait might be poised to disseminate more rapidly among S. aureus and represents a major public health threat.

Daptomycin is a lipopeptide antibiotic that is used clinically against many gram-positive bacterial pathogens and is considered a key frontline bactericidal antibiotic to treat multidrug-resistant enterococci. Emergence of daptomycin resistance during therapy of serious enterococcal infections is a major clinical issue. In this work, we show that deletion of the gene encoding the response regulator, LiaR (a member of the LiaFSR system that controls cell envelope homeostasis), from daptomycin-resistant Enterococcus faecalis not only reversed resistance to 2 clinically available cell membrane–targeting antimicrobials (daptomycin and telavancin), but also resulted in hypersusceptibility to these antibiotics and to a variety of antimicrobial peptides of diverse origin and with different mechanisms of action. The changes in susceptibility to these antibiotics and antimicrobial peptides correlated with in vivo attenuation in a Caenorhabditis elegans model. Mechanistically, deletion of liaR altered the localization of cardiolipin microdomains in the cell membrane. Our findings suggest that LiaR is a master regulator of the enterococcal cell membrane response to diverse antimicrobial agents and peptides; as such, LiaR represents a novel target to restore the activity of clinically useful antimicrobials against these organisms and, potentially, increase susceptibility to endogenous antimicrobial peptides.

To evaluate the molecular characteristics and antibiotic susceptibility in biofilm of vancomycin-resistant Enterococcus faecium (VREF) organisms that had caused catheter-related VREF bacteremia (VREF-CRB), we compared 22 isolates causing bacteremia obtained from patients with VREF-CRB with 30 isolates from control patients with gastrointestinal colonization by VREF. Using pulsed-field gel electrophoresis, we identified 17 unique strains among the 22 VREF-CRB isolates and 23 strains among the gastrointestinal isolates. The esp gene was detected in 53% (9 of 17) of the VREF-CRB and 61% (14 of 23) of the control strains ( P = 0.6). VREF-CRB produced heavier biofilm colonization of silicone disks than did control organisms ( P < 0.001). Daptomycin, minocycline, and quinupristin-dalfopristin were each independently more active than linezolid in reducing biofilm colonization by VREF-CRB ( P < 0.01), with daptomycin being the most active, followed by minocycline. In conclusion, the esp gene in VREF is not associated with heavy biofilm colonization or catheter-related bacteremia. In biofilm, daptomycin and minocycline were the most active antibiotics against VREF, and linezolid was the least active.

Enterococcus faecalis , a common cause of endocarditis and known for its capacity to transfer antibiotic resistance to other pathogens, has recently emerged as an important, multidrug-resistant nosocomial pathogen. However, knowledge of its lineages and the potential of particular clones of this species to disseminate and cause disease is limited. Using a nine-gene multilocus sequence typing (MLST) scheme, we identified an evolving and widespread clonal complex of E. faecalis that has caused outbreaks and life-threatening infections. Moreover, this unusual clonal complex was found to contain isolates of unexpected relatedness, including the first known U.S. vancomycin-resistant enterococcus ( E. faecalis strain V583), the first known penicillinase-producing (Bla + ) E. faecalis isolate, and the previously described widespread clone of penicillinase producers, a trait found in E. faecalis isolates. All members of this clonal cluster (designated as BVE for Bla + Van r endocarditis) were found to contain a previously described putative pathogenicity island (PAI). Further analysis of this PAI demonstrated its dissemination worldwide, albeit with considerable variability, confirmed its association with clinical isolates, and found a common insertion site in different clonal lineages. PAI deletions, MLST, and the uncommon resistances were used to predict the evolution of the BVE clonal cluster. The finding of a virulent and highly successful clonal complex of E. faecalis with different members resistant to the primary therapies of choice, ampicillin and vancomycin, has important implications for the evolution of virulence and successful lineages and for public health monitoring and control.

A transcriptional profile of Enterococcus faecalis V583 (V583) treated with erythromycin is presented. This is the first study describing a complete transcriptional profile of Enterococcus. E. faecalis is a common and nonvirulent bacterium in many natural environments, but also an important cause of nosocomial infections. We have used a genome-wide microarray based on the genome sequence of V583 to study gene expression in cells exposed to erythromycin. V583 is resistant to relatively high concentrations of erythromycin, but growth is retarded by the treatment. The effect of erythromycin treatment on V583 was studied by a time course experiment; samples were extracted at five time points over a period of 90 min. A drastic change in gene transcription was seen with the erythromycin-treated cells compared to the untreated cells. Altogether, 260 genes were down-regulated at one or more time points, while 340 genes were up-regulated. Genes encoding hypothetical proteins and genes encoding transport and binding proteins were the two most dominating groups of differentially expressed genes. The gene encoding ermB (EFA0007) was expressed, but not differentially, which indicated that other genes are important for the survival and growth maintenance of V583 treated with erythromycin. One of these genes is a putative MsrC-like protein, which was up-regulated at all time points studied. Other specific genes that were found to be up-regulated were genes encoding ABC transporters and two-component regulatory systems, and these may be genes that are important for the specific response of V583 to erythromycin.

Our experience with a patient with methicillin-susceptible Staphylococcus aureus aortic native valve endocarditis, who had a relapse involving fever and positive blood culture results while receiving cefazolin, led us to evaluate this organism's ability to hydrolyze cefazolin at high inocula, a previously well-documented phenomenon. Analysis of the infecting strain disclosed a high minimum inhibitory concentration of cefazolin when a large inoculum was used, as well as rapid and complete cefazolin degradation, which was associated with regrowth in a time-kill experiment. DNA sequencing of the beta-lactamase gene showed that it was identical to that of the S. aureus type A beta-lactamase, known to efficiently inactivate cefazolin. A word of caution is given regarding the use of this antibiotic for treatment of endocarditis caused by this type of S. aureus isolate.

A novel glycylcycline agent, tigecycline (GAR-936), was evaluated in vivo in the mouse model of peritonitis against three Enterococcus faecalis and four Enterococcus faecium isolates with different susceptibilities to vancomycin and tetracyclines, all of which were inhibited by ≤0.125 μg of tigecycline/ml. Using a single subcutaneous dose, tigecycline displayed a protective effect (50% protective dose, ≤5.7 mg/kg of body weight) against all strains tested, including two with Tn 925 (from the Tn 916 family), which contains the Tet(M) tetracycline resistance determinant, as well as VanA and VanB strains. As expected, tetracycline and minocycline were ineffective against the isolates carrying Tn 925 .

Eighty-one endocarditis-derived Enterococcus faecalis isolates that were collected from individual patients in the United States between 1974 and 2004 were sequence typed and analyzed for the presence of various genes, including some previously associated with virulence. Overall, using our previously described trilocus sequence typing (TLST), 44 different sequence types (STs) were found within this collection; 26 isolates were singletons (a unique TLST sequence type [ST T ]), some ST T s contained multiple isolates (up to 6 isolates), and 16% of the isolates (13 isolates) could be grouped by additional sequence typing into clonal cluster 21 (CC21). Of note, only four isolates (7%) of the 56 whose multilocus sequence types were determined were found to belong to one of the previously described hospital-associated clonal clusters CC2 and CC9, and only 15% and 37% of all isolates had high-level resistance to gentamicin and streptomycin, respectively, including 10% that were resistant to both. We also found that 64% of the isolates lacked the genes for production of capsule polysaccharide, which has been proposed to enhance the pathogenic potential of the hospital-associated clonal clusters. In summary, while our collection is not a random sample of cases of E. faecalis endocarditis, these results indicate that nonencapsulated strains belonging to non-hospital-associated lineages were predominant among endocarditis E. faecalis isolates recovered during this time period.

During a study to investigate the evolution of ampicillin resistance in Enterococcus faecium , we observed that a number of E. faecium strains, mainly from the recently described subclade A2, showed PBP5 sequences in between PBP5-S and PBP5-R. These hybrid PBP5-S/R patterns reveal a progression of amino acid changes from the S form to the R form of this protein; however, these changes do not strictly correlate with changes in ampicillin MICs.

We report the case of a patient from Brazil with a bloodstream infection caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA) that was susceptible to vancomycin (designated BR-VSSA) but that acquired the vanA gene cluster during antibiotic therapy and became resistant to vancomycin (designated BR-VRSA). Both strains belong to the sequence type (ST) 8 community-associated genetic lineage that carries the staphylococcal chromosomal cassette mec (SCCmec) type IVa and the S. aureus protein A gene (spa) type t292 and are phylogenetically related to MRSA lineage USA300. A conjugative plasmid of 55,706 bp (pBRZ01) carrying the vanA cluster was identified and readily transferred to other staphylococci. The pBRZ01 plasmid harbors DNA sequences that are typical of the plasmid-associated replication genes rep24 or rep21 described in community-associated MRSA strains from Australia (pWBG745). The presence and dissemination of community-associated MRSA containing vanA could become a serious public health concern.

Enterococcus spp. are among the common pathogens causing infective endocarditis (IE). Despite major medical advances and new potent antimicrobial agents, the mortality has not significantly improved for several decades. The usual lack of bactericidal activity of penicillin or ampicillin, the toxicity from the combination of penicillin plus aminoglycosides, and the increased reports of high-level resistance to aminoglycosides have led to the exploration of other regimens for treatment of Enterococcus faecalis IE. As an example, ampicillin plus ceftriaxone is now a well-recognized regimen for this organism. However, the emerging of new drug resistances in Enterococcus faecium dramatically reduces the therapeutic alternatives for this organism in IE which continues to be an immense challenge for clinicians even with the availability of newer antimicrobial agents. This article summarizes the current treatment options for enterococcal endocarditis and reviews of recent publications on the topic.

Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a “last-resort” antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium . Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium , we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120→Ala and Trp73→Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.