Vancomycin Resistance in Enterococcus faecium from the Dallas, Texas, Area Is Conferred Predominantly on pRUM-Like Plasmids.

Vancomycin-resistant E. faecium (VREfm) is a significant public health concern because of limited treatment options. Genomic surveillance can be used to monitor VREfm transmission and evolution. Genomic analysis of VREfm has not been reported for the Dallas/Fort Worth/Arlington, TX, area, which is currently the 4th largest metropolitan area in the United States. Our study aimed to address this gap in knowledge by analyzing the genomes of 46 VREfm strains and 1 vancomycin-sensitive comparator collected during routine fecal surveillance of high-risk patients upon admission to a Dallas, TX, hospital system (August to October 2015). Thirty-one complete and 16 draft genome sequences were generated. The closed VREfm genomes possessed up to 12 extrachromosomal elements each. Overall, 251 closed putative plasmid sequences assigned to previously described and newly defined rep family types were obtained. Phylogenetic analysis identified 10 different sequence types (STs) among the isolates, with the most prevalent being ST17 and ST18. Strikingly, all but three of the VREfm isolates encoded vanA-type vancomycin resistance within Tn1546-like elements on a pRUM-like (rep17) plasmid backbone. Relative to a previously reported typing scheme for the vanA-carrying Tn1546, new variants of the Tn1546 were identified that harbored a combination of 7 insertion sequences (IS), including 3 novel IS elements reported here (ISEfa16, ISEfa17, and ISEfa18). We conclude that pRUM-like plasmids are important vectors for vancomycin resistance in the Dallas, TX, area and should be a focus of plasmid surveillance efforts. IMPORTANCE Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium. Among E. faecium strains, vancomycin resistance genes can be disseminated by plasmids with different host ranges and transfer efficiencies. Surveillance of resistance plasmids is critical to understanding antibiotic resistance transmission. This study analyzed the genome sequences of VREfm isolates collected from the Dallas, TX, area, with particular focus on the mobile elements associated with vancomycin resistance genes. We found that a single plasmid family, the pRUM-like family, was associated with vancomycin resistance in the majority of isolates sampled. Our work suggests that the pRUM-like plasmids should continue to be studied to understand their mechanisms of maintenance, transmission, and evolution in VREfm.

Comparative Genomics of Streptococcus oralis Identifies Large Scale Homologous Recombination and a Genetic Variant Associated with Infection.

The viridans group streptococci (VGS) are a large consortium of commensal streptococci that colonize the human body. Many species within this group are opportunistic pathogens causing bacteremia and infective endocarditis (IE), yet little is known about why some strains cause invasive disease. Identification of virulence determinants is complicated by the difficulty of distinguishing between the closely related species of this group. Here, we analyzed genomic data from VGS that were isolated from blood cultures in patients with invasive infections and oral swabs of healthy volunteers and then determined the best-performing methods for species identification. Using whole-genome sequence data, we characterized the population structure of a diverse sample of Streptococcus oralis isolates and found evidence of frequent recombination. We used multiple genome-wide association study tools to identify candidate determinants of invasiveness. These tools gave consistent results, leading to the discovery of a single synonymous single nucleotide polymorphism (SNP) that was significantly associated with invasiveness. This SNP was within a previously undescribed gene that was conserved across the majority of VGS species. Using the growth in the presence of human serum and a simulated infective endocarditis vegetation model, we were unable to identify a phenotype for the enriched allele in laboratory assays, suggesting a phenotype may be specific to natural infection. These data highlighted the power of analyzing natural populations for gaining insight into pathogenicity, particularly for organisms with complex population structures like the VGS. IMPORTANCE The viridians group streptococci (VGS) are a large collection of closely related commensal streptococci, with many being opportunistic pathogens causing invasive diseases, such as bacteremia and infective endocarditis. Little is known about virulence determinants in these species, and there is a distinct lack of genomic information available for the VGS. In this study, we collected VGS isolates from invasive infections and healthy volunteers and performed whole-genome sequencing for a suite of downstream analyses. We focused on a diverse sample of Streptococcus oralis genomes and identified high rates of recombination in the population as well as a single genome variant highly enriched in invasive isolates. The variant lies within a previously uncharacterized gene, nrdM, which shared homology with the anaerobic ribonucleoside triphosphate reductase, nrdD, and was highly conserved among VGS. This work increased our knowledge of VGS genomics and indicated that differences in virulence potential among S. oralis isolates were, at least in part, genetically determined.

Characterization of presumptive vancomycin-resistant enterococci recovered during infection control surveillance in Dallas, Texas, USA.

Enterococcus faecalis and E. faecium are Gram-positive bacteria that normally inhabit the human gastrointestinal tract. They are also opportunistic pathogens and can cause nosocomial infection outbreaks. To prevent the spread of nosocomial infections, hospitals may rely on screening methods to identify patients colonized with multidrug-resistant organisms including vancomycin-resistant enterococci (VRE). Spectra VRE agar (Remel) contains vancomycin and other medium components that select for VRE and phenotypically differentiate between E. faecalis and E. faecium by colony colour. We obtained 66 de-identified rectal swab cultures on Spectra VRE agar that were obtained during routine patient admission surveillance at a hospital system in Dallas, Texas, USA. We analysed 90 presumptive VRE from 61 of the Spectra VRE agar cultures using molecular and culture methods. Using ddl typing, 55 were found to be E. faecium and 32 were found to be E. faecalis . While most of the E. faecium were positive for the vanA gene by PCR (52 of 55 strains), few of the E. faecalis were positive for either vanA or vanB (five of 32 strains). The 27 E. faecalis vanA- and vanB-negative strains could not be recultured on Spectra VRE agar. Overall, we found that Spectra VRE agar performed robustly for the identification of vancomycin-resistant E. faecium , but presumptive false positives were obtained for vancomycin-resistant E. faecalis .

Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria.

Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria Streptococcus mitis and S. oralis These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 µg/ml) daptomycin resistance rapidly emerges in S. mitis and S. oralis after a single drug exposure. In this study, we found that inactivating mutations in cdsA are associated with high-level daptomycin resistance in S. mitis and S. oralis IE isolates. This is surprising given that cdsA is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of cdsA not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.

Pronounced heterogeneity observed in high-level daptomycin-resistant viridans group streptococci.

Viridans group streptococci (VGS) have demonstrated high-level daptomycin resistance (HLDR) upon daptomycin exposure. This study evaluated the extent of heterogeneity and whether dose escalation or combination therapy could prevent resistance development. Five VGS strains (daptomycin MICs 0.25-2mg/L) were evaluated. In vitro models utilised simulated daptomycin dosages of 4, 6, 8 and 12mg/kg with estimated fCmax of 4.1, 6.6, 8.6 and 12.9mg/L, respectively. Time-kill studies included fCmax simulations of daptomycin alone or combined with ceftriaxone, gentamicin, linezolid, rifampicin or vancomycin. Population analyses were performed on daptomycin-containing and non-containing agar plates. Extreme heterogeneity was observed in four strains with daptomycin population MICs 4-512-fold higher than broth microdilution. Whilst Streptococcus gordonii 1649 did not consistently develop HLDR, its population MIC was above the established daptomycin breakpoint. In vitro modelling demonstrated initial kill by daptomycin in all strains within 8h, with substantial re-growth by 24h despite increasing daptomycin. Daptomycin kill curves also displayed resistance development by 24h. However, synergy or additivity was noted for most regimens and strains. Synergy was most notable with daptomycin plus linezolid or rifampicin. Overall, daptomycin plus ceftriaxone or gentamicin were the most potent regimens. Gentamicin or rifampicin with daptomycin were least additive. For combination regimens with colonies isolated at 24h, HLDR was reduced 16-64-fold (MICs 4-16mg/L). Daptomycin monotherapy for VGS led to rapid development of HLDR likely due to extreme heterogeneity. Combination therapy suppressed or minimised the degree of resistance although the mechanism remains unknown.

Ceftazidime/Avibactam: Who Says You Can't Teach an Old Drug New Tricks?

PURPOSE: Gram-negative resistance continues to rise with treatment options becoming more limited. Ceftazidime/avibactam was recently approved in the United States and Europe, which combines an established third-generation cephalosporin with a new, unique, non-ß-lactam ß-lactamase inhibitor. This review conducts a thorough examination of structure, pharmacology, spectrum of activity, pharmacokinetics/pharmacodynamics, in vitro and clinical efficacy and safety/tolerability of ceftazidime/avibactam, as well as detailed future directions for the agent. METHODS: Pubmed and clinicaltrials.gov searches, as well as abstracts from the 2015 Interscience Conference on Antimicrobial Agents and Chemotherapy/International Society of Chemotherapy (ICAAC/ICC) and ID Week meetings and the 2016 American Society of Microbiology Microbe meeting, were conducted from January 2004 - September 2016. Relevant search terms included ceftazidime, ceftazidime/avibactam, avibactam, NXL104 and AVE1330A. The US package insert for ceftazidime/avibactam (02/2015) and European public assessment report (06/2016) were also reviewed. RESULTS: In vitro susceptibility for ceftazidime/avibactam displayed potent activity against many Enterobacteriaceae including extended-spectrum-ß-lactamase (ESBL) and carbapenemase-producing strains, as well as Pseudomonas aeruginosa. Phase II clinical trials utilized for approval demonstrated comparable safety and efficacy to imipenem/cilistatin for treatment of complicated urinary tract infections (70.4% vs. 71.4%) and combined with metronidazole compared to meropenem in complicated intra-abdominal infections (91.2% vs 93.4%). Phase III data displayed non-inferior efficacy of ceftazidime/avibactam compared to doripenem for complicated urinary tract infections (70.2% vs 66.2%) and combined with metronidazole compared to meropenem in complicated intra-abdominal infections (82.5% vs 84.9%), as well as comparable safety. Ceftazidime/avibactam was well-tolerated but does require renal adjustments. Additionally, 3 case series and a single case report have demonstrated the potential for ceftazidime/avibactam against multidrug resistant organisms for compassionate use or failure after previous therapy. CONCLUSION: By adding avibactam to ceftazidime, clinicians' antimicrobial armamentarium is expanded, potentially increasing the ability to combat multi-drug resistant gram-negative pathogens, particularly ESBL and carbapenemase-producing organisms, as well as Pseudomonas aeruginosa. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Characterization of high-level daptomycin resistance in Viridans group Streptococci developed upon in vitro exposure to daptomycin.

Viridans group streptococci (VGS) are part of the normal flora that may cause bacteremia, often leading to endocarditis. We evaluated daptomycin against four clinical strains of VGS (MICs = 1 or 2 µg/ml) using an in vitro-simulated endocardial vegetation model, a simulated bacteremia model, and kill curves. Daptomycin exposure was simulated at 6 mg/kg of body weight and 8 mg/kg every 24 h for endocardial and bacteremia models. Total drug concentrations were used for analyses containing protein (albumin and pooled human serum), and free (unbound) drug concentrations (93% protein bound) were used for analyses not containing protein. Daptomycin MICs in the presence of protein were significantly higher than those in the absence of protein. Despite MICs below or at the susceptible breakpoint, all daptomycin regimens demonstrated limited kill in both pharmacodynamic models. A reduction of approximately 1 to 2 log10 CFU was seen for all isolates and dosages except daptomycin at 6 mg/kg, which achieved a reduction of 2.7 log10 CFU/g against one strain (Streptococcus gordonii 1649) in the endocardial model. Activity was similar in both pharmacodynamic models in the presence or absence of protein. Similar activity was noted in the kill curves over all multiples of the MIC. Regrowth by 24 h was seen even at 8× MIC. Postexposure daptomycin MICs for both pharmacodynamic models increased to >256 µg/ml for all isolates by 24 and 72 h. Despite susceptibility to daptomycin by standard MIC methods, these VGS developed high-level daptomycin resistance (HLDR) after a short duration following drug exposure not attributed to modification or inactivation of daptomycin. Further evaluation is warranted to determine the mechanism of resistance and clinical implications.

Single-dose pharmacokinetics and tolerability of daptomycin 8 to 10 mg/kg in children aged 2 to 6 years with suspected or proved Gram-positive infections.

A pharmacokinetic analysis was performed for single intravenous doses of daptomycin 8 or 10 mg/kg in subjects aged 2 to 6 years. Proportional increases in maximum plasma concentration (68.4 µg/mL, 79.2 µg/mL) and area under the curve (429.1 µg · h/mL, 549.7 µg · h/mL) were observed for each dose cohort, respectively. Half-life, clearance, and distribution volume were similar between groups. Both doses were well tolerated.

Pharmacokinetics of daptomycin in a critically ill adolescent with vancomycin-resistant enterococcal endocarditis.

Daptomycin is a lipopeptide antibiotic active against multidrug-resistant gram-positive organisms. Our search of the literature found no published pediatric pharmacokinetic data. We report the use of pharmacokinetic analysis of daptomycin in a 13-year-old boy with vancomycin-resistant Enterococcus faecium endocarditis. Pharamcokinetic parameters were found to be significantly different from published adult parameters, such as a faster elimination rate, shorter half-life, and increased clearance. These age-related differences in the pharmacokinetic profile of daptomycin have significant dosing implications. As the use of this drug for off-label indications and in pediatric populations increases, it is important for clinicians to better understand the drug's pharmacokinetic profile in these patient populations.

Gram-positive resistance: pathogens, implications, and treatment options: insights from the Society of Infectious Diseases Pharmacists.

Despite the advent of new antibiotics, resistance in gram-positive pathogens, including staphylococci and enterococci, continues to increase. This is evident with the recent emergence of vancomycin-resistant Staphylococcus aureus . Newer treatment agents are available, including quinupristin-dalfopristin, linezolid, and daptomycin. In addition, investigational agents are being explored. Clinical trials have been conducted for various infections, such as skin and skin structure infections, pneumonia, and bloodstream infections. Antibacterial activity, site of infection, and potential for adverse effects must be taken into account when making decisions regarding therapy.

Urinary tract infection caused by Aerococcus viridans, a case report.

BACKGROUND: Aerococcus viridans organisms are gram-positive, usually airborne cocci that are widely distributed in hospital environments. These bacteria have infrequently been encountered as a human pathogen causing bacteremia, endocarditis and urinary tract infections. The clinical significance of these bacteria may be overlooked due to their fastidious growth and often confused with other strains of streptococci. CASE REPORT: We report a case of urinary tract infection with bacteremia caused by A. viridans in an 87 year-old male nursing home resident. The patient presented with a fever of 103 degrees F, dysuria, hematuria and weakness for three days. Urinalysis showed large amount of blood, more than one hundred white cells/HPF and 4+ bacteria. Laboratory tests revealed, white blood cell count of 2300/cu mm (neutrophils 80%, bands 7%, lymphocytes 11% and monocytes 2%), hemoglobin 15.4 gm/dL, blood urea nitrogen 23 mg/dL and creatinine 1.2 mg/dL. Urine culture yielded growth of 10(5) CFU of A. viridans. The patient was treated for ten days with levofloxacin (both IV + PO). CONCLUSIONS: To date, no clinical case report of this nature has been described implicating A. viridans in urinary tract infections. Increased awareness and more studies of this genus should lead to the identification of their potential role in human infections.

Linezolid, levofloxacin, and vancomycin against vancomycin-tolerant and fluoroquinolone-resistant Streptococcus pneumoniae in an in vitro pharmacodynamic model.

STUDY OBJECTIVE: To compare the pharmacodynamic profiles of linezolid, levofloxacin, and vancomycin against clinical strains of Streptococcus pneumoniae, including vancomycin-tolerant and fluoroquinolone-resistant isolates. DESIGN: In vitro pharmacodynamic model. SETTING: Biosafety level 2, university research laboratory. BACTERIAL STRAINS: Ciprofloxacin-susceptible (79), ciprofloxacin-resistant (R921), and vancomycin-tolerant (P9802-020) clinical strains of S. pneumoniae. INTERVENTION: An in vitro pharmacodynamic model was used to simulate standard dosing regimens of linezolid, levofloxacin, and vancomycin against the isolates 79, R921, and P9802-020. MEASUREMENTS AND MAIN RESULTS: Bacterial density was profiled over 48 hours. Minimum inhibitory concentrations (MICs) for linezolid, levofloxacin, and vancomycin, respectively were 1, 1, 0.5 microg/ml for isolate 79; 1, 4, 0.5 microg/ml for R921; and 0.5, 0.5, 0.5 microg/ml for P9802-020. Vancomycin minimum bactericidal concentration (MBC) values varied across large ranges for the tested strains. Linezolid achieved 99.9% kill against 79 and R921 by 24 and 28 hours, respectively. Levofloxacin achieved 99.9% kill against 79 and P9802-020 by 28 and 4 hours, respectively. Vancomycin achieved 99.9% kill against 79 and R921 by 8 and 24 hours, respectively. Levofloxacin did not demonstrate activity against R921 at the 48-hour end point. Minimal kill (< 2 log) at 48 hours was noted for vancomycin and linezolid against P9802-020. Conclusion. Vancomycin tolerance appeared to be more reliably characterized by persistent viability in time-kill analyses than by MBC:MIC ratios. Vancomycin exhibited bactericidal activity against the non-vancomycin-tolerant strains of S. pneumoniae. Linezolid exhibited both bactericidal and bacteriostatic activity against all three strains tested, whereas levofloxacin demonstrated bactericidal activity against the fluoroquinolone-susceptible isolates. Further investigation of treatment alternatives for infections due to vancomycin-tolerant S. pneumoniae are needed.

Pharmacokinetics and pharmacodynamics of cefepime in patients with various degrees of renal function.

This study evaluated the pharmacokinetics of cefepime in 36 patients with different levels of renal function. Pharmacokinetic and pharmacodynamic parameters were calculated using samples obtained at steady state. Patients with creatinine clearance (CL(CR)) of >100 ml/min had more rapid clearance (CL) and a lower minimum concentration in serum (C(min)). C(min) in this group was found to be 3.3 +/- 3.6 mg/liter (mean and standard deviation), compared to 19.5 +/- 21.5 mg/liter in patients with a CL(CR) of between 60 and 100 ml/min (P = 0.025) and 14.0 +/- 11.5 mg/liter in patients with a CL(CR) of <60 ml/min (P = 0.009). Patient data were also analyzed by the nonparametric expectation maximization method and Bayesian forecasting. The median volume of distribution in the central compartment was 27.08 liters. CL and CL(CR) were highly correlated (P = 0.00033) according to the equation CL= 0.324 liters/h + (0.0551 x CL(CR)). The median rate constants from the central compartment to the peripheral compartment and from the peripheral compartment to the central compartment were 12.58 and 41.09 h(-1), respectively. The time-concentration profiles for 1,000 patients (CL(CR)s, 120, 60, and 30 ml/min) each receiving various dosing regimens were simulated by using Monte Carlo simulations. Standard dosing resulted in a C(min) that was greater than or equal to the MIC in more than 80% of the simulated profiles with MICs < or = 2 mg/liter. Current dosing recommendations may be suboptimal for monotherapy of infections due to less susceptible pathogens (e.g., those for which MICs are > or = 4 mg/liter), particularly when CL(CR) exceeds 120 ml/min.

Pharmacodynamics of cefepime in patients with Gram-negative infections.

We conducted a prospective, open-label study to delineate a relationship between exposure and outcomes in 36 patients treated with cefepime. Twenty patients had documented Gram-negative infections. Timed blood and urine samples were obtained at steady state to determine pharmacokinetic and pharmacodynamic parameters. Microbiological success was significantly correlated with the proportion of the dosing interval that cefepime concentrations exceeded 4.3 x MIC. Our results support in vitro data that suggest bactericidal activity of beta-lactams is optimized at concentrations approximately 4 x MIC. These results should be validated by large prospective clinical trials.