Whole-genome sequencing of carbapenem-resistant Enterobacterales isolates in southeast Louisiana reveals persistent genetic clusters spanning multiple locations.

BACKGROUND: We investigated 51 g-negative carbapenem-resistant Enterobacterales (CRE) isolates collected from 22 patients over a five-year period from six health care institutions in the Ochsner Health network in southeast Louisiana. METHODS: Short genomic reads were generated using Illumina sequencing and assembled for each isolate. Isolates were classified as Enterobacter spp. (n = 20), Klebsiella spp. (n = 30), and Escherichia coli (n = 1) and grouped into 19 different multi-locus sequence types (MLST). Species and patient-specific core genomes were constructed representing ∼50% of the chromosomal genome. RESULTS: We identified two sets of patients with genetically related infections; in both cases, the related isolates were collected > 6 months apart, and in one case, the isolates were collected in different locations. On the other hand, we identified four sets of patients with isolates of the same species collected within 21 days from the same location; however, none had genetically related infections. Genes associated with resistance to carbapenem drugs (blaKPC and/or blaCTX-M-15) were found in 76% of the isolates. We found three blaKPC variants (blaKPC-2, blaKPC-3, and blaKPC-4) associated with four different Enterobacter MLST variants, and two blaKPC variants (blaKPC-2, blaKPC-3) associated with seven different Klebsiella MLST variants. CONCLUSIONS: Molecular surveillance is increasingly becoming a powerful tool to understand bacterial spread in both community and clinical settings. This study provides evidence that genetically related infections in clinical settings do not necessarily reflect temporal associations, and vice versa. Our results also highlight the regional genomic and resistance diversity within related bacterial lineages.

Comparing antimicrobial resistant genes and phenotypes across multiple sequencing platforms and assays for Enterobacterales clinical isolates.

INTRODUCTION: Whole genome sequencing (WGS) of bacterial isolates can be used to identify antimicrobial resistance (AMR) genes. Previous studies have shown that genotype-based AMR has variable accuracy for predicting carbapenem resistance in carbapenem-resistant Enterobacterales (CRE); however, the majority of these studies used short-read platforms (e.g. Illumina) to generate sequence data. In this study, our objective was to determine whether Oxford Nanopore Technologies (ONT) long-read WGS would improve detection of carbapenem AMR genes with respect to short-read only WGS for nine clinical CRE samples. We measured the minimum inhibitory breakpoint (MIC) using two phenotype assays (MicroScan and ETEST) for six antibiotics, including two carbapenems (meropenem and ertapenem) and four non-carbapenems (gentamicin, ciprofloxacin, cefepime, and trimethoprim/sulfamethoxazole). We generated short-read data using the Illumina NextSeq and long-read data using the ONT MinION. Four assembly methods were compared: ONT-only assembly; ONT-only assembly plus short-read polish; ONT + short-read hybrid assembly plus short-read polish; short-read only assembly. RESULTS: Consistent with previous studies, our results suggest that the hybrid assembly produced the highest quality results as measured by gene completeness and contig circularization. However, ONT-only methods had minimal impact on the detection of AMR genes and plasmids compared to short-read methods, although, notably, differences in gene copy number differed between methods. All four assembly methods showed identical presence/absence of the blaKPC-2 carbapenemase gene for all samples. The two phenotype assays showed 100% concordant results for the non-carbapenems, but only 65% concordance for the two carbapenems. The presence/absence of AMR genes was 100% concordant with AMR phenotypes for all four non-carbapenem drugs, although only 22%-50% sensitivity for the carbapenems. CONCLUSIONS: Overall, these findings suggest that the lack of complete correspondence between CRE AMR genotype and phenotype for carbapenems, while concerning, is independent of sequencing platform/assembly method.

Genetic Divergence of Vibrio vulnificus Clinical Isolates with Mild to Severe Outcomes.

The marine bacterium Vibrio vulnificus infects humans via food or water contamination, leading to serious manifestations, including gastroenteritis, wound infections, and septic shock. Previous studies suggest phylogenetic Lineage 1 isolates with the vcgC allele of the vcg gene cause human infections, whereas Lineage 2 isolates with the vcgE allele are less pathogenic. Mouse studies suggest that some variants of the primary toxin could drive more serious infections. A collection of 109 V. vulnificus United States human clinical isolates from 2001 to 2019 with paired clinical outcome data were assembled. The isolates underwent whole-genome sequencing, multilocus-sequence phylogenetic analysis, and toxinotype analysis of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin. In contrast to prior reports, clinical isolates were equally distributed between lineages. We found no correlation between phylogenetic lineage or MARTX toxinotype and disease severity. Infections caused by isolates in Lineage 1 demonstrated a borderline statistically significant higher mortality. Lineage 1 isolates had a trend toward a higher proportion of M-type MARTX toxins compared with Lineage 2, although this was not statistically significant. IMPORTANCE Vibrio vulnificus is an aquatic pathogen that is capable of causing severe disease in humans. Previous studies have suggested that pathogenic isolates were restricted to certain phylogenetic lineages and possibly toxinotype. Our study demonstrated that phylogenetic lineage and multifunctional autoprocessing repeats-in-toxin (MARTX) toxinotype do not predict severity of infection. V. vulnificus strains capable of causing severe human disease are not concentrated in Lineage 1 but are genetically diverse. Thus, food surveillance based on lineage type or toxinotype may not be an appropriate intervention measure to control this rare but serious infection.

Antimicrobial and resource utilization with T2 magnetic resonance for rapid diagnosis of bloodstream infections: systematic review with meta-analysis of controlled studies.

Objectives: To compare antimicrobial and resource utilization with T2 Magnetic Resonance (T2MR) versus blood culture (BC) in patients with suspected bloodstream infection.Methods: We systematically searched MEDLINE, EMBASE, and CENTRAL for randomized trials or observational controlled studies of patients with suspected bloodstream infection receiving a diagnosis with T2MR or BC. Using an inverse variance meta-analysis model, we reported mortality using the risk ratio (RR) and the remaining outcomes as the mean difference (MD).Results: Fourteen studies were included in the meta-analysis. Time to detection (MD = -81 hours; p < 0.001) and time to species identification (MD = -77 hours; p < 0.001) were faster with T2MR. Patients testing positive on T2MR received targeted antimicrobial therapy faster (-42 hours; p < 0.001) and patients testing negative on T2MR were de-escalated from empirical therapy faster (-7 hours; p = 0.02) vs. BC. Length of intensive care unit stay (MD = -5.0 days; p = 0.03) and hospital stay (MD = -4.8 days; p = 0.03) were shorter with T2MR. Mortality rates were comparable between T2MR and BC (28.9% vs. 29.9%, RR = 1.02, p = 0.86).Conclusion: Utilization of T2MR for identification of bloodstream pathogens provides faster time to detection, faster transition to targeted microbial therapy, faster de-escalation of empirical therapy, shorter ICU and hospital stay, and with comparable mortality rate versus BC.

In vitro interaction of fluconazole and trimethoprim-sulfamethoxazole against Candida auris using ETEST and checkerboard methods.

Candida auris was discovered in 2009 and has rapidly emerged as a serious public health threat with cases reported in over 20 countries worldwide. As of May 8, 2020, the Centers for Disease Control and Prevention reported a total of 1122 US cases. C. auris is often multidrug resistant, leaving few options for treatment. Sulfonamides are known to inhibit a bacterial enzyme involved in folate synthesis and may also inhibit yeast organisms by a similar mechanism. The combination of trimethoprim and sulfamethoxazole is more commonly used than either drug alone. The objective of this study was to evaluate the combination of fluconazole and trimethoprim-sulfamethoxazole against C. auris Minimum inhibitory concentrations (MICs) of fluconazole and trimethoprim-sulfamethoxazole were determined by ETEST and broth microdilution for 11 Cauris strains. Fluconazole MICs (µg/mL) were 4->256 by ETEST and 2->256 by broth microdilution (73% resistant); trimethoprim-sulfamethoxazole MICs were >32 by ETEST and 32->128 by broth microdilution (no interpretive guidelines for C. auris). Using our MIC: MIC ETEST method and a checkerboard method, we investigated the interaction of fluconazole and trimethoprim-sulfamethoxazole against all isolates. These interactions were analyzed by calculating the summation fractional inhibitory concentration with synergyof ≤0.5, additivity of >0.5-1.0, indifference of >1-4, and antagonism of >4. The combination of fluconazole and trimethoprim-sulfamethoxazole revealed synergy with three (27%) and additivity with one (9%) isolate. Indifference was found for the remaining seven (64%) isolates. With the checkerboard method, synergy was seen in 1/11 (9%) isolates with fluconazole (½ MIC) plus trimethoprim-sulfamethoxazole (1/64 MIC); additivity, in 7/11 (64%) isolates with fluconazole (1/8 MIC-1×MIC) plus trimethoprim-sulfamethoxazole (1/128 MIC-½ MIC); and indifference in 3/11 (27%) isolates. Regardless, in vitro interactions may or may not correlate with clinical outcomes. Synergy testing with additional drug combinations and isolates should be performed.

Evaluation of the in vitro interaction of fosfomycin and meropenem against metallo-ß-lactamase-producing Pseudomonas aeruginosa using Etest and time-kill assay.

Pseudomonas aeruginosa is a nosocomial pathogen containing various resistance mechanisms. Among them, metallo-ß-lactamase (MBL)-producing Pseudomonas are difficult to treat. Fosfomycin is an older antibiotic that has recently seen increased usage due to its activity against a broad spectrum of multidrug-resistant organisms. Our aim was to evaluate the combination of fosfomycin and meropenem against 20 MBL-producing P. aeruginosa (100% meropenem-resistant and 20% fosfomycin-resistant) using both an Etest minimal inhibitory concentration (MIC): MIC method and time-kill assay. MICs for fosfomycin and meropenem were determined by Etest and by broth microdilution method for the latter. The combination demonstrated synergy by Etest in 3/20 (15%) isolates and 5/20 (25%) isolates by time-kill assay. Results from the Etest method and time-kill assay were in agreement for 14/20 (70%) of isolates. No antagonism was found. Comparing both methods, Etest MIC: MIC method may be useful to rapidly evaluate other antimicrobial combinations.

In vitro synergy with fosfomycin plus doxycyclin against linezolid and vancomycin-resistant Enterococcus faecium.

OBJECTIVE: Linezolid and vancomycin-resistant Enterococcus faecium (LRVREF) is globally emerging as a urinary nosocomial pathogen. A decrease in the amount of successful treatment options has created a necessity for the development of novel antimicrobial therapies. Combination therapy may provide an effective alternative for treatment. Fosfomycin and doxycyclin are currently used individually for the treatment of urinary tract infections. Fosfomycin has recently seen increased use because of its persistent activity against a large spectrum of multidrug-resistant organisms. The purpose of this study was to investigate the interaction of fosfomycin plus doxycyclin against LRVREF isolates. METHODS: MICs for fosfomycin and doxycyclin were determined for 24 unique clinical LRVREF isolates (4%, fosfomycin-susceptible; 92%, doxycyclin-susceptible). In vitro synergy testing with the combination of fosfomycin and doxycyclin was performed using an Etest method and time-kill assay. RESULTS: The combination of fosfomycin and doxycyclin demonstrated synergy with the Etest method in 11/24 (46%) isolates and in 10/24 (42%) isolates by the time-kill assay. Results from the Etest method and time-kill assay were in agreement for 7/24 (29.2%) of isolates. No antagonism was found. CONCLUSIONS: The combination of fosfomycin and doxycyclin should be evaluated further with additional LRVREF isolates. In vivo studies should also be performed before use in clinical situations.

The T2Bacteria Assay Is a Sensitive and Rapid Detector of Bacteremia That Can Be Initiated in the Emergency Department and Has Potential to Favorably Influence Subsequent Therapy.

BACKGROUND: Bacteremia causes a major worldwide burden, in terms of financial and productivity costs, as well the morbidity and mortality it can ultimately cause. Proper treatment of bacteremia is a challenge because of the species-dependent response to antibiotics. The T2Bacteria Panel is a U.S. Food and Drug Administration-cleared and culture-independent assay for detection of bacteremia, including common ESKAPE pathogens-Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa-and provides species identification in as little as 3.6 h directly from blood. OBJECTIVE: Our aim was to evaluate the T2Bacteria assay performance and potential to affect patient care in the emergency department (ED). METHODS: ED patients from a Louisiana and Florida center were enrolled as part of the T2Bacteria Panel clinical study, which was prospective and noninterventional. Blood samples for blood culture (BC) and T2Bacteria were matched in time and anatomic location. RESULTS: Data from 137 ED patients were evaluated. Relative to BC, T2Bacteria showed 100% positive percent agreement and 98.4% negative percent agreement. In addition, for species on the T2Bacteria Panel, the T2Bacteria assay detected 25% more positives associated with infection, and on average identified the infectious species 56.6 h faster. The T2Bacteria assay covered 70.5% of all species detected by BC. Finally, relative to actual care, the T2Bacteria assay could have potentially focused therapy in 8 patients, reduced time to a species-directed therapy in 4 patients, and reduced time to effective therapy in 4 patients. CONCLUSIONS: In this ED population, the T2Bacteria assay was a rapid and sensitive detector of bacteremia from common ESKAPE pathogens and showed the theoretical potential to influence subsequent patient therapy, ranging from antibiotic de-escalation to faster time to effective therapy.

Performance of the T2Bacteria Panel for Diagnosing Bloodstream Infections: A Diagnostic Accuracy Study.

Background: Blood cultures, the gold standard for diagnosing bloodstream infections (BSIs), are insensitive and limited by prolonged time to results. The T2Bacteria Panel (T2 Biosystems) is a direct-from-blood, nonculture test that identifies the most common ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli). Objective: To assess performance of the T2Bacteria Panel in diagnosing suspected BSIs in adults. Design: Prospective patient enrollment (8 December 2015 through 4 August 2017). Setting: Eleven U.S. hospitals. Patients: 1427 patients for whom blood cultures were ordered as standard of care. Intervention: Paired blood culture and T2Bacteria testing. Measurements: Performance of T2Bacteria compared with a single set of blood cultures in diagnosing proven, probable, and possible BSIs caused by T2Bacteria-targeted organisms. Results: Blood culture and T2Bacteria results were positive for targeted bacteria in 3% (39 of 1427) and 13% (181 of 1427) of patients, respectively. Mean times from start of blood culture incubation to positivity and species identification were 38.5 (SD, 32.8) and 71.7 (SD, 39.3) hours, respectively. Mean times to species identification with T2Bacteria were 3.61 (SD, 0.2) to 7.70 (SD, 1.38) hours, depending on the number of samples tested. Per-patient sensitivity and specificity of T2Bacteria for proven BSIs were 90% (95% CI, 76% to 96%) and 90% (CI, 88% to 91%), respectively; the negative predictive value was 99.7% (1242 of 1246). The rate of negative blood cultures with a positive T2Bacteria result was 10% (146 of 1427); 60% (88 of 146) of such results were associated with probable (n = 62) or possible (n = 26) BSIs. If probable BSIs and both probable and possible BSIs were assumed to be true positives missed by blood culture, per-patient specificity of T2Bacteria was 94% and 96%, respectively. Limitation: Low prevalence of positive blood cultures, collection of a single set of culture specimens, and inability of T2Bacteria to detect nontargeted pathogens. Conclusion: The T2Bacteria Panel rapidly and accurately diagnoses BSIs caused by 5 common bacteria. Primary Funding Source: T2 Biosystems.

Synergistic effect between nisin and polymyxin B against pandrug-resistant and extensively drug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen predominantly associated with nosocomial infections. The World Health Organization's data on antibiotic-resistant 'priority pathogens' reports carbapenem-resistant A. baumannii as a pathogen which is in critical need of research and development of new antimicrobials. Emerging resistance against polymyxins, last-resort drugs for carbapenem-resistant A. baumannii, increases the need for new therapeutic approaches such as synergistic combinations. Nisin, an antibacterial peptide produced by the Gram-positive bacteria L. lactis, is a US Food and Drug Administration approved food preservative with bactericidal action predominantly against other Gram-positive bacteria. A 2008 study reported that topical nisin was effective against staphylococcal mastitis in humans. Additionally, nisin has shown activity against Gram-negative bacteria in combination with antimicrobials such as polymyxin B. A recent in vitro study reported that nisin and polymyxin B exhibited synergistic activity against one isolate each of A. baumannii, Acinetobacter lwoffii and Acinetobacter calcoaceticus using time-kill assay and checkerboard technique. We evaluated the synergistic potential of nisin and polymyxin B against 15 unique clinical A. baumannii isolates using time-kill assay. Three of eight (38%) extensively drug-resistant and six of seven (86%) pandrug-resistant A. baumannii isolates showed synergy with one or more combinations of nisin and polymyxin B. The synergy seen with the use of lower concentrations of polymyxin B may help in reducing the dose-dependent side effects. Additional studies involving pharmacokinetics and pharmacodynamics of nisin are required to explore clinical possibilities.

In vitro synergy with fluconazole plus doxycycline or tigecycline against clinical Candida glabrata isolates.

Candida species, traditionally viewed as opportunistic agents, are increasingly seen as a cause of infection in hospitalized patients. Treatment options are limited to a few classes of drugs. Increased resistance, especially by Candida glabrata, is problematic. We investigated the interaction between fluconazole and doxycycline or tigecycline, using clinically unique blood culture C. glabrata isolates. Eighteen isolates were screened using an Etest® MIC:MIC synergy method. With the doxycycline plus fluconazole combination, 28% of isolates showed synergy; tigecycline plus fluconazole showed 94% synergy. No antagonism was seen. The mechanisms of these interactions are unclear. Further research is warranted to assess clinical utility.

Colistin and Polymyxin B Minimal Inhibitory Concentrations Determined by Etest Found Unreliable for Gram-Negative Bacilli.

BACKGROUND: A reliable method of polymyxin B and E (colistin) susceptibility testing remains elusive. These drugs diffuse poorly into agar, creating potentially inaccurate Etest and disk diffusion results, and testing by these methods is not recommended. Broth microdilution is the reference testing method, although it can be sometimes difficult to interpret. Currently, when a colistin susceptibility test is ordered for a patient in the Ochsner Health System, our diagnostic microbiology laboratory performs the Etest. As an in-house quality assessment project, we compared colistin and polymyxin B minimal inhibitory concentrations (MICs) determined by Etest with MICs determined by broth microdilution to evaluate whether colistin MICs are accurately being reported by Etest. METHODS: A total of 143 nonduplicate clinical isolates from Ochsner patients during 2015-2016 were tested: Acinetobacter baumannii (n=60), Pseudomonas aeruginosa (n=44), and Enterobacteriaceae (n=39) (13 Escherichia coli, 15 Klebsiella spp, and 11 Enterobacter spp). Colistin and polymyxin B MICs were determined by Etest and broth microdilution. RESULTS: Using broth microdilution, 16/143 (11%) isolates were nonsusceptible to colistin, and 12/143 (8%) were nonsusceptible to polymyxin B. With Etest, 4/143 (3%) isolates were nonsusceptible to colistin, and 7/143 (5%) were nonsusceptible to polymyxin B. Essential agreement of colistin and polymyxin B MICs between broth microdilution and Etest was 84/143 (59%) and 87/143 (61%), respectively. Categorical agreement for colistin and polymyxin B was 127/143 (89%) and 126/143 (88%), respectively. CONCLUSION: We found a high rate of discrepancy between colistin and polymyxin B Etest and broth microdilution MICs. Very major errors (colistin/polymyxin B-susceptible by Etest, colistin/polymyxin B-resistant by broth microdilution) were detected in 10% of isolates tested with colistin and 8% of polymyxin B-tested isolates. The data from this study confirm that broth microdilution should be performed for susceptibility testing of polymyxins.

Evaluation of the Rapid Polymyxin NP Test for Polymyxin B Resistance Detection Using Enterobacter cloacae and Enterobacter aerogenes Isolates.

Polymyxin resistance is an increasing problem worldwide. Currently, determining susceptibility to polymyxins is problematic and lengthy. Polymyxins diffuse poorly into agar, potentially giving inaccurate disk diffusion and Etest results. A rapid screening test (2 h) for the detection of polymyxin resistance in Enterobacteriaceae, developed by P. Nordmann and L. Poirel (rapid polymyxin NP test) in 2016, detects glucose metabolization in the presence of polymyxin E (PE) and PB via pH-induced color change. The sensitivity and specificity were 99.3 and 95.4%, respectively, with results obtained in ≤2 h. Our goal was to evaluate this test using PB against larger numbers of Enterobacter A total of 143 nonduplicate Enterobacter isolates (102 E. cloacae complex, 41 E. aerogenes) were tested, including 136 collected from Ochsner Health System patients from March to May 2016 and 7 previously determined PB-resistant E. cloacae isolates from JMI Laboratories. MICs were determined via broth microdilution. For the rapid polymyxin NP test, a color change from orange to yellow is positive; a weak/no color change is deemed negative after 4 h. Of 143 Enterobacter isolates, 25 were determined to be PB resistant by broth microdilution (MIC > 2 µg/ml), including all 7 JMI isolates. Of these 25, 7 were positive by the rapid polymyxin NP test (included 3/7 JMI isolates). All 118 isolates determined to be PB susceptible by broth microdilution were NP test negative. The sensitivity and specificity for the rapid polymyxin NP test were 25 and 100%, respectively, compared to broth microdilution. Although the rapid polymyxin NP test is a much faster method (2 to 4 h) for polymyxin resistance determination compared to broth microdilution (16 to 20 h), our study indicates that it may be subject to limitations when testing Enterobacter.

In vitro Synergistic Activity of Caspofungin Plus Polymyxin B Against Fluconazole-Resistant Candida glabrata.

BACKGROUND: Candida species account for most invasive fungal infections, and the emergence of fluconazole and caspofungin resistance is problematic. Overcoming resistance with synergism between 2 drugs may be useful. In a 2013 in vitro study, caspofungin plus colistin (polymyxin E) was found to act synergistically against fluconazole-resistant and susceptible Candida albicans isolates. The purpose of our study was to extend this finding by evaluating caspofungin plus polymyxin B for in vitro synergy against fluconazole-resistant Candida glabrata isolates. MATERIALS AND METHODS: A total of 7 fluconazole-resistant C. glabrata bloodstream infection isolates were obtained from 2010-2011. Of these, 2 isolates were also resistant to caspofungin. Minimum inhibitory concentrations (MICs) for caspofungin and polymyxin B were determined by Etest and broth microdilution. Clinical and Laboratory Standards Institute breakpoints were used for fluconazole and caspofungin MIC interpretations. No interpretive guidelines exist for testing polymyxin B against C. glabrata. Synergy testing with caspofungin (1 × MIC) and polymyxin B (½MIC) was performed using a modified bacterial Etest synergy method and time-kill assay. RESULTS: With the Etest synergy method, 4 out of 7 isolates showed in vitro synergy and 1 out of 7 showed additivity. The remaining isolates (both caspofungin resistant) showed indifference. Using the time-kill assay, 1 out of 7 isolates showed synergy, 1 showed additivity and the remaining 5 (including both caspofungin-resistant isolates) showed indifference. CONCLUSIONS: Caspofungin susceptibility may be required for synergism between caspofungin and polymyxin B. Further synergy testing with caspofungin plus polymyxin B and additional fluconazole-resistant C. glabrata isolates should be performed. In vitro synergy/additivity may or may not correlate with in vivo benefit.

Tuberculous Otomastoiditis.

Tuberculous otitis media and mastoiditis, or tuberculous otomastoiditis, is a rare but well-described infectious process occasionally affecting individuals in the United States but more frequently seen in countries where tuberculosis is endemic. Infection may be primary and occur through mucus aspirated through the Eustachian tube. Alternatively, organisms may secondarily infect the nasopharynx when expectorated from the lungs and, less frequently, may be hematogenously spread. Chronic otorrhea and hearing loss are common symptoms, and extensive perforation of the tympanic membranes and facial nerve paralysis are routinely described. Diagnosis is made by direct culture of Mycobacterium tuberculosis, although more recently, molecular techniques have been used. Successful treatment of tuberculous otomastoiditis routinely involves surgical intervention combined with prolonged antituberculosis therapy.

Detection of a New cfr-Like Gene, cfr(B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program.

Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 µg/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci.

Update on the emerging role of telavancin in hospital-acquired infections.

Telavancin is a lipoglycopeptide that has activity against Gram-positive aerobic and anaerobic bacteria. It has activity against methicillin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus and non-Van-A strains of vancomycin-resistant enterococci. It has been approved by the US Food and Drug Administration (FDA) for complicated skin and skin structure infections and hospital-acquired pneumonia. There is a need for more clinical studies to determine the role of telavancin in treating bacteremia and prosthetic device infections. In this review, we discuss the published data on the use of telavancin in treating hospital-acquired infections and provide an update on new research.

Time-kill assay and Etest evaluation for synergy with polymyxin B and fluconazole against Candida glabrata.

Fluconazole-resistant Candida glabrata is an emerging pathogen that causes fungemia. Polymyxin B, a last-resort antibiotic used to treat multidrug-resistant Gram-negative bacterial infections, has been found to possess in vitro fungicidal activity and showed synergy with fluconazole against a single strain of C. glabrata. Since both agents may be used simultaneously in intensive care unit (ICU) patients, this study was performed to test for possible synergy of this combination against 35 C. glabrata blood isolates, using 2 methods: a time-kill assay and an experimental MIC-MIC Etest method. Thirty-five genetically unique C. glabrata bloodstream isolates were collected from 2009 to 2011, identified using an API 20C system, and genotyped by repetitive sequence-based PCR (rep-PCR). MICs were determined by Etest and broth microdilution methods. Synergy testing was performed using a modified bacterial Etest synergy method and time-kill assay, with final results read at 24 h. The Etest method showed synergy against 19/35 (54%) isolates; the time-kill assay showed synergy against 21/35 (60%) isolates. Isolates not showing drug synergy had an indifferent status. Concordance between methods was 60%. In vitro synergy of polymyxin B and fluconazole against the majority of C. glabrata isolates was demonstrated by both methods. The bacterial Etest synergy method adapted well when used with C. glabrata. Etest was easier to perform than time-kill assay and may be found to be an acceptable alternative to time-kill assay with antifungals.

Comparison of 3 Etest(®) methods and time-kill assay for determination of antimicrobial synergy against carbapenemase-producing Klebsiella species.

Increasing global antibiotic resistance has resulted in more use of antibiotic combinations. There is a lack of a gold standard for in vitro testing of these combinations for synergy or antagonism. Time-kill assay (TKA) may be used but is labor intensive and not practical for clinical use. Etest® synergy methods are more rapid and easier to perform, but there is no agreement regarding which method is best. We tested 31 clinical genetically unique Klebsiella pneumoniae carbapenemase-producing Klebsiella isolates with the combination of meropenem and polymyxin B by TKA and 3 Etest methods, each in triplicate: Method 1, MIC:MIC; Method 2, direct overlay; and Method 3, cross. Overall, testing with Etest synergy methods showed the following agreement with TKA: Method 1: 25/31 (80.6%), Method 2: 7/31 (22.6%), and Method 3: 8/31 (25.8%). The MIC:MIC method had the highest agreement (80.6%, κ = 0.59, P < 0.001) and should be evaluated more extensively.

In Vitro Synergy of Telavancin and Rifampin Against Enterococcus faecium Resistant to Both Linezolid and Vancomycin.

BACKGROUND: An emerging pathogen is Enterococcus faecium resistant to both linezolid and vancomycin (LRVRE). Antimicrobial combinations may be required for therapy and need to be evaluated. The combination of daptomycin and rifampin has demonstrated good in vitro activity against gram-positive bacteria, including E faecium. Telavancin, a newer lipoglycopeptide, has shown in vitro activity against E faecium. We evaluated the combination of telavancin and rifampin and compared the results to the combination of daptomycin and rifampin used previously on the same isolates. METHODS: Twenty-four genetically unique (by pulsed-field gel electrophoresis), clinical LRVRE isolates were collected in the United States from 2001-2004. Etest minimal inhibitory concentrations (MICs) (µg/mL) were 0.064-8 for telavancin, 1-4 for daptomycin, and 0.012 to >32 for rifampin. In vitro synergy testing was performed in triplicate by an Etest MIC:MIC ratio method, and summation fractional inhibitory concentration (ΣFIC) was calculated: synergy ≤0.5; indifference >0.5-4; and antagonism >4. RESULTS: The Etest method showed synergy (ΣFICs of 0.1-0.5) with telavancin + rifampin in 20/24 (83%) isolates and indifference (ΣFICs of 0.6-0.8) in 4/24 (17%) isolates. Similarly, the daptomycin + rifampin combination showed synergy (ΣFICs of 0.1-0.5) in 21/24 (88%) isolates and indifference (ΣFICs of 0.6-1.0) in 3/24 (12%) isolates by the Etest method. No antagonism was found. CONCLUSIONS: In vitro synergy with both combinations (rifampin + telavancin or daptomycin) was 83% and 88%, respectively, by Etest against these LRVRE isolates. Although both daptomycin and telavancin in combination with rifampin showed a high incidence of synergistic activity, further in vitro synergy testing with this combination should be performed against additional E faecium isolates. In vitro synergy may or may not translate into in vivo effectiveness.