土井 洋平

Fosfomycin exhibits broad-spectrum antibacterial activity and is being reevaluated for the treatment of extensively drug-resistant pathogens. Its activity in Gram-negative organisms, however, can be compromised by expression of FosA, a metal-dependent transferase that catalyzes the conjugation of glutathione to fosfomycin, rendering the antibiotic inactive. In this study, we solved the crystal structures of two of the most clinically relevant FosA enzymes: plasmid-encoded FosA3 from Escherichia coli and chromosomally encoded FosA from Klebsiella pneumoniae (FosA(KP)). The structure, molecular dynamics, catalytic activity, and fosfomycin resistance of FosA3 and FosA(KP) were also compared to those of FosA from Pseudomonas aeruginosa (FosA(PA)), for which prior crystal structures exist. E. coli TOP10 transformants expressing FosA3 and FosA(KP) conferred significantly greater fosfomycin resistance (MIC, > 1,024 mu g/ml) than those expressing FosA(PA) (MIC, 16 mu g/ml), which could be explained in part by the higher catalytic efficiencies of the FosA3 and FosA(KP) enzymes. Interestingly, these differences in enzyme activity could not be attributed to structural differences at their active sites. Instead, molecular dynamics simulations and hydrogen-deuterium exchange experiments with FosA(KP) revealed dynamic interconnectivity between its active sites and a loop structure that extends from the active site of each monomer and traverses the dimer interface. This dimer interface loop is longer and more extended in FosA(KP) and FosA3 than in FosA(PA), and kinetic analyses of FosA(KP) and FosA(PA) loop-swapped chimeric enzymes highlighted its importance in FosA activity. Collectively, these data yield novel insights into fosfomycin resistance that could be leveraged to develop new strategies to inhibit FosA and potentiate fosfomycin activity.

Background: Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited. Objectives: To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and - resistant KPC-producing K. pneumoniae clinical strains. Methods: The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS. Results: All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC > 2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-L-arabinose (Ar alpha 4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype. Conclusions: The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ar alpha 4N in clinical settings.

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

The continued rise and spread of antimicrobial resistance among bacterial pathogens pose a serious challenge to global health. Countering antimicrobial-resistant pathogens requires a multifaceted effort that includes the discovery of novel therapeutic approaches. Here, we establish the capacity of the human CXC chemokines CXCL9 and CXCL10 to kill multidrug-resistant Gram-negative bacteria, including New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae and colistin-resistant members of the family Enterobacteriaceae that harbor the mobile colistin resistance protein MCR-1 and thus possess phosphoethanolamine-modified lipid A. Colistin-resistant K. pneumoniae isolates affected by genetic mutation of the PmrA/ PmrB two-component system, a chromosomally encoded regulator of lipopolysaccharide modification, and containing 4-amino-4-deoxy-L-arabinose-modified lipid A were also found to be susceptible to chemokine-mediated antimicrobial activity. However, loss of PhoP/PhoQ autoregulatory control, caused by disruption of the gene encoding the negative regulator MgrB, limited the bactericidal effects of CXCL9 and CXCL10 in a variable, strain-specific manner. Cumulatively, these findings provide mechanistic insight into chemokine-mediated antimicrobial activity, highlight disparities amongst determinants of colistin resistance, and suggest that chemokine-mediated bactericidal effects merit additional investigation as a therapeutic avenue for treating infections caused by multidrug-resistant pathogens. IMPORTANCE As bacterial pathogens become resistant to multiple antibiotics, the infections they cause become increasingly difficult to treat. Carbapenem antibiotics provide an essential clinical barrier against multidrug-resistant bacteria; however, the dissemination of bacterial enzymes capable of inactivating carbapenems threatens the utility of these important antibiotics. Compounding this concern is the global spread of bacteria invulnerable to colistin, a polymyxin antibiotic considered to be a last line of defense against carbapenem-resistant pathogens. As the effectiveness of existing antibiotics erodes, it is critical to develop innovative antimicrobial therapies. To this end, we demonstrate that the chemokines CXCL9 and CXCL10 kill the most concerning carbapenem-and colistin-resistant pathogens. Our findings provide a unique and timely foundation for therapeutic strategies capable of countering antibiotic-resistant "superbugs."

The presence of mcr-1 among Enterobacteriaceae isolates collected from retail vegetables in China between 2015 and 2016 was investigated. Two Raoultella ornithinolytica and seven Escherichia coil strains recovered from lettuce and tomato samples were identified as MCR-1-producers. Similar to isolates from animals and humans, the mcr-1 gene was located on the IncH12/ST3, Inc12, or IncX4 plasmids. The presence of MCR-1-producing organisms in ready -to -eat food samples represents a serious risk for human health.

Bacterial resistance to polymyxin antibiotics has taken on a new and more menacing form. Common are genomically-encoded resistance mechanisms to polymyxins, specifically colistin (polymyxin E), however, the plasmid-borne mobile colistin resistance-1 (mcr-1) gene has recently been identified and poses a new threat to global public health. Within six months of initial identification in Chinese swine in November 2015, the first human clinical isolation in the US was reported (Apr. 2016). Herein we report successful reversion of mcr-1-driven colistin resistance in Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli with adjuvants we previously reported as modulators of chromosomally-encoded colistin resistance. Further screening of our in-house library of nitrogen-dense heterocycles has identified additional chemical scaffolds that actively attenuate colistin resistance. Ultimately, we present a diverse cohort of adjuvants that both sensitize colistin-resistant and colistin-susceptible bacteria to this antibiotic, thus providing a potential avenue to both reduce colistin dosage and toxicity, and overcome colistin resistance. (C) 2017 Elsevier Ltd. All rights reserved.

There are no data comparing outcomes of patients treated with ceftazidime-avibactam versus comparators for carbapenem-resistant Enterobacteriaceae infections. At our center, ceftazidime-avibactam treatment of carbapenem-resistant Klebsiella pneumoniae bacteremia was associated with higher rates of clinical success (P = 0.006) and survival (P = 0.01) than other regimens. Across treatment groups, there were no differences in underlying diseases, severity of illness, source of bacteremia, or strain characteristics (97% produced K. pneumoniae carbapenemase). Aminoglycoside- and colistin-containing regimens were associated with increased rates of nephrotoxicity (P = 0.002).

IncX plasmids are receiving much attention as vehicles of carbapenem and colistin resistance genes, such as bla(NDM), bla(KPC), and mcr-1. Among them, IncX2 subgroup plasmids remain rare. Here, we characterized IncX2 and IncX1-X2 hybrid plasmids coexisting in a FosA6-producing Escherichia coli strain that were possibly generated as a consequence of recombination events between an R6K-like IncX2 plasmid and a pLN126_33-like IncX1 plasmid. Variable multidrug resistance mosaic regions were observed in these plasmids, indicating their potential to serve as flexible carriers of resistance genes. The diversity of IncX group plasmid backbones and accessory genes and the evolution of hybrid IncX plasmids pose a challenge in detecting and classifying them.

Background The best available treatment against carbapenemase-producing Enterobacteriaceae (CPE) is unknown. The objective of this study was to investigate the effect of appropriate therapy and of appropriate combination therapy on mortality of patients with bloodstream infections (BSIs) due to CPE. Methods In this retrospective cohort study, we included patients with clinically significant monomicrobial BSIs due to CPE from the INCREMENT cohort, recruited from 26 tertiary hospitals in ten countries. Exclusion criteria were missing key data, death sooner than 24 h after the index date, therapy with an active antibiotic for at least 2 days when blood cultures were taken, and subsequent episodes in the same patient. We compared 30 day all-cause mortality between patients receiving appropriate (including an active drug against the blood isolate and started in the first 5 days after infection) or inappropriate therapy, and for patients receiving appropriate therapy, between those receiving active monotherapy (only one active drug) or combination therapy (more than one). We used a propensity score for receiving combination therapy and a validated mortality score (INCREMENT-CPE mortality score) to control for confounders in Cox regression analyses. We stratified analyses of combination therapy according to INCREMENT-CPE mortality score (0-7 [low mortality score] vs 8-15 [high mortality score]). INCREMENT is registered with ClinicalTrials.gov, number NCT01764490. Findings Between Jan 1, 2004, and Dec 31, 2013, 480 patients with BSIs due to CPE were enrolled in the INCREMENT cohort, of whom we included 437 (91%) in this study. 343 (78%) patients received appropriate therapy compared with 94 (22%) who received inappropriate therapy. The most frequent organism was Klebsiella pneumoniae (375 [86%] of 437; 291 [85%] of 343 patients receiving appropriate therapy vs 84 [89%] of 94 receiving inappropriate therapy) and the most frequent carbapenemase was K pneumoniae carbapenemase (329 [75%]; 253 [74%] vs 76 [81%]). Appropriate therapy was associated with lower mortality than was inappropriate therapy (132 [38.5%] of 343 patients died vs 57 [60.6%] of 94; absolute difference 22.1% [95% CI 11.0-33.3]; adjusted hazard ratio [HR] 0.45 [95% CI 0.33-0.62]; p< 0.0001). Among those receiving appropriate therapy, 135 (39%) received combination therapy and 208 (61%) received monotherapy. Overall mortality was not different between those receiving combination therapy or monotherapy (47 [35%] of 135 vs 85 [41%] of 208; adjusted HR 1.63 [95% CI 0.67-3.91]; p=0.28). However, combination therapy was associated with lower mortality than was monotherapy in the high-mortality-score stratum (30 [48%] of 63 vs 64 [62%] of 103; adjusted HR 0.56 [0.34-0.91]; p=0.02), but not in the low-mortality-score stratum (17 [24%] of 72 vs 21 [20%] of 105; adjusted odds ratio 1.21 [0.56-2.56]; p=0.62). Interpretation Appropriate therapy was associated with a protective effect on mortality among patients with BSIs due to CPE. Combination therapy was associated with improved survival only in patients with a high mortality score. Patients with BSIs due to CPE should receive active therapy as soon as they are diagnosed, and monotherapy should be considered for those in the low-mortality-score stratum. Funding Spanish Network for Research in Infectious Diseases, European Development Regional Fund, Instituto de Salud Carlos III, and Innovative Medicines Initiative.

Rapid diagnostics that enable identification of infectious agents improve patient outcomes, antimicrobial stewardship, and length of hospital stay. Current methods for pathogen detection in the clinical laboratory include biological culture, nucleic acid amplification, ribosomal protein characterization, and genome sequencing. Pathogen identification from single colonies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of high abundance proteins is gaining popularity in clinical laboratories. Here, we present a novel and complementary approach that utilizes essential microbial glycolipids as chemical fingerprints for identification of individual bacterial species. Gram-positive and negative bacterial glycolipids were extracted using a single optimized protocol. Extracts of the clinically significant ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. were analyzed by MALDI-TOF-MS in negative ion mode to obtain glycolipid mass spectra. A library of glycolipid mass spectra from 50 microbial entries was developed that allowed bacterial speciation of the ESKAPE pathogens, as well as identification of pathogens directly from blood bottles without culture on solid medium and determination of antimicrobial peptide resistance. These results demonstrate that bacterial glycolipid mass spectra represent chemical barcodes that identify pathogens, potentially providing a useful alternative to existing diagnostics.

Fosfomycin is a decades-old antibiotic which is being revisited because of its perceived activity against many extensively drug-resistant Gram-negative pathogens. FosA proteins are Mn2+ and K+ -dependent glutathione S-transferases which confer fosfomycin resistance in Gram-negative bacteria by conjugation of glutathione to the antibiotic. Plasmid-borne fosA variants have been reported in fosfomycin-resistant Escherichia coli strains. However, the prevalence and distribution of fosA in other Gram-negative bacteria are not known. We systematically surveyed the presence of fosA in Gram-negative bacteria in over 18,000 published genomes from 18 Gram-negative species and investigated their contribution to fosfomycin resistance. We show that FosA homologues are present in the majority of genomes in some species (e.g., Klebsiella spp., Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa), whereas they are largely absent in others (e.g., E. coli, Acinetobacter baumannii, and Burkholderia cepacia). FosA proteins in different bacterial pathogens are highly divergent, but key amino acid residues in the active site are conserved. Chromosomal fosA genes conferred high-level fosfomycin resistance when expressed in E. coli, and deletion of chromosomal fosA in S. marcescens eliminated fosfomycin resistance. Our results indicate that FosA is encoded by clinically relevant Gram-negative species and contributes to intrinsic fosfomycin resistance. IMPORTANCE There is a critical need to identify alternate approaches to treat infections caused by extensively drug-resistant (XDR) Gram-negative bacteria. Fosfomycin is an old antibiotic which is routinely used for the treatment of urinary tract infections, although there is substantial interest in expanding its use to systemic infections caused by XDR Gram-negative bacteria. In this study, we show that fosA genes, which encode dimeric Mn2+-and K+-dependent glutathione S-transferase, are widely distributed in the genomes of Gram-negative bacteria-particularly those belonging to the family Enterobacteriaceae-and confer fosfomycin resistance. This finding suggests that chromosomally located fosA genes represent a vast reservoir of fosfomycin resistance determinants that may be transferred to E. coli. Furthermore, they suggest that inhibition of FosA activity may provide a viable strategy to potentiate the activity of fosfomycin against XDR Gram-negative bacteria.

Background. Data on the use of ceftolozane-tazobactam and emergence of ceftolozane-tazobactam resistance during multidrug resistant (MDR)-Pseudomonas aeruginosa infections are limited. Methods. We performed a retrospective study of 21 patients treated with ceftolozane-tazobactam for MDR-P. aeruginosa infections. Whole genome sequencing and quantitative real-time polymerase chain reaction were performed on longitudinal isolates. Results. Median age was 58 years; 9 patients (43%) were transplant recipients. Median simplified acute physiology score-II (SAPS-II) was 26. Eighteen (86%) patients were treated for respiratory tract infections; others were treated for bloodstream, complicated intraabdominal infections, or complicated urinary tract infections. Ceftolozane-tazobactam was discontinued in 1 patient (rash). Thirty-day all-cause and attributable mortality rates were 10% (2/21) and 5% (1/21), respectively; corresponding 90-day mortality rates were 48% (10/21) and 19% (4/21). The ceftolozane-tazobactam failure rate was 29% (6/21). SAPS-II score was the sole predictor of failure. Ceftolozane-tazobactam resistance emerged in 3 (14%) patients. Resistance was associated with de novo mutations, rather than acquisition of resistant nosocomial isolates. ampC overexpression and mutations were identified as potential resistance determinants. Conclusions. In this small study, ceftolozane-tazobactam was successful in treating 71% of patients with MDR-P. aeruginosa infections, most of whom had pneumonia. The emergence of ceftolozane-tazobactam resistance in 3 patients is worrisome and may be mediated in part by AmpC-related mechanisms. More research on treatment responses and resistance during various types of MDR-P. aeruginosa infections is needed to define ceftolozane-tazobactam's place in the armamentarium.

mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceae. mcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli. Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa. Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii. Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.

Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as a major threat. Commonly used antibiotics are generally inactive against CRE. Therefore, timely detection of CRE is of paramount importance. Among CRE, those producing carbapenem-hydrolyzing beta-lactamase enzymes (carbapenemase-producing Enterobacteriaceae) are particularly of concern because they tend to spread, and treatment is difficult. The carbapenemase groups most commonly encountered include KPC, NDM, and OXA-48. Treatment options are limited and include combinations of polymyxins, tigecycline, aminoglycosides, or carbapenems; newer agents with activity against CRE and better safety profiles are becoming available and will likely emerge as the preferred therapy.

We identified rmtE1, an uncommon 16S ribosomal methyltransferase gene, in an aminoglycoside- and cephalosporin-resistant Escherichia colt sequence type 448 clinical strain co-harboring bla(CMY-2). Long-read sequencing revealed insertion of a 101,257-bp fragment carrying both resistance genes to the chromosome. Our findings underscore E. con sequence type 448 as a potential high-risk multidrug-resistant clone.

Background The mcr-1 gene confers transferable colistin resistance. mcr-1-positive Enterobacteriaceae (MCRPE) have attracted substantial medical, media, and political attention; however, so far studies have not addressed their clinical impact. Herein, we report the prevalence of MCRPE in human infections and carriage, clinical associations of mcr-1positive Escherichia coli (MCRPEC) infection, and risk factors for MCRPEC carriage. Methods We undertook this study at two hospitals in Zhejiang and Guangdong, China. We did a retrospective cross-sectional assessment of prevalence of MCRPE infection from isolates of Gram-negative bacteria collected at the hospitals from 2007 to 2015 (prevalence study). We did a retrospective case-control study of risk factors for infection and mortality after infection, using all MCRPEC from infection isolates and a random sample of mcr-1-negative E coli infections from the retrospective collection between 2012 and 2015 (infection study). We also did a prospective case-control study to assess risk factors for carriage of MCRPEC in rectal swabs from inpatients with MCRPEC and mcr1 negative at the hospitals and collected between May and December, 2015, compared with mcr-1-negative isolates from rectal swabs of inpatients (colonisation study). Strains were analysed for antibiotic resistance, plasmid typing, and transfer analysis, and strain relatedness. Findings We identified 21 621 non-duplicate isolates of Enterobacteriaceae, Acinetobacter spp, and Pseudomonas aeruginosa from 18 698 inpatients and 2923 healthy volunteers. Of 17 498 isolates associated with infection, mcr-1 was detected in 76 (1%) of 5332 E coli isolates, 13 (< 1%) of 348 Klebsiella pneumoniae, one (< 1%) of 890 Enterobacter cloacae, and one (1%) of 162 Enterobacter aerogenes. For the infection study, we included 76 mcr-1-positive clinical E coli isolates and 508 mcr-1-negative isolates. Overall, MCRPEC infection was associated with male sex (209 [41%] vs 47 [63%], adjusted p= 0 similar to 011), immunosuppression (30 [6%] vs 11 [15%], adjusted p= 0 similar to 011), and antibiotic use, particularly carbapenems (45 [9%] vs 18 [24%], adjusted p= 0 similar to 002) and fluoroquinolones (95 [19%] vs 23 [30%], adjusted p= 0 similar to 017), before hospital admission. For the colonisation study, we screened 2923 rectal swabs from healthy volunteers, of which 19 were MCRPEC, and 1200 rectal swabs from patients, of which 35 were MCRPEC. Antibiotic use before hospital admission (p< 0 similar to 0001) was associated with MCRPEC carriage in 35 patients compared with 378 patients with mcr-1-negative E coli colonisation, whereas living next to a farm was associated with mcr-1-negative E coli colonisation (p= 0 similar to 03, univariate test). mcr-1 could be transferred between bacteria at high frequencies (10-1 to 10-3), and plasmid types and MCRPEC multi-locus sequence types (MLSTs) were more variable in Guangdong than in Zhejiang and included the human pathogen ST131. MCRPEC also included 17 unreported ST clades. Interpretation In 2017, colistin will be formally banned from animal feeds in China and switched to human therapy. Infection with MRCPEC is associated with sex, immunosuppression, and previous antibiotic exposure, while colonisation is also associated with antibiotic exposure. MLST and plasmid analysis shows that MCRPEC are diversely spread throughout China and pervasive in Chinese communities. Funding National Key Basic Research Program of China, National Natural Science Foundation of China/Zhejiang, National Key Research and Development Program, and MRC, UK.

OBJECTIVE To describe the investigation and control of a rare cluster of Klebsiella pneumoniae carbapenemase-producing Citrobacter freundii in a hospital in southern Florida. METHODS An epidemiologic investigation, review of infection prevention procedures, and molecular studies including whole genome sequencing were conducted. RESULTS An outbreak of K. pneumoniae carbapenemase-3-producing C. freundii was identified at a tertiary hospital in Florida in 2014. Of the 6 cases identified, 3 occurred in the same intensive care unit and were caused by the same clone. For 2 of the 3 remaining cases, the isolates had low carbapenem minimum inhibitory concentrations and were unrelated by whole genome sequencing. As a response to the outbreak, supplementary environmental cleaning was implemented, including closure and terminal cleaning of the unit where the 3 cases clustered, in addition to the infection control bundle already in place at the time. No further cases were identified after these additional interventions. CONCLUSIONS Although C. freundii is not a species that commonly demonstrates carbapenem resistance, our findings suggest that carbapenemase-producing C. freundii may be underdetected even when active surveillance is in place and has a potential to cause hospital outbreak. Infect Control Hosp Epidemiol 2017;38:320-326

Background. Polymyxins including colistin are an important "last-line" treatment for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKp). Increasing use of colistin has led to resistance to this cationic antimicrobial peptide. Methods. A cohort nested within the Consortium on Resistance against Carbapenems in Klebsiella pneumoniae (CRACKLE) was constructed of patients with infection, or colonization with CRKp isolates tested for colistin susceptibility during the study period of December, 2011 to October, 2014. Reference colistin resistance determination as performed by broth macrodilution was compared to results from clinical microbiology laboratories (Etest) and to polymyxin resistance testing. Each patient was included once, at the time of their first colistin-tested CRKp positive culture. Time to 30-day in-hospital all-cause mortality was evaluated by Kaplan-Meier curves and Cox proportional hazard modeling. Results. In 246 patients with CRKp, 13% possessed ColR CRKp. ColR was underestimated by Etest (very major error rate = 35%, major error rate = 0.4%). A variety of rep-PCR strain types were encountered in both the ColS and the ColR groups. Carbapenem resistance was mediated primarily by bla(KPC-2) (46%) and bla(KPC-3) (50%). ColR was associated with increased hazard for in-hospital mortality (aHR 3.48; 95% confidence interval, 1.73-6.57; P < .001). The plasmid-associated ColR genes, mcr-1 and mcr-2 were not detected in any of the ColR CRKp. Conclusions. In this cohort, 13% of patients with CRKp presented with ColR CRKp. The apparent polyclonal nature of the isolates suggests de novo emergence of ColR in this cohort as the primary factor driving ColR. Importantly, mortality was increased in patients with ColR isolates.

Ceftazidime-avibactam is a novel beta lactam/ beta-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Kleb-siella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidimeavibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant K. pneumoniae emerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Wholegenome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant K. pneumoniae isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne bla(KPC-3), which were not present in baseline isolates. bla(KPC-3) mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in K. pneumoniae, plasmid transfer, and blaKPC cloning into competent Escherichia coli. In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs >= 4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced >= 4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of bla(KPC-3) encoding D179Y/T243M and D179Y variants was diminished compared to bla(KPC-3) expression in baseline isolates. In conclusion, the development of resistance-conferring bla(KPC-3) mutations in K. pneumoniae within 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.

BACKGROUND. Endoscope-associated infections are reported despite following proper reprocessing methods. Microbiological testing can confirm the adequacy of endoscope reprocessing. Multiple controversies related to the method and interpretation of microbiological testing cultures have arisen that make their routine performance a complex target. OBJECTIVE. We conducted a pilot study using disposable bronchoscopes (DBs) to simulate different reprocessing times and soaking times and to compare high-level disinfection versus ethylene oxide sterilization. We also reviewed the time to reprocessing and duration of the procedures. METHODS. Bronchoscopes were chosen because an alternative disposable scope is commercially available and because bronchoscopes are more prone to delays in processing. Disposable bronchoscopes were contaminated using a liquid bacterial suspension and were then incubated for 1-4 hours. Standard processing and high-level disinfection were performed on 36 endoscopes. Ethylene oxide sterilization was performed on 21 endoscopes. Endoscope cultures were performed using the standard "brush, flush, brush" technique. RESULTS. After brushing was performed, a final water-flush culture procedure was the most effective method of detecting bacterial persistence on the disposable scopes. Klebsiella pneumoniae was the most commonly recovered organism after reprocessing. Ethylene oxide sterilization did not result in total elimination of viable bacteria. CONCLUSION. Routine endoscopy cultures may be required to assess the adequacy of endoscopic processing.

We detected the mcr-1 gene in 21(14.8%) Salmonella isolates from pigs at slaughter; 19 were serovar Typhimurium sequence type 34. The gene was located on IncH12-like plasmids that also harbored IncF replicons and lacked a conjugative transfer region. These findings highlight the need to prevent further spread of colistin resistance in animals and humans.

Carbapenemase-producing Enterobacteriaceae (CPE) are an important and increasing threat to global health. Both clonal spread and plasmid-mediated transmission contribute to the ongoing rise in incidence of these bacteria. Among the 4 classes of -lactamases defined by the Ambler classification system, the carbapenemases that confer carbapenem resistance in Enterobacteriaceae belong to 3 of them: Class A (K. pneumoniae carbapenemases, KPC), Class B (metallo--lactamases, MBL including New Delhi metallo--lactamases, NDM) and Class D (OXA-48-like carbapenemases). KPC-producing CPE are the most commonly occurring CPE in the United States. MBL-producing CPE have been most commonly associated with the Indian Subcontinent as well as with specific countries in Europe, including Romania, Denmark, Spain, and Hungary. The epicenter of OXA-48-like-producing is in Turkey and surrounding countries. Detailed knowledge of the epidemiology and molecular characteristics of CPE is essential to stem the spread of these pathogens.

Thirty-seven carbapenem-resistant Enterobacteriaceae (CRE)-infected patients were treated with ceftazidime-avibactam. Clinical success and survival rates at 30 days were 59% (22/37) and 76% (28/37), respectively. In 23% (5/22) of clinical successes, CRE infections recurred within 90 days. Microbiologic failure rate was 27% (10/37). Ceftazidime-avibactam resistance was detected in 30% (3/10) of microbiologic failures.

Treatment options for infections due to carbapenem-resistant Acinetobacter baumannii are extremely limited. Minocycline is a semisynthetic tetracycline derivative with activity against this pathogen. This study compared susceptibility testing methods that are used in clinical microbiology laboratories (Etest, disk diffusion, and Sensititre broth microdilution methods) for testing of minocycline, tigecycline, and doxycycline against 107 carbapenem-resistant A. baumannii clinical isolates. Susceptibility rates determined with the standard broth microdilution method using cation-adjusted Mueller-Hinton (MH) broth were 77.6% for minocycline and 29% for doxycycline, and 92.5% of isolates had tigecycline MICs of <= 2 mu g/ml. Using MH agar from BD and Oxoid, susceptibility rates determined with the Etest method were 67.3% and 52.3% for minocycline, 21.5% and 18.7% for doxycycline, and 71% and 29.9% for tigecycline, respectively. With the disk diffusion method using MH agar from BD and Oxoid, susceptibility rates were 82.2% and 72.9% for minocycline and 34.6% and 34.6% for doxycycline, respectively, and rates of MICs of <= 2 mu g/ml were 46.7% and 23.4% for tigecycline. In comparison with the standard broth microdilution results, very major rates were low (similar to 2.8%) for all three drugs across the methods, but major error rates were higher (similar to 5.6%), especially with the Etest method. For minocycline, minor error rates ranged from 14% to 37.4%. For tigecycline, minor error rates ranged from 6.5% to 69.2%. The majority of minor errors were due to susceptible results being reported as intermediate. For minocycline susceptibility testing of carbapenem-resistant A. baumannii strains, very major errors are rare, but major and minor errors overcalling strains as intermediate or resistant occur frequently with susceptibility testing methods that are feasible in clinical laboratories.

Klebsiella pneumoniae remains an important cause of intrapulmonary infection and invasive disease worldwide. K. pneumoniae can evade serum killing and phagocytosis primarily through the expression of a polysaccharide capsule, but its pathogenicity is also influenced by host factors. We examined whether CD36, a scavenger receptor that recognizes pathogen and modified self ligands, is a host determinant of K. pneumoniae pathogenicity. Despite differences in serum sensitivity and virulence of 3 distinct K. pneumoniae (hypermucoviscous K1, research K2, and carbapenemase-producing ST258) strains, the absence of CD36 significantly increased host susceptibility to acute intrapulmonary infection by K. pneumoniae, regardless of strain. We demonstrate that CD36 enhances LPS responsiveness to K. pneumoniae to increase downstream cytokine production and macrophage phagocytosis that is independent of polysaccharide capsular antigen. Our study provides new insights into host determinants of K. pneumoniae pathogenicity and raises the possibility that functional mutations in CD36 may predispose individuals to K. pneumoniae syndromes.

The extended-spectrum beta-lactamase gene bla(CTX-M-2) is mainly associated with ISCR1 embedded in complex sul1-type integrons, but information on the genetic context of plasmids harboring the ISCR1-blaCTX-M-2 module remains limited. In this study, a bla(CTX-M-2)-harboring plasmid (pYD786-1) belonging to the sequence type 2(ST2)-IncHI2 plasmid type and isolated from an Escherichia coli ST410 clinical strain was sequenced and analyzed. pYD786-1 belongs to the APEC-O1-R- type IncHI2 plasmids, which are widely distributed in human, poultry, and livestock strains. It contains a multidrug resistance mosaic region (MRR) consisting of a Tn21:: In2 transposon backbone augmented by acquisition of duplicate ISCR1- blaCTX-M- 2 modules. Tn2411, a Tn21:: In2 precursor, likely played a role in the generation of the MRR in pN13- 01290_ 23, the putative progenitor plasmid of pYD786- 1, found in a foodborne Salmonella strain. Tn21/ Tn2411:: In:: ISCR1- bla(CTX-M-2) derivatives, including pYD786- 1, have been identified in strains from Europe, South America, and the United States, suggesting potential global dissemination of the bla(CTX-M-2) modules mediated by this vehicle.

Objective: To develop a score to predict mortality in patients with bloodstream infections (BSIs) due to carbapenemase-producing Enterobacteriaceae (CPE). Patients and Methods: A multinational retrospective cohort study (INCREMENT project) was performed from January 1, 2004, through December 31, 2013. Patients with clinically relevant monomicrobial BSIs due to CPE were included and randomly assigned to either a derivation cohort (DC) or a validation cohort (VC). The variables were assessed on the day the susceptibility results were available, and the predictive score was developed using hierarchical logistic regression. The main outcome variable was 14-day all-cause mortality. The predictive ability of the model and scores were measured by calculating the area under the receiver operating characteristic curve. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated for different cutoffs of the score. Results: The DC and VC included 314 and 154 patients, respectively. The final logistic regression model of the DC included the following variables: severe sepsis or shock at presentation (5 points); Pitt score of 6 or more (4 points); Charlson comorbidity index of 2 or more (3 points); source of BSI other than urinary or biliary tract (3 points); inappropriate empirical therapy and inappropriate early targeted therapy (2 points). The score exhibited an area under the receiver operating characteristic curve of 0.80 (95% CI, 0.74-0.85) in the DC and 0.80 (95% CI, 0.73-0.88) in the VC. The results for 30-day all-cause mortality were similar. Conclusion: A validated score predictive of early mortality in patients with BSIs due to CPE was developed. (C) 2016 Mayo Foundation for Medical Education and Research

The objectives of this study were to elucidate the genetic context of a novel plasmid-mediated fosA variant, fosA6, conferring fosfomycin resistance and to characterize the kinetic properties of FosA6. The genome of fosfomycin-resistant Escherichia coli strain YD786 was sequenced. Homologues of FosA6 were identified through BLAST searches. FosA6 and FosA(ST258) were purified and characterized using a steady-state kinetic approach. Inhibition of FosA activity was examined with sodium phosphonoformate. Plasmid-encoded glutathione-S-transferase (GST) FosA6 conferring high-level fosfomycin resistance was identified in a CTX-M-2-producing E. coli clinical strain at a US hospital. fosA6 was carried on a self-conjugative, 69 kb IncFII plasmid. The Delta lysR-fosA6-Delta yjiR_1 fragment, located between IS10R and Delta IS26, was nearly identical to those on the chromosomes of some Klebsiella pneumoniae strains (MGH78578, PMK1 and KPPR1). FosA6 shared > 99% identity with chromosomally encoded FosA(PMK1) in K. pneumoniae of various STs and 98% identity with FosA(ST258), which is commonly found in K. pneumoniae clonal complex (CC) 258 including ST258. FosA6 and FosA(ST258) demonstrated robust GST activities that were comparable to each other. Sodium phosphonoformate, a GST inhibitor, reduced the fosfomycin MICs by 6- to 24-fold for K. pneumoniae and E. coli strains carrying fosA genes on the chromosomes and plasmids, respectively. fosA6, probably captured from the chromosome of K. pneumoniae, conferred high-level fosfomycin resistance in E. coli. FosA6 functioned as a GST and inactivated fosfomycin efficiently. K. pneumoniae may serve as a reservoir of fosfomycin resistance for E. coli.

OBJECTIVE. To concomitantly determine the differential degrees of air and environmental contamination by Acinetobacter baumannii based on anatomic source of colonization and type of ICU layout (single-occupancy vs open layout). DESIGN. Longitudinal prospective surveillance study of air and environmental surfaces in patient rooms. SETTING. A 1,500-bed public teaching hospital in Miami, Florida. PATIENTS. Consecutive A. baumannii colonized patients admitted to our ICUs between October 2013 and February 2014. METHODS. Air and environmental surfaces of the rooms of A. baumannii colonized patients were sampled daily for up to 10 days. Pulsed field gel electrophoresis (PFGE) was used to type and match the matching air, environmental, and clinical A. baumannii isolates. RESULTS. A total of 25 A. baumannii colonized patients were identified during the study period; 17 were colonized in the respiratory tract and 8 were colonized in the rectum. In rooms with rectally colonized patients, 38.3% of air samples were positive for A. baumannii; in rooms of patients with respiratory colonization, 13.1% of air samples were positive (P = .0001). In rooms with rectally colonized patients, 15.5% of environmental samples were positive for A. baumannii; in rooms of patients with respiratory colonization, 9.5% of environmental samples were positive (P = .02). The rates of air contamination in the open-layout and single-occupancy ICUs were 17.9% and 21,8%, respectively (P = .5). Environmental surfaces were positive in 9.5% of instances in open-layout ICUs versus 13.4% in single-occupancy ICUs (P = .09). CONCLUSIONS. Air and environmental surface contaminations were significantly greater among rectally colonized patients; however, ICU layout did not influence the rate of contamination.

Novel combination therapies are desperately needed for combating lung infections caused by bacterial "superbugs". This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the cystic fibrosis (CF) drugs KALYDECO (ivacaftor) and ORKAMBI (ivacaftor + lumacaftor) against Gram-negative pathogens that commonly colonize the CF lung, in particular, the problematic Pseudomonas aeruginosa. The in vitro synergistic activity of polymyxin B combined with ivacaftor or lumacaftor was assessed using checkerboard and static time-kill assays against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa isolates from the lungs of CF patients. Polymyxin B, ivacaftor, and lumacaftor were ineffective when used individually against polymyxin-resistant (MIC >= 4 mg/L) isolates. However, when used together, the combination of clinically relevant concentrations of polymyxin B (2 mg/L) combined with ivacaftor (8 mg/L) or ivacaftor (8 mg/L) + lumacaftor (8 mg/L) displayed synergistic killing activity against polymyxin-resistant P. aeruginosa isolates as demonstrated by a 100-fold decrease in the bacterial count (CFU/mL) even after 24 h. The combinations also displayed excellent antibacterial activity against P. aeruginosa under CF relevant conditions in a sputum medium assay. The combination of lumacaftor (alone) with polymyxin B showed additivity against P. aeruginosa. The potential antimicrobial mode of action of the combinations against P. aeruginosa was investigated using different methods. Treatment with the combinations induced cytosolic GFP release from P. aeruginosa cells and showed permeabilizing activity in the nitrocefin assay, indicating damage to both the outer and inner Gram-negative cell membranes. Moreover, scanning and transmission electron micrographs revealed that the combinations produce outer membrane damage to P. aeruginosa cells that is distinct from the effect of each compound per se. Ivacaftor was also shown to be a weak inhibitor of the bacterial DNA gyrase and topoisomerase IV with no effect on either human type I or type Ha topoisomerases. Lumacaftor displayed the ability to increase the cellular production of damaging reactive oxygen species. In summary, the combination of polymyxin B with KALYDECO or ORKAMBI exhibited synergistic activity against highly polymyxin-resistant P. aeruginosa CF isolates and can be potentially useful for otherwise untreatable CF lung infections.

Polymyxins, an old class of antibiotics, are currently used as the last resort for the treatment of multidrug-resistant (MDR) Acinetobacter baumannii. However, recent pharmacokinetic and pharmacodynamic data indicate that monotherapy can lead to the development of resistance. Novel approaches are urgently needed to preserve and improve the efficacy of this last-line class of antibiotics. This study examined the antimicrobial activity of novel combination of polymyxin B with anthelmintic closantel against A. baumannii. Closantel monotherapy (16 mg 1(-1)) was ineffective against most tested A. baumannii isolates. However, closantel at 4-16 mg l(-1) with a clinically achievable concentration of polymyxin B (2 mg l(-1)) successfully inhibited the development of polymyxin resistance in polymyxin-susceptible isolates, and provided synergistic killing against polymyxin-resistant isolates (MIC. 4 mg l(-1)). Our findings suggest that the combination of polymyxin B with closantel could be potentially useful for the treatment of MDR, including polymyxin-resistant, A. baumannii infections. The repositioning of non-antibiotic drugs to treat bacterial infections may significantly expedite discovery of new treatment options for bacterial 'superbugs'.

Aminoglycoside-producing Actinobacteria are known to protect themselves from their own aminoglycoside metabolites by producing 16S ribosomal RNA methyltransferase (16S-RMTase), which prevents them from binding to the 16S rRNA targets. Ten acquired 16S-RMTases have been reported from gram-negative pathogens. Most of them posttranscriptionally methylate residue G1405 of 16S rRNA resulting in high-level resistance to gentamicin, tobramycin, amikacin, and plazomicin. Strains that produce 16S-RMTase are frequently multidrug-resistant or even extensively drug resistant. Although the direct clinical impact of high-level aminoglycoside resistance resulting from production of 16S-RMTase is yet to be determined, ongoing spread of this mechanism will further limit treatment options for multidrug-resistant and extensively drug-resistant gram-negative infections.