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Carbapenemase-producing Enterobacterales (CPEs) are one of the top priority antimicrobial-resistant pathogens. Among CPEs, those producing acquired metallo-β-lactamases (MBLs) are considered particularly problematic as few agents are active against them. Imipenemase (IMP) is the most frequently encountered acquired MBL in Japan, but comprehensive assessment of clinical and microbiological features of IMP-producing Enterobacterales infection remains scarce. Here, we retrospectively evaluated 62 patients who were hospitalized at a university hospital in Japan and had IMP-producing Enterobacterales from a clinical culture. The isolates were either Enterobacter cloacae complex or Klebsiella pneumoniae , and most of them were isolated from sputum. The majority of K. pneumoniae, but not E. cloacae complex isolates, were susceptible to aztreonam. Sequence type (ST) 78 and ST517 were prevalent for E. cloacae complex and K. pneumoniae , respectively, and all isolates carried bla IMP-1 . Twenty-four of the patients were deemed infected with IMP-producing Enterobacterales . Among the infected patients, therapy varied and largely consisted of conventional β-lactam agents, fluoroquinolones, or combinations. Three (13%), five (21%), and nine (38%) of them died by days 14, 30, and 90, respectively. While incremental mortality over 90 days was observed in association with underlying comorbidities, active conventional treatment options were available for most patients with IMP-producing Enterobacterales infections, distinguishing them from more multidrug-resistant CPE infections associated with globally common MBLs, such as New Delhi metallo-β-lactamase (NDM) and Verona integron-encoded metallo-β-lactamase (VIM)., Competing Interests: The authors declare no conflict of interest.

Carbapenemase-producing Escherichia coli sequence type (ST) 648 strains were isolated from two international visitors without previous medical exposure from Southeast Asian countries in a hospital in Japan. One isolate, FUJ80154, carried bla NDM-5 in a complex class 1 integron on an IncFIB/FII plasmid; the other isolate, FUJ80155, carried two copies of bla OXA-48 on the chromosome flanked by IS 1R on both sides. The core-genome based-phylogenetic analysis with publicly available genome data of E. coli ST648 carrying bla NDM-5 or bla OXA-48-like demonstrated high genetic similarity between FUJ80154 and NDM-5-prooducing E. coli ST648 strains isolated in South and Southeast Asian countries. On the other hand, no closely related isolates of FUJ80155 were identified. In the absence of prior hospitalization overseas, neither patient had qualified for routine screening of multidrug-resistant organisms, and the isolates were incidentally identified in cultures ordered at the discretion of the treating physician. IMPORTANCE Although patients with history of international hospitalization are often subject to screening for multidrug-resistant organisms, it is unclear whether patients who reside in countries where carbapenemase-producing Enterobacterales (CPE) is endemic but have no history of local hospitalization contribute to the transmission of CPE. In this study, NDM-5-producing and OXA-48-producing Escherichia coli sequence type (ST) 648, a recently recognized high-risk, multidrug-resistant clone, were detected from two overseas visitors without previous medical exposure. The findings of this study suggest that active surveillance culture on admission to hospital may be considered for travelers from countries with endemicity of carbapenem-resistant organisms even without history of local hospitalization and underscore the need to monitor cross-border transmission of high-risk clones, such as carbapenemase-producing E. coli ST648.

Rapid detection and reporting of carbapenemase-producing Enterobacterales (CPE) is one of the top priorities of clinical microbiology laboratories. The Clinical and Laboratory Standards Institute recommends the modified carbapenem inactivation method (mCIM) as the preferred method for this purpose, but it requires a broth incubation process which can be cumbersome. Here, we compared the performance of mCIM with three alternative rapid CPE detection methods against a collection of genetically defined CPE, with most carrying bla IMP , and non-CPE clinical isolates. The sensitivities of mCIM, simplified carbapenem inactivation method (sCIM), Rapidec Carba NP, and NG-Test Carba 5 were 98.0%, 54.9%, 90.2%, and 72.5%, whereas the specificities were 89.5%, 84.2%, 89.5%, and 100%, respectively. Modification of the interpretive criteria of sCIM increased its sensitivity to 88.2% and specificity to 89.5%. The results suggest that mCIM is currently the optimal method for CPE detection in an epidemiological setting where CPE-producing IMP group carbapenemase is predominant. While sCIM is easier to perform, it requires further validation before it can be widely adopted as an alternative to mCIM in the clinical laboratory. IMPORTANCE Simple identification methods for carbapenemase-producing Enterobacterales are required for the clinical laboratory. The simplified carbapenem inactivation method (sCIM) is a carbapenemase detection method that can be performed with less hands-on time than mCIM, but its sensitivity and specificity were suboptimal compared with other phenotypic detection methods when tested against a collection of IMP-producing CPE. Insufficient inactivation of imipenem from inadequate inoculation was suspected as the cause. While sCIM is easier to perform, it requires optimization before it can be widely adopted as an alternative to mCIM in the clinical laboratory.

Background: Pseudomonas aeruginosa (PA) is a common cause of respiratory infection and morbidity. Pseudomonas elastase is an important virulence factor regulated by the lasR gene. Whether PA elastase activity is associated with worse clinical outcomes in ICU patients is unknown., Research Question: Is there an association between PA elastase activity and worse host outcomes in a cohort of ICU patients?, Methods: PA respiratory isolates from 238 unique ICU patients from two tertiary-care centers within the University of Pittsburgh Medical Center health system were prospectively collected and screened for total protease and elastase activity, biofilm production, antimicrobial resistance, and polymicrobial status. The association between pathogen characteristics and 30-day and 90-day mortality was calculated using logistic regression. For subgroup analysis, two patterns of early (≤72 h) and late sample (>72 h) collection from the index ICU admission were distinguished using a finite mixture model. Lung inflammation and injury was evaluated in a mouse model using a PA high elastase vs low elastase producer., Results: PA elastase activity was common in ICU respiratory isolates representing 75% of samples and was associated with increased 30-day mortality (adjusted OR [95% CI]: 1.39 [1.05-1.83]). Subgroup analysis demonstrated that elastase activity was a risk factor for 30- and 90-day mortality in the early sample group, whereas antimicrobial resistance was a risk factor for 90-day mortality in the late sample group. Whole genome sequencing of high and low elastase producers showed that predicted loss-of-function lasR genotypes were less common among high elastase producers. Mice infected with a high elastase producer showed increased lung bacterial burden and inflammatory profile compared with mice infected with a low elastase producer., Interpretation: Elastase activity is associated with 30-day ICU mortality. A high elastase producing clinical isolate confers increased lung tissue inflammation compared with a low elastase producer in vivo., (Copyright © 2021 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

OXA-232 is an OXA-48-group class D β-lactamase that hydrolyzes expanded-spectrum cephalosporins and carbapenems at low levels. Clinical strains producing OXA-232 are sometimes susceptible to carbapenems, making it difficult to identify them in the clinical microbiology laboratory. We describe the development of carbapenem resistance in sequential clinical isolates of Raoultella ornithinolytica carrying bla OXA-232 in a hospitalized patient, where the ertapenem MIC increased from 0.5 μg/ml to 512 μg/ml and the meropenem MIC increased from 0.125 μg/ml to 32 μg/ml during the course of ertapenem therapy. Whole-genome sequencing (WGS) analysis identified loss-of-function mutations in ompC and ompF in carbapenem-resistant isolates that were not present in the initial carbapenem-susceptible isolate. Complementation of a carbapenem-resistant isolate with an intact ompF gene resulted in 16- to 32-fold reductions in carbapenem MICs, whereas complementation with intact ompC resulted in a 2-fold reduction in carbapenem MICs. Additionally, bla OXA-232 expression increased 2.9-fold in a carbapenem-resistant isolate. Rapid development of high-level carbapenem resistance in initially carbapenem-susceptible OXA-232-producing R. ornithinolytica under selective pressure from carbapenem therapy highlights the diagnostic challenges in detecting Enterobacteriaceae strains producing this inefficient carbapenemase., (Copyright © 2019 American Society for Microbiology.)

Understanding the nuances of AmpC β-lactamase-mediated resistance can be challenging, even for the infectious diseases specialist. AmpC resistance can be classified into 3 categories: (1) inducible chromosomal resistance that emerges in the setting of a β-lactam compound, (2) stable derepression due to mutations in ampC regulatory genes, or (3) the presence of plasmid-mediated ampC genes. This review will mainly focus on inducible AmpC resistance in Enterobacteriaceae. Although several observational studies have explored optimal treatment for AmpC producers, few provide reliable insights into effective management approaches. Heterogeneity within the data and inherent selection bias make inferences on effective β-lactam choices problematic. Most experts agree it is prudent to avoid expanded-spectrum (ie, third-generation) cephalosporins for the treatment of organisms posing the greatest risk of ampC induction, which has best been described in the context of Enterobacter cloacae infections. The role of other broad-spectrum β-lactams and the likelihood of ampC induction by other Enterobacteriaceae are less clear. We will review the mechanisms of resistance and triggers resulting in AmpC expression, the species-specific epidemiology of AmpC production, approaches to the detection of AmpC production, and treatment options for AmpC-producing infections., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

We report patient-to-patient transmission of Enterobacter hormaechei isolates with reduced susceptibility to ceftazidime-avibactam due to production of KPC-40, a variant of KPC-3 with a two-amino-acid insertion in the Ω-loop region (L167_E168dup). The index patient had received a prolonged course of ceftazidime-avibactam therapy, whereas the second patient had not received the agent and still became colonized with the KPC-40-producing strain. The complex dynamics of KPC ( Klebsiella pneumoniae carbapenemase) described here highlight several key diagnostic and therapeutic considerations., (Copyright © 2019 American Society for Microbiology.)

Carbapenem resistant Enterobacteriaceae are a significant public health concern, and genes encoding the Klebsiella pneumoniae carbapenemase (KPC) have contributed to the global spread of carbapenem resistance. In the current study, we used whole-genome sequencing to investigate the diversity of bla KPC -containing plasmids and antimicrobial resistance mechanisms among 26 bla KPC -containing Escherichia coli, and 13 bla KPC -containing Enterobacter asburiae, Enterobacter hormaechei, K. pneumoniae, Klebsiella variicola, Klebsiella michiganensis, and Serratia marcescens strains, which were isolated from the same patients as the bla KPC -containing E. coli. A bla KPC -containing IncN and/or IncFII K plasmid was identified in 77% (30/39) of the E. coli and other bacterial species analyzed. Complete genome sequencing and comparative analysis of a bla KPC -containing IncN plasmid from one of the E. coli strains demonstrated that this plasmid is present in the K. pneumoniae and S. marcescens strains from this patient, and is conserved among 13 of the E. coli and other bacterial species analyzed. Interestingly, while both IncFII K and IncN plasmids were prevalent among the strains analyzed, the IncN plasmids were more often identified in multiple bacterial species from the same patients, demonstrating a contribution of this IncN plasmid to the inter-genera dissemination of the bla KPC genes between the E. coli and other bacterial species analyzed.

Background: fosA3 is the most commonly reported plasmid-mediated fosfomycin resistance gene among Enterobacteriaceae., Objectives: To identify the origin of fosA3., Methods: The chromosome of Kluyvera georgiana clinical strain YDC799 was fully sequenced with single-molecule real-time sequencing. Comparative genetic analysis was performed for K. georgiana YDC799, K. georgiana type strain ATCC 51603 and representative fosA3-carrying plasmids. fosA genes were cloned in Escherichia coli to confirm function., Results: K. georgiana YDC799 harboured fosA (designated fosAKG) and blaCTX-M-8 on the chromosome. The genetic environments surrounding fosA3 and bounded by IS26 were nearly identical with the corresponding regions of K. georgiana YDC799 and ATCC 51603. The amino acid sequence of FosAKG from YDC799 and K. georgiana ATCC 51603 shared 99% and 94% identity with FosA3, respectively. Cloned FosAKG conferred fosfomycin resistance with an MIC of >1024 mg/L for E. coli., Conclusions: The plasmid-mediated fosA3 gene was likely mobilized from the chromosome of K. georgiana by an IS26-mediated event., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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 Mn 2+ 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 Mn 2+ - 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., (Copyright © 2017 Ito et al.)

Ceftazidime-avibactam is a novel β-lactam/β-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidime-avibactam 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. Whole-genome 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 bla KPC 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., (Copyright © 2017 American Society for Microbiology.)

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., Trial Registration: clinicaltrials.gov Identifier: NCT01 764490., (Copyright © 2016 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

We compared the in vitro activities of gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and plazomicin (PLZ) against 13 Enterobacter isolates possessing both Klebsiella pneumoniae carbapenemase and extended-spectrum β-lactamase (KPC+/ESBL+) with activity against 8 KPC+/ESBL-, 6 KPC-/ESBL+, and 38 KPC-/ESBL- isolates. The rates of resistance to GEN and TOB were higher for KPC+/ESBL+ (100% for both) than for KPC+/ESBL- (25% and 38%, respectively), KPC-/ESBL+ (50% and 17%, respectively), and KPC-/ESBL- (0% and 3%, respectively) isolates. KPC+/ESBL+ isolates were more likely than others to possess an aminoglycoside-modifying enzyme (AME) (100% versus 38%, 67%, and 5%; P = 0.007, 0.06, and <0.0001, respectively) or multiple AMEs (100% versus 13%, 33%, and 0%, respectively; P < 0.01 for all). KPC+/ESBL+ isolates also had a greater number of AMEs (mean of 4.6 versus 1.5, 0.9, and 0.05, respectively; P < 0.01 for all). GEN and TOB MICs were higher against isolates with >1 AME than with ≤1 AME. The presence of at least 2/3 of KPC, SHV, and TEM predicted the presence of AMEs. PLZ MICs against all isolates were ≤4 μg/ml, regardless of KPC/ESBL pattern or the presence of AMEs. In conclusion, GEN and TOB are limited as treatment options against KPC+ and ESBL+ Enterobacter PLZ may represent a valuable addition to the antimicrobial armamentarium. A full understanding of AMEs and other aminoglycoside resistance mechanisms will allow clinicians to incorporate PLZ rationally into treatment regimens. The development of molecular assays that accurately and rapidly predict antimicrobial responses among KPC- and ESBL-producing Enterobacter spp. should be a top research priority., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: The objective of this study was to conduct a comparative analysis with reported IncR plasmids of a Klebsiella pneumoniae IncR plasmid carrying an MDR region., Methods: MDR K. pneumoniae isolates were serially identified from two inpatients at a hospital in the USA in 2014. MDR plasmid pYDC676 was fully sequenced, annotated and compared with related plasmids. Antimicrobial susceptibility testing, PFGE and MLST were also conducted., Results: The K. pneumoniae isolates were identical by PFGE, belonged to ST37 and harboured an identical ∼50 kb IncR plasmid (pYDC676). pYDC676 possessed the backbone and multi-IS loci closely related to IncR plasmids reported from aquatic bacteria, as well as animal and human K. pneumoniae strains, and carried an MDR region consisting of armA, blaDHA-1 and qnrB4, a combination that has been reported in IncR plasmids from K. pneumoniae ST11 strains in Europe and Asia. A plasmid with the identical IncR backbone and a similar MDR region containing blaDHA-1 and qnrB4 has also been reported in ST37 strains from Europe, suggesting potential dissemination of this lineage of IncR plasmids in K. pneumoniae ST37., Conclusions: K. pneumoniae ST37 strains with an MDR IncR plasmid carrying armA, blaDHA-1 and qnrB4 were identified in a hospital in the USA, where these resistance genes remain rare. The IncR backbone may play a role in the global dissemination of these resistance genes., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Enterobacter cloacae strain G6809 with reduced susceptibility to carbapenems was identified from a patient in a long-term acute care hospital in Kentucky. G6809 belonged to sequence type (ST) 88 and carried two carbapenemase genes, bla(KPC-18) and bla(VIM-1). Whole-genome sequencing localized bla(KPC-18) to the chromosome and bla(VIM-1) to a 58-kb plasmid. The strain was highly resistant to ceftazidime-avibactam. Insidious coproduction of metallo-β-lactamase with KPC-type carbapenemase has implications for the use of next-generation β-lactam-β-lactamase inhibitor combinations., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

A high fosfomycin resistance rate was observed in Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) in our previous study, but little is known about its mechanisms. In this study, we explored the prevalence of plasmid-mediated fosfomycin resistance determinants among fosfomycin-resistant KPC-KP strains from a Chinese university hospital and determined the complete sequence of a novel fosA3-carrying plasmid isolated from an epidemic K. pneumoniae sequence type (ST) 11 strain. A total of 97 KPC-KP strains were studied, of which 57 (58.8%) were resistant to fosfomycin, including 44 (45.4%) harboring fosA3 and 1 harboring fosA. All fosA3-positive strains belonged to the dominant ST11-pulse type (PT) A clone according to multilocus sequence typing and pulsed-field gel electrophoresis, suggesting clonal dissemination. The fosA-positive isolate belonged to ST11-PTE. The fosA3-carrying plasmid pKP1034 is 136,848 bp in length and is not self-transmissible. It is a multireplicon plasmid belonging to IncR-F33:A-: B-. Besides fosA3, a variety of other resistance determinants, including blaKPC-2, rmtB, blaCTX-M-65, and blaSHV-12, are identified in pKP1034, which would allow for coselection of fosA3 by most β-lactams and/or aminoglycosides and facilitate its dissemination despite limited use of fosfomycin in China. Detailed comparisons with related plasmids revealed that pKP1034 is highly mosaic and might have evolved from alarming recombination of the blaKPC-2-carrying plasmid pKPC-LK30 from Taiwan and the epidemic fosA3-carrying plasmid pHN7A8 from mainland China., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Increased incidence of infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum β-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single molecule, real-time sequencing. Plasmid diversity was assessed by endonuclease digestion. Genomic and epidemiologic data were used in conjunction to investigate the outbreak source. Two clusters of Kp patient isolates were genetically related to endoscope isolates by PFGE. A subset of patient isolates were collected post-ERCP, suggesting ERCP endoscopes as a possible source. A phylogeny of 7 Kp genomes from patient and endoscope isolates supported ERCP as a potential source of transmission. Differences in gene content defined 5 ST258 subclades and identified 2 of the subclades as outbreak-associated. A novel KPC-encoding plasmid, pKp28 helped define and track one endoscope-associated ST258 subclade. WGS demonstrated high genetic relatedness of patient and ERCP endoscope isolates suggesting ERCP-associated transmission of ST258 KPC-Kp. Gene and plasmid content discriminated the outbreak from endemic ST258 populations and assisted with the molecular epidemiologic investigation of an extended KPC-Kp outbreak.

Carbapenem-resistant Klebsiella pneumoniae strains that produce K. pneumoniae carbapenemase (KPC) have spread globally in the last decade. Colistin is a key agent in treating infections caused by this pathogen. In this issue of the Journal of Clinical Microbiology, Giani et al. (T. Giani, F. Arena, G. Vaggelli, V. Conte, A Chiarell, L. H. De Angelis, R. Fornaini, M. Grazzini, F. Niccolini, P. Pecile, and G. M. Rossolini, J Clin Microbiol 53:3341-3344, 2015, http://dx.doi.org/10.1128/JCM.01017-15) describe a sustained outbreak of colistin-resistant KPC-producing K. pneumoniae., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Carbapenemase-producing Enterobacteriaceae (CPE) were almost nonexistent up to the 1990s, but are today encountered routinely in hospitals and other healthcare facilities in many countries including the United States. KPC-producing Klebsiella pneumoniae was the first to emerge and spread globally and is endemic in the United States, Israel, Greece, and Italy. Recently, NDM-producing Enterobacteriaceae and OXA-48-producing K. pneumoniae appear to be disseminating from South Asia and Northern Africa, respectively. They are almost always resistant to all β-lactams including carbapenems and many other classes. Mortality from invasive CPE infections reaches up to 40%. To obtain the maximal benefit from the limited options available, dosing of antimicrobial agents should be optimized based on pharmacokinetic data, especially for colistin and carbapenems. In addition, multiple observational studies have associated combination antimicrobial therapy with lower mortality compared with monotherapy for these infections. The outcomes appear to be especially favorable when patients are treated with a carbapenem and a second agent such as colistin, tigecycline, and gentamicin, but the best approach is yet to be defined., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

The aminoglycoside-modifying enzyme AAC(6')-Ib is common among carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strains. We investigated amikacin (AMK) activity against 20 AAC(6')-Ib-producing CR-Kp strains. MICs clustered at 16 to 32 μg/ml. By the time-kill study, AMK (1× and 4× the MIC) was bactericidal against 30% and 85% of the strains, respectively. At achievable human serum concentrations, however, the majority of strains showed regrowth, suggesting that AAC(6')-Ib confers intermediate AMK resistance. AMK and trimethoprim-sulfamethoxazole (TMP-SMX) were synergistic against 90% of the strains, indicating that the combination may overcome resistance., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We sequenced a novel conjugative blaKPC-2-harboring IncN plasmid, pYD626E, from an Escherichia coli sequence type 648 strain previously identified in Pittsburgh, Pennsylvania. pYD626E was 72,800 bp long and carried four β-lactamase genes, blaKPC-2, blaSHV-12, blaLAP-1, and blaTEM-1. In addition, it harbored qnrS1 (fluoroquinolone resistance) and dfrA14 (trimethoprim resistance). The plasmid profile and clinical history supported the in vivo transfer of this plasmid between Klebsiella pneumoniae and Escherichia coli., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Microbiological data regarding Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter spp. are scarce. In this study, 11 unique KPC-producing Enterobacter isolates were identified among 44 ertapenem-nonsusceptible Enterobacter isolates collected between 2009 and 2013 at a hospital system in Western Pennsylvania. All cases were healthcare-associated and occurred in medically complex patients. While pulsed-field gel electrophoresis showed diverse restriction patterns overall, multilocus sequence typing identified Enterobacter cloacae isolates with sequence types 93 and 171 from 2 hospitals each. The levels of carbapenem minimum inhibitory concentrations were highly variable. All isolates remained susceptible to colistin and tigecycline, and the majority, to amikacin and doxycycline. A blaKPC-carrying IncN plasmid conferring trimethoprim-sulfamethoxazole resistance was identified in 3 of the isolates. Spread of blaKPC in Enterobacter spp. appears to be due to a combination of plasmid-mediated and clonal processes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The whole-genome sequence of a carbapenem-resistant Klebsiella pneumoniae strain, PittNDM01, which coproduces NDM-1 and OXA-232 carbapenemases, was determined in this study. The use of single-molecule, real-time (SMRT) sequencing provided a closed genome in a single sequencing run. K. pneumoniae PittNDM01 has a single chromosome of 5,348,284 bp and four plasmids: pPKPN1 (283,371 bp), pPKPN2 (103,694 bp), pPKPN3 (70,814 bp), and pPKPN4 (6,141 bp). The contents of the chromosome were similar to that of the K. pneumoniae reference genome strain MGH 78578, with the exception of a large inversion spanning 23.3% of the chromosome. In contrast, three of the four plasmids are unique. The plasmid pPKPN1, an IncHI1B-like plasmid, carries the blaNDM-1, armA, and qnrB1 genes, along with tellurium and mercury resistance operons. blaNDM-1 is carried on a unique structure in which Tn125 is further bracketed by IS26 downstream of a class 1 integron. The IncFIA-like plasmid pPKPN3 also carries an array of resistance elements, including blaCTX-M-15 and a mercury resistance operon. The ColE-type plasmid pPKPN4 carrying blaOXA-232 is identical to a plasmid previously reported from France. SMRT sequencing was useful in resolving the complex bacterial genomic structures in the de novo assemblies., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Of 20 Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli isolates identified at hospitals in western Pennsylvania, 60% belonged to the epidemic ST131-fimH30 subclone. IncFIIk was the most common replicon type for the blaKPC-carrying plasmids (n = 8). All IncFIIk plasmids possessed a scaffold similar to that of pKpQIL, and seven of them were borne by ST131-fimH30 isolates. IncN plasmids conferred resistance to trimethoprim-sulfamethoxazole, and IncA/C plasmids conferred resistance to gentamicin. Three blaKPC-carrying plasmids (IncA/C and IncN) possessed blaSHV-7/12 and qnrA1 or qnrS1., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Doripenem-colistin exerts synergy against some, but not all, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains in vitro. We determined if doripenem MICs and/or ompK36 porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of doripenem (8 μg/ml) and colistin (2 μg/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 μg/ml. Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutant ompK36, respectively. The most common mutations were IS5 promoter insertions (n = 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD; n = 8), which were associated with higher doripenem MICs than other mutations or wild-type ompK36 (all P values ≤ 0.04). Bactericidal activity (24 h) was achieved by doripenem-colistin against 12%, 43%, and 75% of ins aa134-135 GD, IS5, and wild-type/other mutants, respectively (P = 0.04). Doripenem-colistin was more active in time-kill studies than colistin at 12 and 24 h if the doripenem MIC was ≤8 μg/ml (P = 0.0007 and 0.09, respectively), but not if the MIC was >8 μg/ml (P = 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins aa134-135 GD or IS5 mutants (P = 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS5 mutations was the only independent predictor of doripenem-colistin responses at 24 h (P = 0.002). In conclusion, ompK36 genotypes identified ST258 KPC-K. pneumoniae strains that were most likely to respond to doripenem-colistin.

We characterized carbapenem resistance mechanisms among 12 Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (referred to here as KPC K. pneumoniae) clinical isolates and evaluated their effects on the activity of 2- and 3-drug combinations of colistin, doripenem, and ertapenem. All isolates were resistant to ertapenem and doripenem; 75% (9/12) were resistant to colistin. Isolates belonged to the ST258 clonal group and harbored blaKPC-2, blaSHV-12, and blaTEM-1. As determined by time-kill assays, doripenem (8 μg/ml) and ertapenem (2 μg/ml) were inactive against 92% (11/12) and 100% (12/12) of isolates, respectively. Colistin (2.5 μg/ml) exerted some activity (range, 0.39 to 2.5 log10) against 78% (7/9) of colistin-resistant isolates. Colistin-ertapenem, colistin-doripenem, and colistin-doripenem-ertapenem exhibited synergy against 42% (5/12), 50% (6/12), and 67% (8/12) of isolates, respectively. Expression of ompK35 and ompK36 porins correlated with each other (R(2) = 0.80). Levels of porin expression did not correlate with colistin-doripenem or colistin-ertapenem synergy. However, synergy with colistin-doripenem-ertapenem was more likely against isolates with high porin expression than those with low expression (100% [8/8] versus 0% [0/4]; P = 0.002). Moreover, bactericidal activity (area under the bacterial killing curve) against isolates with high porin expression was greater for colistin-doripenem-ertapenem than colistin-doripenem or colistin-ertapenem (P ≤ 0.049). In conclusion, colistin-carbapenem combinations may provide optimal activity against KPC K. pneumoniae, including colistin-resistant isolates. Screening for porin expression may identify isolates that are most likely to respond to a triple combination of colistin-doripenem-ertapenem. In the future, molecular characterization of KPC K. pneumoniae isolates may be a practical tool for identifying effective combination regimens.

Klebsiella pneumoniae strain MRSN2404 was isolated from the chronic wound of a soldier who had been wounded in Iraq in 2006. The strain displayed very high MICs of all aminoglycosides, including arbekacin. A gene encoding a novel 16S rRNA methyltransferase, now designated RmtH, was identified. RmtH had 64% identity with RmtB1 and RmtB2. rmtH was bracketed by two copies of ISCR2, which may have played a role in its mobilization.

Background: Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae has become endemic in many US hospitals. On the other hand, KPC-producing Escherichia coli remains rare., Methods: We studied infection or colonization due to KPC-producing E. coli identified at our hospital between September 2008 and February 2011. A case-control study was conducted to document clinical features associated with this organism. Susceptibility testing, sequencing of β-lactamase genes, pulsed-field gel electrophoresis, multilocus sequence typing, and plasmid analysis were performed for characterization of the isolates., Results: Thirteen patients with KPC-producing E. coli were identified. The patients had multiple comorbid conditions and were in hospital for variable periods of time before KPC-producing E. coli was identified. The presence of liver diseases was independently associated with recovery of KPC-producing E. coli when compared with extended-spectrum β-lactamase-producing E. coli. The isolates showed variable susceptibility to carbapenems. Seven isolates belonged to sequence type (ST) 131, which is the international epidemic, multidrug-resistant clone, but their plasmid profiles were diverse. KPC-producing organisms other than E. coli were isolated within 1 month from 5 of the patients. The KPC-encoding plasmids were highly related in 3 of them, suggesting the occurrence of their interspecies transfer., Conclusions: KPC-producing E. coli infections occur in severely ill patients who are admitted to the hospital. Acquisition of the KPC-encoding plasmids by the ST 131 clone, reported here for the first time to our knowledge in the United States, seems to represent multiple independent events. These plasmids are often shared between E. coli and other species.

Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been associated with serious infections and high mortality. The optimal antimicrobial therapy for infection due to KPC-producing K. pneumoniae is not well established. We conducted a retrospective cohort study to evaluate the clinical outcome of patients with bacteremia caused by KPC-producing K. pneumoniae. A total of 41 unique patients with blood cultures growing KPC-producing K. pneumoniae were identified at two medical centers in the United States. Most of the infections were hospital acquired (32; 78%), while the rest of the cases were health care associated (9; 22%). The overall 28-day crude mortality rate was 39.0% (16/41). In the multivariate analysis, definitive therapy with a combination regimen was independently associated with survival (odds ratio, 0.07 [95% confidence interval, 0.009 to 0.71], P = 0.02). The 28-day mortality was 13.3% in the combination therapy group compared with 57.8% in the monotherapy group (P = 0.01). The most commonly used combinations were colistin-polymyxin B or tigecycline combined with a carbapenem. The mortality in this group was 12.5% (1/8). Despite in vitro susceptibility, patients who received monotherapy with colistin-polymyxin B or tigecycline had a higher mortality of 66.7% (8/12). The use of combination therapy for definitive therapy appears to be associated with improved survival in bacteremia due to KPC-producing K. pneumoniae.

ADC-56, a novel extended-spectrum AmpC (ESAC) β-lactamase, was identified in an Acinetobacter baumannii clinical isolate. ADC-56 possessed an R148Q change compared with its putative progenitor, ADC-30, which enabled it to hydrolyze cefepime. Molecular modeling suggested that R148 interacted with Q267, E272, and I291 through a hydrogen bond network which constrained the H-10 helix. This permitted cefepime to undergo conformational changes in the active site, with the carboxyl interacting with R340, likely allowing for better binding and turnover.

Five cases of infection due to colistin-resistant, Klebsiella pneumoniae carbapenemase-producing K. pneumoniae belonging to the international epidemic clone ST258 occurred over a 4-month period. These cases likely represented both emergence of resistance and transmission of resistant organism. The colistin-resistant isolates were able to persist in the absence of selective pressure in vitro.

Three Acinetobacter baumannii isolates that possess OXA-40 group carbapenemase genes were identified. They belonged to novel sequence types (ST122, ST123, and ST124) and harbored bla(OXA-160), bla(OXA-72), and bla(OXA-40), respectively. OXA-160 is a novel variant of OXA-40 with a P227S substitution. An isogenic Escherichia coli clone producing OXA-160 was more susceptible to carbapenems than a clone producing OXA-40. The genetic environment of bla(OXA-160) and bla(OXA-40) beyond the putative XerC/XerD recombination sites was distinct from the scaffold reported previously.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, Escherichia coli, and Serratia marcescens were sequentially identified in a patient who underwent small bowel transplantation. Molecular typing and plasmid analysis suggested that the KPC gene was acquired by E. coli, most likely from K. pneumoniae, and was subsequently transferred to S. marcescens.

Background: Since 2001 there have been several reported outbreaks due to carbapenem-resistant Klebsiella pneumoniae (Kp), particularly in the northeastern states., Methods: Carbapenemase-producing Enterobacteriaceae from healthcare facilities in Northeast Florida were phenotypically identified and confirmed using PCR amplification and sequencing of the blaKPC gene., Results: Results from PFGE analysis of these isolates demonstrated possible horizontal spread from two possible "outbreak" strains during the study period., Conclusions: We present the first published cluster of Kp and Escherichia coli (Ec) cases in Florida carrying the KPC-2 or KPC-3 gene.

The X-ray crystal structure of AmpC beta-lactamase (AmpC(D)) with a tripeptide deletion (Gly286-Ser287-Asp288) produced by Escherichia coli HKY28, a ceftazidime-resistant strain, was determined at a resolution of 1.7 A. The structure of AmpC(D) suggests that the tripeptide deletion at positions 286-288 located in the H10 helix causes a structural change of the Asn289-Asn294 region from the alpha-helix present in the native AmpC beta-lactamase of E. coli to a loop structure, which results in a widening of the substrate-binding site.

An outbreak of cephalosporin-resistant Klebsiella pneumoniae occurred in a neonatal intensive care unit in São Paulo, Brazil. Of the 10 pulsotypes identified during the outbreak and follow-up periods, nine produced CTX-M-2 or its new variant CTX-M-59 and one produced SHV-5. bla(CTX-M-2/59) genes were located on closely related plasmids that were transferable.

Escherichia coli HKY28, a ceftazidime-resistant strain isolated from a urine specimen in Japan, produced an inhibitor-sensitive AmpC beta-lactamase variant. The deduced amino acid sequence of the enzyme contained a number of substitutions and a tripeptide deletion (Gly286-Ser287-Asp288) compared with the sequence of native AmpC of E. coli. When the deletion was reverted by a 9-base insertion at the relevant site of ampC in the clone, the typical inhibitor-resistant phenotype of AmpC was restored, while at the same time the levels of resistance to ceftazidime, cefpirome, and cefepime were reduced eightfold or more. Molecular modeling studies indicated that a structural change took place in the H-10 helix as a result of the deletion, and this change caused an alteration of the substrate binding site, leading to a unique phenotype analogous to that of inhibitor-sensitive class A extended-spectrum beta-lactamases. The degree of inhibition was greater with sulbactam and tazobactam than with clavulanic acid. To our knowledge, this is the first report to have characterized an E. coli ampC that encodes chromosomal AmpC beta-lactamase sensitive to the available beta-lactamase inhibitors.

Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori-mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited gamma-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-l-norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial gamma-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant gamma-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that gamma-glutamyl transpeptidase plays a significant role in H. pylori-mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial gamma-glutamyl transpeptidase function.

We describe an outbreak caused by Serratia marcescens carrying blaKPC-3 that was sourced to a long-term care facility in Florida, USA. Whole-genome sequencing and plasmid profiling showed involvement of 3 clonal lineages of S. marcescens and 2 blaKPC-3-carrying plasmids. Determining the resistance mechanism is critical for timely implementation of infection control measures. [ABSTRACT FROM AUTHOR]

Background: fosA3 is the most commonly reported plasmid-mediated fosfomycin resistance gene among Enterobacteriaceae.Objectives: To identify the origin of fosA3.Methods: The chromosome of Kluyvera georgiana clinical strain YDC799 was fully sequenced with single-molecule real-time sequencing. Comparative genetic analysis was performed for K. georgiana YDC799, K. georgiana type strain ATCC 51603 and representative fosA3-carrying plasmids. fosA genes were cloned in Escherichia coli to confirm function.Results: K. georgiana YDC799 harboured fosA (designated fosAKG) and blaCTX-M-8 on the chromosome. The genetic environments surrounding fosA3 and bounded by IS26 were nearly identical with the corresponding regions of K. georgiana YDC799 and ATCC 51603. The amino acid sequence of FosAKG from YDC799 and K. georgiana ATCC 51603 shared 99% and 94% identity with FosA3, respectively. Cloned FosAKG conferred fosfomycin resistance with an MIC of >1024 mg/L for E. coli.Conclusions: The plasmid-mediated fosA3 gene was likely mobilized from the chromosome of K. georgiana by an IS26-mediated event. [ABSTRACT FROM AUTHOR]

Objectives: The objective of this study was to conduct a comparative analysis with reported IncR plasmids of a Klebsiella pneumoniae IncR plasmid carrying an MDR region.Methods: MDR K. pneumoniae isolates were serially identified from two inpatients at a hospital in the USA in 2014. MDR plasmid pYDC676 was fully sequenced, annotated and compared with related plasmids. Antimicrobial susceptibility testing, PFGE and MLST were also conducted.Results: The K. pneumoniae isolates were identical by PFGE, belonged to ST37 and harboured an identical ∼50 kb IncR plasmid (pYDC676). pYDC676 possessed the backbone and multi-IS loci closely related to IncR plasmids reported from aquatic bacteria, as well as animal and human K. pneumoniae strains, and carried an MDR region consisting of armA, blaDHA-1 and qnrB4, a combination that has been reported in IncR plasmids from K. pneumoniae ST11 strains in Europe and Asia. A plasmid with the identical IncR backbone and a similar MDR region containing blaDHA-1 and qnrB4 has also been reported in ST37 strains from Europe, suggesting potential dissemination of this lineage of IncR plasmids in K. pneumoniae ST37.Conclusions: K. pneumoniae ST37 strains with an MDR IncR plasmid carrying armA, blaDHA-1 and qnrB4 were identified in a hospital in the USA, where these resistance genes remain rare. The IncR backbone may play a role in the global dissemination of these resistance genes. [ABSTRACT FROM AUTHOR]