A phase I trial of cyclosporine for hospitalized patients with COVID-19.

BACKGROUNDCOVID-19 remains a global health emergency with limited treatment options, lagging vaccine rates, and inadequate healthcare resources in the face of an ongoing calamity. The disease is characterized by immune dysregulation and cytokine storm. Cyclosporine A (CSA) is a calcineurin inhibitor that modulates cytokine production and may have direct antiviral properties against coronaviruses.METHODSTo test whether a short course of CSA was safe in patients with COVID-19, we treated 10 hospitalized, oxygen-requiring, noncritically ill patients with CSA (starting at a dose of 9 mg/kg/d). We evaluated patients for clinical response and adverse events, measured serum cytokines and chemokines associated with COVID-19 hyperinflammation, and conducted gene-expression analyses.RESULTSFive participants experienced adverse events, none of which were serious; transaminitis was most common. No participant required intensive care unit-level care, and all patients were discharged alive. CSA treatment was associated with significant reductions in serum cytokines and chemokines important in COVID-19 hyperinflammation, including CXCL10. Following CSA administration, we also observed a significant reduction in type I IFN gene expression signatures and other transcriptional profiles associated with exacerbated hyperinflammation in the peripheral blood cells of these patients.CONCLUSIONShort courses of CSA appear safe and feasible in patients with COVID-19 who require oxygen and may be a useful adjunct in resource-limited health care settings.TRIAL REGISTRATIONThis trial was registered on ClinicalTrials.gov (Investigational New Drug Application no. 149997; ClinicalTrials.gov NCT04412785).FUNDINGThis study was internally funded by the Center for Cellular Immunotherapies.

A Ceftazidime-Avibactam-Resistant and Carbapenem-Susceptible Klebsiella pneumoniae Strain Harboring blaKPC-14 Isolated in New York City.

Ceftazidime-avibactam is a potent antibiotic combination against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae Here, we describe a unique ceftazidime-avibactam-resistant and carbapenem-susceptible K. pneumoniae strain harboring a novel blaKPC-14 variant. This strain was isolated from a New York City patient in 2003, which predates the introduction of avibactam. Despite resistance to ceftazidime-avibactam, the strain was susceptible to imipenem-relebactam and meropenem-vaborbactam. Comprehensive genomic sequencing revealed that blaKPC-14 is harbored on an ST6 IncN plasmid associated with the early spread of blaKPCIMPORTANCE KPC is currently the most common carbapenemase identified in the United States. More than 40 KPC variants have been described, of which KPC-2 and KPC-3 are the most frequent clinical variants. However, our understanding of the genetic structures and ß-lactam resistance profiles of other novel KPC variants remains incomplete. Here, we report a novel blaKPC variant (blaKPC-14) and the complete genome sequence of blaKPC-14-harboring K. pneumoniae strain BK13048, which is susceptible to carbapenems but resistant to ceftazidime-avibactam. To the best of our knowledge, this is one of the earliest KPC-producing K. pneumoniae strains exhibiting resistance to ceftazidime-avibactam.

Piperacillin-Tazobactam-Resistant/Third-Generation Cephalosporin-Susceptible Escherichia coli and Klebsiella pneumoniae Isolates: Resistance Mechanisms and In vitro-In vivo Discordance.

We previously reported the detection of Escherichia coli and Klebsiella pneumoniae that displayed in vitro piperacillin-tazobactam (TZP) resistance but were susceptible to third-generation cephalosporins (TZP-R/Ceph3-S). In this study, we assessed the phenotypic and genotypic profiles of 12 clinical non-clonal TZP-R/Ceph3-S E. coli and K. pneumoniae isolates derived from bloodstream infections. Whole-genome sequencing revealed that most of the TZP-R/Ceph3-S E. coli and K. pneumoniae isolates examined harbored blaTEM-1 and blaSHV-1 genes, respectively, but none harbored extended-spectrum ß-lactamase, AmpC ß-lactamase or carbapenemase genes. Increasing the tazobactam concentration from 4 mg/L to 16 mg/L restored TZP in vitro susceptibility among E. coli isolates expressing TEM-1, but had minimal impact on the susceptibility of K. pneumoniae to TZP. Real-time qPCR analysis showed that blaTEM-1 expression was amplified in TZP-R E. coli upon incubation with sub-inhibitory TZP concentrations. Using an immunocompetent murine septicemia model, the efficacy of TZP against TZP-R/Ceph3-S isolates was assessed using TZP doses that mimicked human plasma exposures following intravenous (IV) administration of TZP 4.5 g q6h over 0.5 h for 24 h. Efficacy was assessed by survival through 96 h. There was high mortality in untreated control mice for all tested isolates. Compared with controls, TZP human-simulated exposure significantly improved survival for all TZP-R/Ceph3-S E. coli and K. pneumoniae isolates examined (P < 0.05). Thus, TZP was associated with remarkable in vivo activity against TZP-R/Ceph3-S E. coli and K. pneumoniae despite the observed resistance in vitro.

In vitro selection of aztreonam/avibactam resistance in dual-carbapenemase-producing Klebsiella pneumoniae.

OBJECTIVES: To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. METHODS: The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. RESULTS: We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. CONCLUSIONS: Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.

First Report of blaVIM-4- and mcr-9-Coharboring Enterobacter Species Isolated from a Pediatric Patient.

An Enterobacter hormaechei isolate harboring blaVIM-4 and mcr-9 was recovered from a pediatric patient in a U.S. hospital. The blaVIM-4 and mcr-9 genes are carried on the same IncH12 plasmid, pME-1a. The isolate tested susceptible to colistin, without observed induction of colistin resistance. The mcr-9 gene is located between two insertion elements, IS903 and IS1, but lacks the downstream regulatory genes (qseC and qseB) found in other isolates that harbor mcr-9IMPORTANCE We describe the complete genome assembly and sequence of a clinical Enterobacter isolate harboring both blaVIM-4 and mcr-9 recovered from a pediatric patient in the United States with a history of travel to Egypt. Moreover, to the best of our knowledge, this is the first report of an Enterobacter isolate harboring both blaVIM-4 and mcr-9 from the United States. The blaVIM-4 and mcr-9 genes are carried on the same IncH12 plasmid, pME-1a. The isolate tested susceptible to colistin, without observed induction of colistin resistance. The mcr-9 gene is located between two insertion elements, IS903 and IS1, but lacks the downstream regulatory genes (qseC and qseB) found in other isolates that harbor mcr-9.

CG258 Klebsiella pneumoniae isolates without ß-lactam resistance at the onset of the carbapenem-resistant Enterobacteriaceae epidemic in New York City.

Objectives: To examine the epidemiology of ß-lactam resistance in 'clonal group 258' (CG258), a successful KPC clonal group, over 14 years. Methods: Isolates were collected from 1999 to 2013 for a study of antibiotic resistance in Enterobacteriaceae in New York City; 515 bloodstream isolates had antibiotic susceptibility data available and 436 were available for a CG258 PCR assay. The 56 resulting CG258 isolates were characterized by MLST, capsular type and ESBL and KPC carriage. KPC-positive isolates were assessed for common KPC plasmid types, KPC subtype and Tn4401 isoform. Results: RT-PCR revealed 56 isolates were CG258. Seventeen of the 56 CG258 isolates were phenotypically susceptible to all carbapenems (all KPC negative). Five out of 17 susceptible isolates were of the cps-2 (wzi154) capsule type; none was cps-1 (wzi29). Nineteen out of 28 KPC-2 isolates were cps-1 (wzi29) and 8/10 KPC-3 isolates carried cps-2 (wzi154); however, cps-2 (wzi154) predominated among KPC-2-positive isolates in 2003 and 2004. KPC-2 was first detected in 2003 and KPC-3 was first detected in 2006. KPC-harbouring plasmids pKpQIL (all Tn4401a) and pBK30683 (all Tn4401d) were detected in 16/38 and 6/38 carbapenem-resistant isolates, respectively. Discussion: CG258-lineage Klebsiella pneumoniae isolates were completely absent in 1999, but common in 2003. Twenty-one percent of CG258 isolates were susceptible to carbapenems in addition to lacking both common ESBL and blaKPC-mediated resistance. The cps-2 (wzi154) capsule type was common in both these susceptible isolates and in early KPC-2-harbouring isolates, suggesting it was the initial capsule type in CG258. Carbapenem-resistant isolates carried common KPC-harbouring plasmids with the same KPC and Tn4401 isoforms, suggesting frequent clonal spread.

Epidemiology of Bloodstream Infections Caused by Escherichia coli and Klebsiella pneumoniae That Are Piperacillin-Tazobactam-Nonsusceptible but Ceftriaxone-Susceptible.

BACKGROUND: Piperacillin-tazobactam-nonsusceptible (TZP-NS) Enterobacteriaceae are typically also resistant to ceftriaxone. We recently encountered bacteremias due to Escherichia coli (Ec) and Klebsiella pneumoniae (Kp) that were TZP-NS but ceftriaxone-susceptible (CRO-S). METHODS: We reviewed all Ec and Kp bacteremias from 2011 to 2015 at our center and assessed the prevalence, antimicrobial susceptibilities, genetic profiles, patient characteristics, treatments, and outcomes of TZP-NS/CRO-S infections. We identified risk factors for TZP-NS/CRO-S infections compared with Ec and Kp bacteremias that were TZP-S and CRO-S (Control Group 1) and compared outcomes of patients with TZP-NS/CRO-S bacteremias, Control Group 1, and patients bacteremic with extended-spectrum ß-lactamase (ESBL)-producing Ec and Kp. RESULTS: There were 1857 Ec and Kp bacteremia episodes, of which 78 (4.2%) were TZP-NS/CRO-S (Ec: 50/1227 [4.1%]; Kp: 28/630 [4.4%]). All TZP-NS/CRO-S isolates were also ampicillin-sulbactam-NS. Of 32 TZP-NS/CRO-S isolates that were sequenced, 28 (88%) harbored bla TEM-1 or bla SHV-1, none had an ESBL or AmpC ß-lactamase gene, and many sequence types were represented. Independent risk factors for TZP-NS/CRO-S bacteremia were exposure to ß-lactam/ß-lactamase inhibitors (BL/BLIs; adjusted odds ratio [aOR], 5.5; P < .001) and cephalosporins (aOR, 3.0; P = .04). Thirty-day mortality after TZP-NS/CRO-S bacteremia was 25%, which was similar to control groups and was similar in patients treated empirically with BL/BLIs compared with those treated with cephalosporins or carbapenems. Targeted therapy with cephalosporins did not yield a higher 30-day mortality rate than carbapenem therapy. CONCLUSIONS: TZP-NS/CRO-S Ec and Kp are emerging causes of bacteremia, and further research is needed to better understand the epidemiology, resistance mechanisms, and clinical impact of these strains.

Coidentification of mcr-4.3 and blaNDM-1 in a Clinical Enterobacter cloacae Isolate from China.

We describe the first report of a clinical colistin-resistant ST84 Enterobacter cloacae isolate coharboring mcr-4.3 (previously named mcr-4.2) and blaNDM-1 from a patient in China. The blaNDM-1-harboring IncX3 plasmid and the novel mcr-4.3-harboring ColE plasmid were completely sequenced. Although this isolate showed a high level of resistance to colistin, mcr-4.3 plasmid transformation, gene subcloning, susceptibility testing, and lipid A matrix-assisted laser desorption ionization mass spectrometry analysis indicated that mcr-4.3 itself does not confer resistance to colistin.

Colonization With Levofloxacin-resistant Extended-spectrum ß-Lactamase-producing Enterobacteriaceae and Risk of Bacteremia in Hematopoietic Stem Cell Transplant Recipients.

Background: Bacteremia caused by extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) is associated with inadequate empirical therapy and substantial mortality in neutropenic patients. Strategies are needed to identify neutropenic patients at high risk of these infections. Methods: From April 2014 to September 2016, we collected perianal swabs, both at admission and weekly thereafter, from patients undergoing hematopoietic stem cell transplantation (HSCT). Patients received prophylactic levofloxacin while neutropenic. Swabs were plated onto selective agar, colonies were identified and underwent antimicrobial susceptibility testing, and phenotypic ESBL testing and polymerase chain reaction for ß-lactamase genes were performed on ceftriaxone-resistant Enterobacteriaceae. We then determined the prevalence of pre-transplant ESBL-E colonization and risk of ESBL-E bacteremia. Colonizing and bloodstream isolates from patients with ESBL-E bacteremia underwent multilocus sequence typing and pulsed-field gel electrophoresis. Results: We analyzed 312 patients, including 212 allogeneic and 100 autologous HSCT recipients. Ten percent (31/312) of patients had pre-transplant ESBL-E colonization. Susceptibility rates of colonizing ESBL-E were: levofloxacin, 25%; cefepime, 9%; piperacillin-tazobactam, 84%; and meropenem, 97%. Of 31 patients colonized with ESBL-E pre-transplant, 10 (32%) developed ESBL-E bacteremia during their transplant admission, compared to 1 (0.4%) of 281 patients not colonized with ESBL-E (P < .001). All bloodstream ESBL-E were levofloxacin-resistant and colonizing and bloodstream isolates from individual patients had identical genotypic profiles. Conclusions: HSCT recipients who are colonized with levofloxacin-resistant ESBL-E pre-transplant and receive levofloxacin prophylaxis have high rates of bacteremia from their colonizing strain during neutropenia. Assessing for ESBL-E colonization in neutropenic patients could lead to optimization of empirical antibacterial therapy.

Genomic Characterization of VIM Metallo-ß-Lactamase-Producing Alcaligenes faecalis from Gaza, Palestine.

Carbapenemase-producing Gram-negative bacteria (CP-GNB) have increasingly spread worldwide, and different families of carbapenemases have been identified in various bacterial species. Here, we report the identification of five VIM metallo-ß-lactamase-producing Alcaligenes faecalis isolates associated with a small outbreak in a large hospital in Gaza, Palestine. Next-generation sequencing analysis showed blaVIM-2 is harbored by a chromosomal genomic island among three strains, while blaVIM-4 is carried by a novel plasmid in two strains.

Genomic Characterization of Two KPC-Producing Klebsiella Isolates Collected in 1997 in New York City.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have now become a global public health threat. However, the origin of this pandemic and the characterization of pre-2003 blaKPC-harboring plasmids remain unknown. Here we used next-generation sequencing to characterize two KPC-2-producing K. pneumoniae and Kmichiganensis isolates collected from a New York City hospital in 1997. Although identified in two different Klebsiella species, the blaKPC-2 gene was harbored by Tn4401b transposons on two highly similar IncN plasmids.

Emergence of Ceftazidime-Avibactam Resistance Due to Plasmid-Borne blaKPC-3 Mutations during Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections.

Ceftazidime-avibactam is a novel ß-lactam/ß-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 blaKPC-3, which were not present in baseline isolates. blaKPC-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 blaKPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to blaKPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring blaKPC-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.

Comprehensive Genome Analysis of Carbapenemase-Producing Enterobacter spp.: New Insights into Phylogeny, Population Structure, and Resistance Mechanisms.

Knowledge regarding the genomic structure of Enterobacter spp., the second most prevalent carbapenemase-producing Enterobacteriaceae, remains limited. Here we sequenced 97 clinical Enterobacter species isolates that were both carbapenem susceptible and resistant from various geographic regions to decipher the molecular origins of carbapenem resistance and to understand the changing phylogeny of these emerging and drug-resistant pathogens. Of the carbapenem-resistant isolates, 30 possessed blaKPC-2, 40 had blaKPC-3, 2 had blaKPC-4, and 2 had blaNDM-1 Twenty-three isolates were carbapenem susceptible. Six genomes were sequenced to completion, and their sizes ranged from 4.6 to 5.1 Mbp. Phylogenomic analysis placed 96 of these genomes, 351 additional Enterobacter genomes downloaded from NCBI GenBank, and six newly sequenced type strains into 19 phylogenomic groups-18 groups (A to R) in the Enterobacter cloacae complex and Enterobacter aerogenes Diverse mechanisms underlying the molecular evolutionary trajectory of these drug-resistant Enterobacter spp. were revealed, including the acquisition of an antibiotic resistance plasmid, followed by clonal spread, horizontal transfer of blaKPC-harboring plasmids between different phylogenomic groups, and repeated transposition of the blaKPC gene among different plasmid backbones. Group A, which comprises multilocus sequence type 171 (ST171), was the most commonly identified (23% of isolates). Genomic analysis showed that ST171 isolates evolved from a common ancestor and formed two different major clusters; each acquiring unique blaKPC-harboring plasmids, followed by clonal expansion. The data presented here represent the first comprehensive study of phylogenomic interrogation and the relationship between antibiotic resistance and plasmid discrimination among carbapenem-resistant Enterobacter spp., demonstrating the genetic diversity and complexity of the molecular mechanisms driving antibiotic resistance in this genus. IMPORTANCE: Enterobacter spp., especially carbapenemase-producing Enterobacter spp., have emerged as a clinically significant cause of nosocomial infections. However, only limited information is available on the distribution of carbapenem resistance across this genus. Augmenting this problem is an erroneous identification of Enterobacter strains because of ambiguous typing methods and imprecise taxonomy. In this study, we used a whole-genome-based comparative phylogenetic approach to (i) revisit and redefine the genus Enterobacter and (ii) unravel the emergence and evolution of the Klebsiella pneumoniae carbapenemase-harboring Enterobacter spp. Using genomic analysis of 447 sequenced strains, we developed an improved understanding of the species designations within this complex genus and identified the diverse mechanisms driving the molecular evolution of carbapenem resistance. The findings in this study provide a solid genomic framework that will serve as an important resource in the future development of molecular diagnostics and in supporting drug discovery programs.

Evaluation of a Multiplex PCR Assay To Rapidly Detect Enterobacteriaceae with a Broad Range of ß-Lactamases Directly from Perianal Swabs.

We developed and evaluated multiplexed molecular beacon probes in a real-time PCR assay to identify prominent extended-spectrum-ß-lactamase, plasmid-mediated AmpC ß-lactamase (pAmpC) and carbapenemase genes directly from perianal swab specimens within 6 h. We evaluated this assay on 158 perianal swabs collected from hematopoietic stem cell transplant recipients and found that this assay was highly sensitive and specific for detection of CTX-M-, pAmpC-, and KPC-producing Enterobacteriaceae compared to culture on chromogenic agar.

Colistin- and Carbapenem-Resistant Escherichia coli Harboring mcr-1 and blaNDM-5, Causing a Complicated Urinary Tract Infection in a Patient from the United States.

UNLABELLED: Colistin is increasingly used as an antibiotic of last resort for the treatment of carbapenem-resistant Gram-negative infections. The plasmid-borne colistin resistance gene mcr-1 was initially identified in animal and clinical samples from China and subsequently reported worldwide, including in the United States. Of particular concern is the spread of mcr-1 into carbapenem-resistant bacteria, thereby creating strains that approach pan-resistance. While several reports of mcr-1 have involved carbapenem-resistant strains, no such isolates have been described in the United States. Here, we report the isolation and identification of an Escherichia coli strain harboring both mcr-1 and carbapenemase gene blaNDM-5 from a urine sample in a patient without recent travel outside the United States. The isolate exhibited resistance to both colistin and carbapenems, but was susceptible to amikacin, aztreonam, gentamicin, nitrofurantoin, tigecycline, and trimethoprim-sulfamethoxazole. The mcr-1- and blaNDM-5-harboring plasmids were completely sequenced and shown to be highly similar to plasmids previously reported from China. The strain in this report was first isolated in August 2014, highlighting an earlier presence of mcr-1 within the United States than previously recognized. IMPORTANCE: Colistin has become the last line of defense for the treatment of infections caused by Gram-negative bacteria resistant to multiple classes of antibiotics, in particular carbapenem-resistant Enterobacteriaceae (CRE). Resistance to colistin, encoded by the plasmid-borne gene mcr-1, was first identified in animal and clinical samples from China in November 2015 and has subsequently been reported from numerous other countries. In April 2016, mcr-1 was identified in a carbapenem-susceptible Escherichia coli strain from a clinical sample in the United States, followed by a second report from a carbapenem-susceptible E. coli strain originally isolated in May 2015. We report the isolation and identification of an E. coli strain harboring both colistin (mcr-1) and carbapenem (blaNDM-5) resistance genes, originally isolated in August 2014 from urine of a patient with recurrent urinary tract infections. To our knowledge, this is the first report in the United States of a clinical bacterial isolate with both colistin and carbapenem resistance, highlighting the importance of active surveillance efforts for colistin- and carbapenem-resistant organisms.

Detection of the mcr-1 Colistin Resistance Gene in Carbapenem-Resistant Enterobacteriaceae from Different Hospitals in China.

The spread of the plasmid-mediated colistin resistance gene, mcr-1, into carbapenem-resistant Enterobacteriaceae (CRE) clinical isolates poses a significant threat to global health. Here we report the identification of three mcr-1-harboring carbapenem-resistant Escherichia coli strains, collected from three patients in two provinces in China. Our results show that mcr-1-harboring CRE strains have started to spread in different hospitals in China. In addition, this report presents the first description of chromosomal integration of mcr-1 into a carbapenem-resistant E. coli strain.

Molecular Diversity and Plasmid Analysis of KPC-Producing Escherichia coli.

The emergence and spread of Klebsiella pneumoniae carbapenemase (KPC) among Enterobacteriaceae presents a major public health threat to the world. Although not as common as in K. pneumoniae, KPC is also found in Escherichia coli strains. Here, we genetically characterized 9 carbapenem-resistant E. coli strains isolated from six hospitals in the United States and completely sequenced their blaKPC-harboring plasmids. The nine strains were isolated from different geographical locations and belonged to 8 different E. coli sequence types. Seven blaKPC-harboring plasmids belonged to four different known incompatibility groups (IncN, -FIA, -FIIK2, and -FIIK1) and ranged in size from ∼16 kb to ∼241 kb. In this analysis, we also identified two plasmids that have novel replicons: (i) pBK28610, which is similar to p34978-3 with an insertion of Tn4401b, and (ii) pBK31611, which does not have an apparent homologue in the GenBank database. Moreover, we report the emergence of a pKP048-like plasmid, pBK34397, in E. coli in the United States. Meanwhile, we also found examples of interspecies spread of blaKPC plasmids, as pBK34592 is identical to pBK30683, isolated from K. pneumoniae In addition, we discovered examples of acquisition (pBK32602 acquired an ∼46-kb fragment including a novel replication gene, along with Tn4401b and other resistance genes) and/or loss (pKpQIL-Ec has a 14.5-kb deletion compared to pKpQIL-10 and pBK33689) of DNA, demonstrating the plasticity of these plasmids and their rapid evolution in the clinic. Overall, our study shows that the spread of blaKPC-producing E. coli is largely due to horizontal transfer of blaKPC-harboring plasmids and related mobile elements into diverse genetic backgrounds.

Complete Sequences of mcr-1-Harboring Plasmids from Extended-Spectrum-ß-Lactamase- and Carbapenemase-Producing Enterobacteriaceae.

Here we completely sequenced four mcr-1-haboring plasmids, isolated from two extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli and two carbapenemase-producing Klebsiella pneumoniae clinical isolates. The mcr-1-harboring plasmids from an E. coli sequence type 2448 (ST2448) isolate and two K. pneumoniae ST25 isolates were identical (all pMCR1-IncX4), belonging to the IncX4 incompatibility group, while the plasmid from an E. coli ST2085 isolate (pMCR1-IncI2) belongs to the IncI2 group. A nearly identical 2.6-kb mcr-1-pap2 element was found to be shared by all mcr-1-carrying plasmids.

Genomic Characterization of Enterobacter cloacae Isolates from China That Coproduce KPC-3 and NDM-1 Carbapenemases.

Here, we report twoEnterobacter cloacaesequence type 231 isolates coproducing KPC-3 and NDM-1 that have caused lethal infections in a tertiary hospital in China. TheblaNDM-1-harboring plasmids carry IncA/C2and IncR replicons, showing a mosaic plasmid structure, and theblaNDM-1is harbored on a novel class I integron-like element.blaKPC-3is located on a Tn3-ΔblaTEM-1-blaKPC-3-ΔTn1722element, flanked by two 9-bp direct-repeat sequences and harbored on an IncX6 plasmid.

A Two-Year Surveillance in Five Colombian Tertiary Care Hospitals Reveals High Frequency of Non-CG258 Clones of Carbapenem-Resistant Klebsiella pneumoniae with Distinct Clinical Characteristics.

The global spread of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) has been largely associated with sequence type 258 (ST258) and its related variants (clonal group 258 [CG258]). Here we describe the molecular epidemiology of CR-Kp from five tertiary care hospitals in Medellín, the second largest city in Colombia. All CR-Kp-infected patients admitted from June 2012 to June 2014 were included (n = 193). Patients' clinical information was obtained from medical records. Carbapenemase KPC, VIM, IMP, NDM, and OXA-48 genes were detected by PCR. A CG258-tonB79 cluster-specific real-time PCR (targeting the multilocus sequence type [MLST] tonB79 allele), pulsed-field gel electrophoresis (PFGE), and MLST analysis were performed for typing. Remarkably, 62.2% (n = 120) of isolates were from STs unrelated to CG258 (non-CG258). KPC-3 predominated in CG258 isolates (86.3%), while KPC-2 prevailed in non-CG258 isolates (75.5%) (P < 0.001). Multidrug resistance (MDR) frequency was significantly higher in CG258 strains (91.4% versus 56.1%; P < 0.001). ST512 (a single-locus variant of ST258) is the main ST in CG258 (96.3%), and isolates in this group showed closely related pulsotype and similar resistance gene profiles, suggesting the clonal spread of this strain. In contrast, high heterogeneity of STs (34/54), including eight novel STs, was found in non-CG258 isolates. Among non-CG258 isolates, ST14 (13.3%; n = 16) and ST307 (14.2%; n = 17) were the most frequent, and they showed distinct molecular and clinical characteristics in comparison to CG258 isolates. Our results suggest that the dissemination of carbapenem resistance in Medellín is due to heterogeneous K. pneumoniae clones, likely the result of horizontal transmission of KPC in different unrelated lineages, further highlighting the challenge in CR-Kp infection control and the need for a multifocal intervention.