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.

ABSTRACT 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 blaKPC. IMPORTANCE 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 β-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.

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 β-lactamase, AmpC β-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.

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.

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 (4mg/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 bla(NDM-1) gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of bla(CMY-16) mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of bla(CMY-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.

Objectives To examine the epidemiology of β-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.

Abstract 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 β-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 blaTEM-1 or blaSHV-1, none had an ESBL or AmpC β-lactamase gene, and many sequence types were represented. Independent risk factors for TZP-NS/CRO-S bacteremia were exposure to β-lactam/β-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.

We describe the first report of a clinical colistin-resistant ST84 Enterobacter cloacae isolate coharboring mcr-4.3 (previously named mcr-4.2) and bla(NDM-1) from a patient in China. The bla(NDM-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.

BackgroundBacteremia caused by extended-spectrum β-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.MethodsFrom 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 β-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.ResultsWe 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.ConclusionsHSCT 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.

Background. Infections caused by carbapenem-resistant Enterobacter cloacae (CR-Ecl) have been increasingly reported in the clinical setting; here we describe the clinical and molecular characteristics of CR-Ecl infections in a KPC endemic region.Methods. A cross-sectional study was conducted in five tertiary-care hospitals in Medellin-Colombia. All patients infected by CR-Ecl from June-2012 to June-2014 were included. Sociodemographics and clinical information was retrieved from medical records. Antimicrobial susceptibility testing, phenotypic and molecular carbapenemase detection were performed. Analysis of hsp60 and PFGE was done in a subset of isolates. Results. Of 109 patients enrolled, 60.55% (66/109) were infected with non-carbapenemase-producing-Ecl (non-CP-Ecl). CP-Ecl patients were frequently hospitalized in the ICU (37.21% vs 12.12%) and had exposure to carbapenems (39.53% vs 15.15%) compared to non-CP-Ecl infected patients. All-cause 30-day mortality was higher in CP-Ecl than non-CP-Ecl infected patients (27.91% vs 19.70%). CP-Ecl harbored KPC-2 (83.72%) and KPC-3 (6.97%). Analysis of hsp60 showed that CP-Ecl belonged primarily to cluster-VI of Enterobacter xiangfangensis (12/34) and cluster-XI (12/34) corresponding to E. cloacae subsp. cloacae. Non-CP-Ecl isolates belonged to cluster-VII/VIII (45/54), of E. hormaechi subsp. steigerwaltii. PFGE revealed isolates in cluster VII/VIII and XI were closely related within their own clusters.Conclusions: The results revealed a high frequency of non-CP-Ecl among the CR-Ecl infections in a KPC endemic region, displaying distinct clinical and molecular characteristics in comparison to CP-Ecl. The study highlights a significant contribution of non-CP-Ecl to the prevalence of CR-Ecl. Infection control measures to curtail dissemination of CR-Ecl should not only focus on CP-Ecl but should also include non-CP-Ecl.

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 bla(KPC)-harboring plasmids remain unknown. Here we used next-generation sequencing to characterize two KPC-2producing K. pneumoniae and K. michiganensis isolates collected from a New York City hospital in 1997. Although identified in two different Klebsiella species, the bla(KPC)-(2) gene was harbored by Tn4401b transposons on two highly similar IncN plasmids.

ABSTRACT 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-β-lactamase-producing Alcaligenes faecalis isolates associated with a small outbreak in a large hospital in Gaza, Palestine. Next-generation sequencing analysis showed bla VIM-2 is harbored by a chromosomal genomic island among three strains, while bla VIM-4 is carried by a novel plasmid in two strains.

ABSTRACT 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.

Bacteremia caused by ceftriaxone-resistant Enterobacteriaceae (CRO-R-E) is associated with inadequate empirical therapy and high mortality in neutropenic patients. Increased knowledge of rates of CRO-R-E colonization and risk of bacteremia in these patients is needed. From November 2015 to Apr 2017, we collected stool or perianal swab samples from patients with acute leukemia upon initiation of induction chemotherapy and weekly thereafter during neutropenia. Patients with less than 7 days of neutropenia while an inpatient were excluded. We plated samples onto ESBL screening agar, identified isolated bacteria, performed antimicrobial susceptibility testing, and screened CRO-R-E for β-lactamase genes by PCR. We then determined the prevalence of CRO-R-E colonization at the onset of chemotherapy and the incidence of acquiring CRO-R-E or developing CRO-R-E bacteremia during the hospitalization. We analyzed 82 patients. Their median age was 65 years, 76% had acute myeloid leukemia, and the median duration of neutropenia was 19 days. Nineteen patients (23%) were initially colonized with CRO-R-E, including 13 (20%) of 66 patients with newly diagnosed leukemia and 6 (38%) of 16 patients with relapsed or refractory disease. Only one of the colonized patients received prophylactic levofloxacin. There were 23 colonizing CRO-R-E, of which Escherichia coli (n = 13), Klebsiella pneumoniae (n = 4) and Citrobacter spp. (n = 4) were most common. Antimicrobial susceptibility rates were ceftazidime (13%), cefepime (26%), piperacillin-tazobactam (70%), and meropenem (91%). Thirteen (56%) were ESBL-producers, 8 (35%) produced AmpC β-lactamases, and 2 (9%) produced K. pneumoniae carbapenemase (KPC). Of patients not initially colonized with CRO-R-E, 5% acquired CRO-R-E. Two (11%) of the 19 patients colonized with CRO-R-E developed CRO-R-E bacteremia (one CTX-M-producing E. coli and one KPC-producing K. pneumoniae), whereas 6 (32%) developed bacteremia due to CRO-susceptible Enterobacteriaceae. By comparison, only 1 (2%) of 63 patients not initially colonized with CRO-R-E developed CRO-R-E bacteremia (P = 0.13). Patients with acute leukemia had high rates of colonization with CRO-R-E. However, the risk of CRO-R-E bacteremia in colonized patients was modest in the absence of levofloxacin prophylaxis. M. Satlin, Hardy Diagnostics: Investigator, Research support. Allergan: Grant Investigator, Research grant. Merck: Grant Investigator, Grant recipient. S. G. Jenkins, Cormedix: Consultant, Consulting fee. Bayer: Consultant, Consulting fee. Merck: Grant Investigator and Scientific Advisor, Research grant. T. J. Walsh, The Medicines Company: Consultant and Investigator, Consulting fee and Research grant. Astellas: Consultant and Investigator, Consulting fee and Research grant. Allergan: Consultant and Investigator, Consulting fee and Research grant. Merck: Consultant and Investigator, Consulting fee and Research grant

Bacterial pneumonia is a common complication in hospitalized patients and it is associated with high morbidity and mortality. Standard culture-based methods may take 2–3 days to identify the etiologies of HAP, leading to delays in appropriate therapy. A rapid molecular assay that could diagnose the etiology of bacterial pneumonia directly from BAL samples within a few hours could facilitate faster and more directed administration of antimicrobial therapy. BAL samples were collected from hospitalized patients with suspected pneumonia, including ventilated patients, from December 2016 through April 2017. Genomic DNA was isolated from BAL samples using NucliSENS® easyMAG®. A panel of target-specific molecular beacon probes in a real time PCR assay (MB-PCR) was used to identify the following pathogens: universal bacterial Identification (16S rRNA), E. coli (uidA), K. pneumoniae (gapA), S. aureus (spa), P. aeruginosa (rpsL), A. baumannii (Ab-ITS) and the following resistance determinants: ESBLs (CTX-M, TEM and SHV), carbapenemases (NDM, VIM, IMP, OXA-48 and KPC) and mecA. The results of MB-PCR were then compared with quantitative culture results performed by the clinical microbiological lab. We evaluated 53 BAL samples to identify the bacterial pathogen and key resistance determinants. Thirty-one samples yielded growth of ≥1 × 104 CFU/mL of bacteria by quantitative culture. The bacterial identification using MB-PCR for 16S rRNA correctly identified the presence of bacteria in all 31 samples (100% sensitivity). The MB-PCR identified P. aeruginosa (n = 5), S. aureus (n = 5), E. coli (n = 1), A. baumannii (n = 1), and K. pneumoniae (n = 1) in BAL samples that yielded ≥1 × 104 CFU/mL of the same pathogen by culture (100% sensitivity). The MB-PCR also identified blaTEM-harboring E. coli that grew ampicillin-resistant E. coli by culture. The specificity of the16S rRNA probe was 70%, as 7/53 BAL were false positive, whereas the specificity for the MB-PCR was 100% for P. aeruginosa, S. aureus, E. coli, and A. baumannii, and 98% for K. pneumoniae. Multiplex MB-PCR assay is a rapid, sensitive and specific tool for detection of common bacterial causes of nosocomial pneumonia and important resistance determinants directly from BAL samples. M. Satlin, Hardy Diagnostics: Investigator, Research support; S. G. Jenkins, Cormedix: Consultant, Consulting fee; Bayer: Consultant, Consulting fee Merck: Grant Investigator and Scientific Advisor, Research grant; T. J. Walsh, The Medicines Company: Consultant and Investigator, Consulting fee and Research grant Astellas: Consultant and Investigator, Consulting fee and Research grant Allergan: Consultant and Investigator, Consulting fee and Research grant Merck: Consultant and Investigator, Consulting fee and Research grant

ABSTRACT Enterobacteriaceae with bla NDM-7 are relatively uncommon and had previously been described in Europe, India, the United States, and Japan. This study describes the characteristics of Enterobacteriaceae ( Klebsiella pneumoniae [ n = 2], Escherichia coli [ n = 2], Serratia marcescens [ n = 1], and Enterobacter hormaechei [ n = 1] isolates) with bla NDM-7 obtained from 4 patients from Calgary, Canada, from 2013 to 2014. The 46,161-bp IncX3 plasmids with bla NDM-7 are highly similar to other bla NDM -harboring IncX3 plasmids and, interestingly, showed identical structures within the different isolates. This finding may indicate horizontal transmission within our health region, or it may indicate contact with individuals from areas of endemicity within the hospital setting. Patients infected or colonized with bacteria containing bla NDM-7 IncX3 plasmids generate infection control challenges. Epidemiological and molecular studies are required to better understand the dynamics of transmission, the risk factors, and the reservoirs for bacteria harboring bla NDM-7 . To the best of our knowledge, this is the first report of S. marcescens and E. hormaechei with bla NDM-7 .

ABSTRACT Here, we report two Enterobacter cloacae sequence type 231 isolates coproducing KPC-3 and NDM-1 that have caused lethal infections in a tertiary hospital in China. The bla NDM-1 -harboring plasmids carry IncA/C 2 and IncR replicons, showing a mosaic plasmid structure, and the bla NDM-1 is harbored on a novel class I integron-like element. bla KPC-3 is located on a Tn 3 -Δ bla TEM-1 - bla KPC-3 -ΔTn 1722 element, flanked by two 9-bp direct-repeat sequences and harbored on an IncX6 plasmid.

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.

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.

Here we completely sequenced four mcr-1-haboring plasmids, isolated from two extended-spectrum-β-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.