Carbapenem- and Colistin-Resistant Enterobacter cloacae from Delta,Colorado, in 2015

Resistance to carbapenems in Enterobacteriaceae is a clinical problem of growing significance. Difficulty in treating multidrug-resistant Gram-negative organisms with conventional antibiotics has led to a renewed and increasing use of polymyxin compounds, such as colistin. Here, we report the isolation of carbapenem- and colistin-resistant Enterobacter cloacae from a polymicrobial lower extremity wound in an ambulatory patient. Whole-genome sequencing demonstrated the presence of chromosomal bla_IMI-1 and bla_AmpC, as well as numerous efflux pump genes.     

Characterization of an Enterobacter cloacae Strain Producing both KPC and NDM Carbapenemases by Whole-Genome Sequencing

A carbapenem-resistant Enterobacter cloacae strain, WCHECl-14653, causing a fatal bloodstream infection, was characterized by genome sequencing and conjugation experiments. The strain carried two carbapenemase genes, bla_NDM-1 and bla_KPC-2, on separate IncF plasmids. The coexistence of bla_NDM-1 and bla_KPC-2 conferred slightly higher-level carbapenem resistance compared with that of bla_NDM-1 or bla_KPC-2 alone, and the coexistence of two IncF plasmids may generate new platforms for spreading carbapenemase genes.     

Genetic and Biochemical Characterization of FRI-1, a Carbapenem-Hydrolyzing Class A β-Lactamase from Enterobacter cloacae

An Enterobacter cloacae isolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI-1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified β-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (IC₅₀s) of clavulanic acid and tazobactam were 10-fold higher than those found for Klebsiella pneumoniae carbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to β-lactamase inhibitors. The bla_FRI-1 gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5′ end of the β-lactamase gene was identified, leading to inducible expression of the bla_FRI-1 gene.     

Occurrence of Enterobacter hormaechei carrying blaNDM-1 and blaKPC-2 in China

Three carbapenem-resistant clinical isolates of the Enterobacter cloacae complex (ECC) were recovered from different patients in a hospital. All 3 isolates carried 2 carbapenemase genes bla_KPC-2 and bla_NDM-1. A study was performed to characterize their relatedness and to investigate possible links among the patients. Whole genome sequencing revealed that the isolates were Enterobacter hormaechei and belonged to ST177 of the ECC. There were 19–142 single nucleotide polymorphisms (SNPs) between the isolates, suggesting that the isolates were likely from a central reservoir, which might have existed for some time. bla_KPC-2 and bla_NDM-1 were carried on 2 different IncF-type plasmids in the isolates. The 3 bla_NDM-1-carrying plasmids were almost identical and were self-transmissible, while the bla_KPC-2-carrying plasmids were only transmissible in the presence of the bla_NDM-1-carrying plasmid. The source of and direct links among them were not identified, suggesting a hospital transmission of a common multidrug resistant strain.     

Novel Ambler Class A Carbapenem-Hydrolyzing β-Lactamase from a Pseudomonas fluorescens Isolate from the Seine River,Paris, France

A Pseudomonas fluorescens isolate (PF-1) resistant to carbapenems was recovered during an environmental survey performed with water from the Seine River (Paris). It expressed a novel Ambler class A carbapenemase, BIC-1, sharing 68 and 59% amino acid identities with β-lactamases SFC-1 from Serratia fonticola and the plasmid-encoded KPC-2, respectively. β-Lactamase BIC-1 hydrolyzed penicillins, carbapenems, and cephalosporins except ceftazidime and monobactams. The bla_BIC-1 gene was chromosomally located and was also identified in two other P. fluorescens strains isolated from the Seine River 3 months later.Pseudomonas fluorescens is a psychrotrophic bacterium that expresses a chromosomally encoded and inducible Ambler class C β-lactamase (10, 15). Carbapenemases (serine- or metallo-β-lactamases) remain the most common mechanism of resistance to carbapenems in Gram-negative organisms (22, 23). To date, most acquired metallo-β-lactamase (MBL)-encoding genes (bla_IMP or bla_VIM variants) have been reported from Pseudomonas aeruginosa and very rarely from P. fluorescens (8, 12). Class A carbapenemases are either chromosome encoded or plasmid encoded and remain rarely identified in Gram-negative organisms. Indeed, several class A carbapenemases are chromosomally encoded (NMC-A, SFC-1, SME-1 to -3, IMI-1), with the exception of the emerging KPC β-lactamases, which are plasmid encoded (24). The chromosomal location of the bla_SFC-1 gene could be the result of a horizontal gene transfer into an environmental Serratia fonticola isolate (5). The bla_SME genes from Serratia marcescens are presumed to be chromosomal. Genes encoding NMC-A and IMI β-lactamases have been found sporadically, either in clinical or in environmental isolates of Enterobacter cloacae and Enterobacter asburiae from rivers (2). The bla_NMC-A gene was found chromosomally located in several clinical isolates. The bla_IMI-1 gene was chromosomally located, whereas the bla_IMI-2 genes were identified on plasmids. The imiR-imi-2 gene tandem in E. cloacae and E. asburiae appeared to be flanked by transposable elements (2). Class A carbapenemases such as GES-2, GES-5, and KPC-2 have been recently reported from P. aeruginosa (16) but not from other Pseudomonas species.Here, we characterize a novel class A carbapenemase identified from a P. fluorescens environmental isolate. This identification occurred during a survey aimed to study the spread of multidrug-resistant Gram-negative organisms in the environment.     

Fecal carriage and molecular epidemiologic characteristics of carbapenemase-producing Enterobacterales in primary care hospital in a Japanese city

BackgroundThe worldwide spread of organisms with antimicrobial resistance is of concern, especially the trend of significantly increasing carbapenemase-producing Enterobacterales (CPE). In this study, we investigated the annual trend of intestinal CPE carriage rates in inpatients and healthy adults in a primary care hospital in Tenri, Japan.MethodsWe collected 551 samples of feces from inpatients in our institution and 936 samples from healthy people living in Tenri city from December 2012 to April 2015. All samples were cultured on MacConkey agar plates containing 4 μg/mL ceftazidime for screening test. The colonies grown on the screening medium were detected for carbapenemase genes (bla_IMP-1, bla_IMP-2, bla_VIM, bla_KPC, bla_GES, bla_NDM, and bla_OXA-48 groups) by multiplex PCR, and CPE were identified by MALDI-TOF MS. Plasmid replicon typing and pulsed-field gel electrophoresis (PFGE) were performed on PCR-positive strains.ResultsThe CPE carriage rate was 1.6% (9/551) in the inpatient group and 0% (0/936) in the healthy adults group. The numbers of strains positive for the carbapenemase gene were 4 for Enterobacter cloacae, 2 for Klebsiella pneumoniae, 1 for Citrobacter freundii, 1 for Raoultella ornithinolytica and 1 for Escherichia coli. In all CPE strains, the carbapenemase gene was bla _IMP-6 and the plasmid replicon type was IncN. The 4 E. cloacae strains showed a similar pattern in PFGE.ConclusionIn the same city in Japan, CPE intestinal carriers were detected only in the inpatient group in this study but not in a healthy adults, suggesting that the spread of asymptomatic CPE carriers was confined to inpatients.     

Characterization of a Novel Putative Xer-Dependent Integrative Mobile Element Carrying the blaNMC-A Carbapenemase Gene,Inserted into the Chromosome of Members of the Enterobacter cloacae Complex

An Enterobacter ludwigii strain was isolated during routine screening of a Japanese patient for carriage of carbapenem-resistant Enterobacteriaceae. PCR analysis revealed the bla_NMC-A carbapenemase gene. Whole-genome sequencing revealed that bla_NMC-A was inserted in the chromosome and associated with a novel 29.1-kb putative Xer-dependent integrative mobile element, named EludIMEX-1. Bioinformatic analysis identified similar elements in the genomes of an Enterobacter asburiae strain and of other Enterobacter cloacae complex strains, confirming the mobile nature of this element.     

Predominance of KPC-3 in a Survey for Carbapenemase-Producing Enterobacteriaceae in Portugal

Among the 2,105 Enterobacteriaceae tested in a survey done in Portugal, 165 were nonsusceptible to carbapenems, from which 35 (26 Klebsiella pneumoniae, 3 Escherichia coli, 2 Enterobacter aerogenes, and 3 Enterobacter cloacae isolates and 1 Klebsiella oxytoca isolate) were confirmed to be carbapenemase producers by the presence of 30 Tn4401d-bla_KPC-3, 4 intI3-bla_GES-5, and one intI1-bla_VIM-2 gene, alone or in combination with other bla genes. The dissemination of bla_KPC-3 gene carried by an IncF plasmid suggests lateral gene transfer as a major mechanism of dissemination.     

Emergence of sequence type 252 Enterobacter cloacae producing GES-5 carbapenemase in a Czech hospital

ST252 Enterobacter cloacae, producing GES-5 carbapenemase, was isolated in a Czech hospital. bla_GES-5 was part of a novel class 1 integron, In1406, which also included a new allele of the aadA15 gene cassette. In1406 was located on a ColE2-like plasmid, pEcl-35771cz (6953 bp).     

Genetic and Biochemical Characterization of OXA-405, an OXA-48-Type Extended-Spectrum β-Lactamase without Significant Carbapenemase Activity

The epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceae in many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescens isolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a bla_OXA-48-type gene. Sequencing identified a novel OXA-48-type β-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The bla_OXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M bla_OXA-48-carrying plasmid except for a ca. 16.4-kb deletion in the tra operon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like β-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceae may be based on the biochemical detection of carbapenemase activity in clinical settings.     

Dominance of IMP-4-Producing Enterobacter cloacae among Carbapenemase-Producing Enterobacteriaceae in Australia

The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) has been increasing worldwide. bla_IMP has been reported to be the predominant carbapenemase-encoding gene within Enterobacteriaceae in Australia. However, there are limited data currently available on CPE from Queensland, Australia. A total of 58 CPE isolates were isolated between July 2009 and March 2014 from Queensland hospitals. The clonality of isolates was determined by Diversilab repetitive sequence-based PCR. The isolates were investigated for the resistance mechanisms carbapenemase, extended-spectrum β-lactamase, and AmpC β-lactamase and for aminoglycoside resistance and plasmid-mediated quinolone resistance genes by PCR. The plasmid types associated with carbapenemase-encoding genes were characterized. The majority of the CPE were Enterobacter cloacae (n = 29). The majority of Queensland CPE isolates were IMP producers and comprised 11 species (n = 48). Nine NDM-producing Enterobacteriaceae were identified. One NDM-producing Klebsiella pneumoniae isolate coproduced OXA-48. One K. pneumoniae isolate was an OXA-181 producer. The incidence of IMP producers increased significantly in 2013. bla_IMP-4 was found in all IMP-producing isolates. bla_TEM, qnrB, and aacA4 were common among IMP-4 producers. The HI2 (67%) and L/M (21%) replicons were associated with bla_IMP-4. All HI2 plasmids were of sequence type 1 (ST1). All but one of the NDM producers possessed bla_CTX-M-15. The 16S rRNA methylase genes found among NDM producers were armA, rmtB, rmtC, and rmtF. The substantial increase in the prevalence of CPE in Queensland has been associated mainly with the emergence E. cloacae strains possessing HI2 plasmids carrying bla_IMP-4 over the past 2 years. The importation of NDM producers and/or OXA-48-like producers in patients also contributed to the increased emergence of CPE.     

Increasing prevalence of blaOXA-23-carrying Acinetobacter baumannii and the emergence of blaOXA-182-carrying Acinetobacter nosocomialis in Korea

Carbapenem-resistant Acinetobacter spp. have been increasingly reported worldwide with the production of OXA-type carbapenemases as the main mechanism of carbapenem resistance. The prevalent bla_OXA genes are known to vary significantly depending on time and place of isolation. We investigated the prevalence of bla_OXA genes by PCR in Acinetobacter spp. isolated in Korea. Among a total of 336 isolates collected from Hospital A from 2002 to 2011, the overall proportion of bla_OXA-23-like, ISAba1-associated bla_OXA-51-like, and bla_OXA-182 genes were 44.0%, 49.7%, and 5.1%, respectively. The bla_OXA-58-like gene was detected in only 1 isolate. A drastic increase in Acinetobacter isolates with bla_OXA-23-like genes and a decrease in isolates harboring ISAba1-associated bla_OXA-51-like genes have been observed since the mid-2000s. The bla_OXA-23-like genes were detected in all carbapenem-nonsusceptible isolates collected in 2011 from 9 hospitals. The OXA-182, which belongs to the fifth group of OXA-type carbapenemase, was detected in Acinetobacter baumannii isolates recovered as early as 2002. It is worrisome results that bla_OXA-182-carrying Acinetobacter nosocomialis has emerged and caused outbreaks of infection.     

Emergence of a carbapenem-resistant and colistin-heteroresistant Enterobacter cloacae clinical isolate in Japan

A carbapenem-resistant and colistin-heteroresistant clinical isolate of Enterobacter cloacae was obtained from an inpatient in Okinawa, Japan. The minimum inhibitory concentrations of both imipenem and meropenem were 32 μg/mL. The isolate showed heteroresistance to colistin using the Etest method and resistance to colistin using the broth microdilution method. It had a disrupted ompC and a mutation in the promoter region of bla_ACT-2, but did not harbor any genes encoding carbapenemase. The disruption of ompC and the mutation in bla_ACT-2 was associated with the carbapenem resistance of this isolate. This isolate also had mutations in pmrAB and phoPQ encoding two-component regulatory systems, which may be associated with colistin heteroresistance.     

NDM-1 encoded by a pNDM-HN380-like plasmid pNDM-BJ03 in clinical Enterobacter cloacae

A carbapenemase-producing Enterobacter cloacae hhy03 with a bla_NDM-1 and bla_SHV-12-coharboring plasmid was isolated from a sputum specimen of a patient. This is the third nucleotide sequence report of bla_NDM-1-harboring plasmid from Enterobacter cloacae that have caused lethal infections in China, indicating the spread of NDM-1 by IncX3 plasmid between Enterobacteriaceae.     

Clonal Dissemination of OXA-370-Producing Klebsiella pneumoniae in Rio de Janeiro,Brazil

Enzymes of the OXA-48 family have become some of the most important beta-lactamases in the world. A new OXA-48 variant (OXA-370) was first described for an Enterobacter hormaechei strain isolated in Rio Grande do Sul (southern region of Brazil) in 2013. Here we report detection of the bla_OXA-370 gene in 24 isolates belonging to three Enterobacteriaceae species (22 Klebsiella pneumoniae isolates, 1 Enterobacter cloacae isolate, and 1 Enterobacter aerogenes isolate) collected from five hospitals in Rio de Janeiro, Brazil, in 2013 and 2014. The isolates showed a multidrug resistance profile, and 12.5% were resistant to polymyxin B. Besides bla_OXA-370, no other carbapenemase genes were observed by PCR, whereas bla_OXA-1 was found in all isolates and 22 isolates (91.6%) possessed bla_CTX-M-15. Molecular typing of the K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) showed the presence of two clonal groups, i.e., KpA (21 isolates) and KpB (1 isolate). KpA was characterized as sequence type 16 (ST16) and KpB as ST1041 by multilocus sequence typing (MLST). ST16 has been observed for KPC-producing K. pneumoniae in Rio de Janeiro. Plasmid analysis performed with six representative OXA-370-producing isolates showed plasmids harboring the bla_OXA-370 gene in all strains, ranging from 25 kb to 150 kb. This study suggests that there is an urgent need to investigate the presence of OXA-370 and dissemination of the K. pneumoniae ST16 clone carrying this gene in Brazil.     

Molecular characterization of carbapenemase producing Klebsiella pneumoniae strains by multiplex PCR and PFGE methods: The first K.pneumoniae isolates co-producing OXA-48/KPC and KPC/NDM in Turkey

IntroductionCarbapenems are frequently used in the treatment of multidrug-resistant infections caused by Klebsiella pneumoniae. The aim of the study is to definition and incidence of transferable carbapenemase genes of carbapenem resistant K. pneumoniae (CRKP) and to determine clonal relatedness of these strains in tertiary care hospital in Turkey.MethodsIdentification of all 100 K. pneumoniae isolates and low sensitivity to any of the carbapenem group antibiotics were determined by Vitek-2 (BioMérieux, France). The frequency of carbapenemase genes (bla_OXA-48, bla_NDM, bla_KPC, bla_VIM, bla_IMP) and extended spectrum beta-lactamase (ESBL) genes (bla_CTX-M, bla_SHV, bla_TEM) which frequently detected in Turkey, have been investigated by multiplex polymerase chain reaction (PCR). Clonal relatedness was determined using Pulsed-field gel electrophoresis(PFGE).ResultsNinety five isolates carried at least one of the carbapenemase genes (81.05% bla_OXA-48, 38.9% bla_NDM, 9.47% bla_KPC,1.05% bla_VIM). One isolate was carried the bla_OXA-48+KPC and the two isolates were carried the bla_KPC+NDM. PFGE demonstrated the presence of 24 pulse types and 63.09% of the isolates were in four main pulse types.ConclusionsThis study demonstrated the incidence of bla_NDM is beginning to reach endemic levels, in addition to bla_OXA-48 found endemic in Turkey. To our knowledge, this is the first report of the co-production of these two genes (bla_{KPC + NDM} and bla_{OXA-48 + KPC}) in CRKP isolates.     

Antimicrobial activity of plazomicin against Enterobacteriaceae-producing carbapenemases from 50 Brazilian medical centers

Plazomicin is a next-generation aminoglycoside with activity against Enterobacteriaceae, including carbapenemase-producing Enterobacteriaceae (CPE). The aim of this study was to evaluate the activity of plazomicin against CPE (Klebsiella spp., Escherichia coli, Serratia spp., Enterobacter spp., Citrobacter spp., Morganella spp., Proteus spp., Providencia spp.) from different Brazilian hospitals. A total of 4000 carbapenem-resistant Enterobacteriaceae isolates were collected from clinical samples in 50 Brazilian hospitals during 2013–2015. Of these, 499 carbapenem-resistant isolates (CLSI criteria) were selected for further evaluation via broth microdilution to assess for the activity of plazomicin, colistin, tigecycline, meropenem, amikacin, and gentamicin. Additionally, the isolates were assessed for the presence of carbapenemase genes (bla_KPC, bla_NDM, bla_OXA-48-like, bla_IMP, bla_BKC, bla_GES, and bla_VIM) by polymerase chain reaction (PCR). When PCR was positive to bla_OXA-48-like, bla_IMP, bla_GES, and bla_VIM, the carbapenemase genes were sequenced. bla_KPC was the most prevalent carbapenemase gene found (n = 397), followed by bla_NDM (n = 81), bla_OXA-48 (n = 12), and bla_IMP-1 (n = 3). Other genes were identified in only 1 isolate each: bla_BKC-1, bla_GES-16, bla_GES-1, bla_OXA-370, and bla_VIM-1. One isolate had 2 carbapenemase genes (bla_KPC and bla_NDM). Thirty-three percent of the isolates were nonsusceptible to colistin, 24% to tigecycline, 97% to meropenem, 51% to amikacin, and 81% to gentamicin (via EUCAST criteria). The plazomicin MIC_50/90 was 0.5/64 mg/L, with 85% of MICs ≤2 mg/L and 87% of MICs ≤4 mg/L. Elevated MICs to plazomicin were not associated with a specific carbapenemase or bacterial species. The MICs of plazomicin against CPE were lower than those of other aminoglycosides. Plazomicin is a promising drug for the treatment of CPE infections.     

Resistome Analysis of Enterobacter cloacae CY01, an Extensively Drug-Resistant Strain Producing VIM-1 Metallo-β-Lactamase from China

Resistome analysis of clinical VIM-1-producing Enterobacter cloacae strain CY01 from China revealed the presence of multiple resistance determinants. Two resistance plasmids were identified in CY01. The pCY-VIM plasmid was 14 kb in size and possessed a replicase gene (repA), a gene cluster encoding the partitioning function (parABC), and a carbapenemase gene (bla_VIM-1). Another 5.9-kb plasmid, pCY-MdT, with an aac(6′)-Ib gene, was very closely related (13 nucleotide differences) to pMdT1, a ColE1 plasmid carrying aac(6′)-Ib-cr4.     

南昌4家教学医院碳青霉烯类耐药肺炎克雷伯菌耐药机制及同源性分析

目的探讨临床分离的碳青霉烯类耐药肺炎克雷伯菌(CRKP)的耐药机制及同源性。方法收集南昌地区4家教学医院耐碳青霉烯类抗菌药物(亚胺培南和/或美罗培南)的肺炎克雷伯菌29株,双纸片增效法检测超广谱β-内酰胺酶(ESBLs)、三维实验检测AmpC酶、改良Hodge实验和双纸片协同法检测碳青霉烯酶,PCR扩增耐药基因,并对扩增产物测序,确定其基因型。脉冲场凝胶电泳(PFGE)对其进行同源性分析。结果 29株分离菌除对阿米卡星的耐药率较低(37.9%)外,对其他13种抗菌药物的耐药率均大于69%,其中对头孢呋辛、亚胺培南的耐药率为100%,多重耐药肺炎克雷伯菌的检出率为62.1%(18/29)。29株分离菌中有19株携带碳青霉烯酶基因,占65.5%(19/29),以blaKPC-2为主(44.8%,13/29),其次为blaNDM-1、blaIMP-26及blaIMP-4,携带率分别为17.2%(5/29)、10.3%(3/29)及6.9%(2/29),另有3株同时携带blaKPC-2和blaIMP-26,1株同时携带blaKPC-2和blaIMP-4。17株除携带碳青霉烯酶基因外,还携带ESBLs基因和(或)AmpC基因,占58.6%。未检测到VIM、GES、SPM及OXA等其他碳青霉烯酶基因。29株分离菌中有27株被PFGE成功分型,分别属于20个不同克隆,其余2株分型不成功。属于同一克隆型且携带blaKPC-2基因的4株菌来自同一医院。结论 CRKP耐药及多重耐药现象严重;携带碳青霉烯酶基因是肺炎克雷伯菌耐碳青霉烯类药物的主要原因,blaKPC-2携带率高,且在局部有短暂流行。本地区已监测到携带blaNDM的肺炎克雷伯菌,值得关注。