Expression of AraC/XylS stress response regulators in two distinct carbapenem-resistant Enterobacter cloacae ST89 biotypes

2020 ◽  
Vol 75 (5) ◽  
pp. 1146-1150
Author(s):  
Piotr Majewski ◽  
Anna Gutowska ◽  
Pawel Sacha ◽  
Thamarai Schneiders ◽  
Mariola Talalaj ◽  
...  
Keyword(s):  
Membrane Permeability ◽  
Molecular Mechanisms ◽  
Carbapenem Resistance ◽  
Enterobacter Cloacae ◽  
Modern Medicine ◽  
Regulatory Genes ◽  
Global Regulator ◽  
Response Regulators ◽  
Bacterial Membranes ◽  
Carbapenem Resistant

Abstract Background The growing incidence of MDR Gram-negative bacteria is a rapidly emerging challenge in modern medicine. Objectives We sought to establish the role of intrinsic drug-resistance regulators in combination with specific genetic mutations in 11 Enterobacter cloacae isolates obtained from a single patient within a 7 week period. Methods The molecular characterization of eight carbapenem-resistant and three carbapenem-susceptible E. cloacae ST89 isolates included expression-level analysis and WGS. Quantitative PCR included: (i) chromosomal cephalosporinase gene (ampC); (ii) membrane permeability factor genes, e.g. ompF, ompC, acrA, acrB and tolC; and (iii) intrinsic regulatory genes, e.g. ramA, ampR, rob, marA and soxS, which confer reductions in antibiotic susceptibility. Results In this study we describe the influence of the alterations in membrane permeability (ompF and ompC levels), intrinsic regulatory genes (ramA, marA, soxS) and intrinsic chromosomal cephalosporinase AmpC on reductions in carbapenem susceptibility of E. cloacae clinical isolates. Interestingly, only the first isolate possessed the acquired VIM-4 carbapenemase, which has been lost in subsequent isolates. The remaining XDR E. cloacae ST89 isolates presented complex carbapenem-resistance pathways, which included perturbations in permeability of bacterial membranes mediated by overexpression of ramA, encoding an AraC/XylS global regulator. Moreover, susceptible isolates differed significantly from other isolates in terms of marA down-regulation and soxS up-regulation. Conclusions Molecular mechanisms of resistance among carbapenem-resistant E. cloacae included production of acquired VIM-4 carbapenemase, significant alterations in membrane permeability due to increased expression of ramA, encoding an AraC/XylS global regulator, and the overproduction of chromosomal AmpC cephalosporinase.

scholarly journals Characterization of resistance mechanisms of Enterobacter cloacae Complex co-resistant to carbapenem and colistin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shixing Liu ◽  
Renchi Fang ◽  
Ying Zhang ◽  
Lijiang Chen ◽  
Na Huang ◽  
...  
Keyword(s):  
Lipid A ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Carbapenem Resistance ◽  
Enterobacter Cloacae ◽  
Resistance Mechanisms ◽  
Colistin Resistance ◽  
Carbapenem Resistant ◽  
Horizontal Spread

Abstract Background The emergence of carbapenem-resistant and colistin-resistant ECC pose a huge challenge to infection control. The purpose of this study was to clarify the mechanism of the carbapenems and colistin co-resistance in Enterobacter cloacae Complex (ECC) strains. Results This study showed that the mechanisms of carbapenem resistance in this study are: 1. Generating carbapenemase (7 of 19); 2. The production of AmpC or ESBLs combined with decreased expression of out membrane protein (12 of 19). hsp60 sequence analysis suggested 10 of 19 the strains belong to colistin hetero-resistant clusters and the mechanism of colistin resistance is increasing expression of acrA in the efflux pump AcrAB-TolC alone (18 of 19) or accompanied by a decrease of affinity between colistin and outer membrane caused by the modification of lipid A (14 of 19). Moreover, an ECC strain co-harboring plasmid-mediated mcr-4.3 and blaNDM-1 has been found. Conclusions This study suggested that there is no overlap between the resistance mechanism of co-resistant ECC strains to carbapenem and colistin. However, the emergence of strain co-harboring plasmid-mediated resistance genes indicated that ECC is a potential carrier for the horizontal spread of carbapenems and colistin resistance.

scholarly journals A Prolonged Outbreak of KPC-3-Producing Enterobacter cloacae and Klebsiella pneumoniae Driven by Multiple Mechanisms of Resistance Transmission at a Large Academic Burn Center

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Hajime Kanamori ◽  
Christian M. Parobek ◽  
Jonathan J. Juliano ◽  
David van Duin ◽  
Bruce A. Cairns ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Molecular Mechanisms ◽  
Enterobacter Cloacae ◽  
The United States ◽  
Multidrug Resistant ◽  
Control Measures ◽  
Content Type ◽  
Burn Center ◽  
Infection Control Measures ◽  
Carbapenem Resistant

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter cloacae has been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in health care settings, including carbapenem-resistant Enterobacteriaceae (CRE). We experienced a prolonged outbreak of CRE E. cloacae and K. pneumoniae over a 3-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates by using WGS. Twenty-two clinical isolates of CRE, including E. cloacae (n = 15) and K. pneumoniae (n = 7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacae and K. pneumoniae. This outbreak was primarily maintained by a clonal expansion of E. cloacae sequence type 114 (ST114) with distribution of multiple resistance determinants. Plasmid and transposon analyses suggested that the majority of bla KPC-3 was transmitted via an identical Tn4401b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of the strain and/or plasmid in association with a transposon, highlighting the versatility of KPC-producing Enterobacteriaceae in their ability to utilize multiple modes to resistance gene propagation.

scholarly journals Detection of carbapenemase-producing Pseudomonas aeruginosa by phenotypic and genotypic methods in a tertiary care hospital of East India

2019 ◽  
Vol 11 (04) ◽  
pp. 287-291 ◽  
Author(s):  
Nishu Verma ◽  
Ashok Prahraj ◽  
Baijayantimala Mishra ◽  
Bijayini Behera ◽  
Kavita Gupta
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Tertiary Care ◽  
Carbapenem Resistance ◽  
Hospital Infection ◽  
High Rate ◽  
Care Hospital ◽  
Carbapenem Resistant ◽  
Hospital Infection Control ◽  
Synergy Test

Abstract BACKGROUND: Carbapenemase-producing Pseudomonas aeruginosa is a serious threat in hospital infection due to its multidrug resistance. AIM: The aim of the study was to determine the frequency of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa and detect the presence of carbapenemase enzymes in carbapenem-resistant P. aeruginosa (CRPA) isolates by phenotypic and genotypic methods. MATERIAL AND METHODS: Double-disk synergy test [DDST] and combined disk synergy test [CDST]) was performed in CRPA isolates and the prevalence ofblaKPC,blaNDM-1,blaIMP,blaVIM,blaSIM,blaSPM,blaGIM, andblaOXA-48 was determined. RESULTS: Of 559 isolates included in the study, a total of 102 isolates were resistant to carbapenem that accounted for overall 18.24% (102/559) prevalence. Of these 102 isolates, 89 (87.25%) isolates were positive by DDST and 95 (93.17%) isolates were positive by CDST. Of 102 CRPA isolates,blaVIM was detected in 30 isolates (30/102, 29.1%), followed byblaNDM-1 in 29 (29/102, 28.4%) isolates andblaSIM andblaGIM in 6 isolates each (6/102, 5.8%). A combination of two carbapenemase genes was detected in 12 isolates, with six (6/102, 5.88%) CRPA isolates harboring with bothblaVIM andblaNDM-1 genes. Four isolates were found to harbor a combination of three carbapenem-resistant genes. CONCLUSION: A high rate of carbapenemase production was observed in P. aeruginosa. Coproducers of multiple carbapenemases are also a cause of concern. An in-depth understanding of molecular mechanisms of resistance will be helpful in optimizing patient management and hospital infection control.

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

2015 ◽  
Vol 59 (10) ◽  
pp. 6625-6628 ◽  
Author(s):  
Wenjing Wu ◽  
Yu Feng ◽  
Alessandra Carattoli ◽  
Zhiyong Zong
Keyword(s):  
Whole Genome Sequencing ◽  
Bloodstream Infection ◽  
Genome Sequencing ◽  
Carbapenem Resistance ◽  
Enterobacter Cloacae ◽  
Whole Genome ◽  
Content Type ◽  
Carbapenem Resistant

ABSTRACTA carbapenem-resistantEnterobacter cloacaestrain, WCHECl-14653, causing a fatal bloodstream infection, was characterized by genome sequencing and conjugation experiments. The strain carried two carbapenemase genes,blaNDM-1andblaKPC-2, on separate IncF plasmids. The coexistence ofblaNDM-1andblaKPC-2conferred slightly higher-level carbapenem resistance compared with that ofblaNDM-1orblaKPC-2alone, and the coexistence of two IncF plasmids may generate new platforms for spreading carbapenemase genes.

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

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Kalyan D. Chavda ◽  
Liang Chen ◽  
Derrick E. Fouts ◽  
Granger Sutton ◽  
Lauren Brinkac ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Molecular Mechanisms ◽  
Genomic Structure ◽  
Genomic Analysis ◽  
Carbapenem Resistance ◽  
Phylogenomic Analysis ◽  
Drug Resistant ◽  
Limited Information ◽  
Evolutionary Trajectory ◽  
Carbapenem Resistant

ABSTRACT 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 bla KPC-2 , 40 had bla KPC-3 , 2 had bla KPC-4 , and 2 had bla NDM-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 bla KPC -harboring plasmids between different phylogenomic groups, and repeated transposition of the bla KPC 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 bla KPC -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.

scholarly journals Diversity of Molecular Mechanisms Conferring Carbapenem Resistance toPseudomonas aeruginosaIsolates from Saudi Arabia

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Mohamed H. Al-Agamy ◽  
Katy Jeannot ◽  
Taghrid S. El-Mahdy ◽  
Hassan A. Samaha ◽  
Atef M. Shibl ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Carbapenem Resistance ◽  
Tertiary Hospital ◽  
Carbapenem Resistant ◽  
Extended Spectrum ◽  
Resistance Patterns ◽  
O 15 ◽  
O 11

Background.This study described various molecular and epidemiological characters determining antibiotic resistance patterns inPseudomonas aeruginosaisolates.Methods.A total of 34 carbapenem-resistantP. aeruginosaclinical isolates were isolated from samples collected at a tertiary hospital in Riyadh, Saudi Arabia, from January to December 2011. Susceptibility testing, serotyping, molecular characterization of carbapenem resistance, and pulsed-field gel electrophoresis (PFGE) were performed.Results.All isolates were resistant to ceftazidime, and more than half were highly resistant (minimum inhibitory concentration (MIC) > 256 mg/L). Fifteen isolates had MIC values ≥64 mg/L for any of the carbapenems examined. Vietnamese extended-spectrumβ-lactamase (VEB-1) (n=16/34) and oxacillinase (OXA-10) (n=14/34) were the most prevalent extended-spectrumβ-lactamase and penicillinase, respectively. Verona imipenemase (VIM-1, VIM-2, VIM-4, VIM-11, and VIM-28) and imipenemase (IMP-7) variants were found in metallo-β-lactamase producers. A decrease in outer membrane porin gene (oprD) expression was seen in nine isolates, and an increase in efflux pump gene (MexAB) expression was detected in five isolates. Six serotypes (O:1, O:4, O:7, O:10, O:11, and O:15) were found among the 34 isolates. The predominant serotype was O:11 (16 isolates), followed by O:15 (nine isolates). PFGE analysis of the 34 carbapenem-resistantP. aeruginosaisolates revealed 14 different pulsotypes.Conclusions.These results revealed diverse mechanisms conferring carbapenem resistance toP. aeruginosaisolates from Saudi Arabia.

scholarly journals Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant Klebsiella pneumoniae and Enterobacter cloacae

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tae Hwan Kim ◽  
Xun Tao ◽  
Bartolome Moya ◽  
Yuanyuan Jiao ◽  
Kari B. Basso ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Enterobacter Cloacae ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Outer Membrane Permeability

ABSTRACT Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae. Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Both clinical isolates harbored a blaKPC-2 and several other β-lactamases. The OM permeability of each antibiotic was studied separately (“discrete assay”) and simultaneously (“cassette assay”) by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our β-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of β-lactam antibiotics. IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While β-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of β-lactams leads to high drug concentrations at their periplasmic target sites, allowing β-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of β-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five β-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem β-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of β-lactams and combat carbapenem-resistant isolates.

scholarly journals Characterization of fosfomycin resistance and molecular epidemiology among carbapenem-resistant Klebsiella pneumoniae strains from two tertiary hospitals in China

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Haichen Wang ◽  
Changhang Min ◽  
Jun Li ◽  
Ting Yu ◽  
Yongmei Hu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Molecular Mechanisms ◽  
Genomic Analysis ◽  
Carbapenem Resistance ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Its Gene ◽  
Tertiary Hospitals ◽  
Resistant Strains

Abstract Background Fosfomycin has been proven to be a vital choice to treat infection caused by multidrug resistance bacteria, especially carbapenem-resistant Klebsiella pneumoniae (CRKP). However, fosfomycin resistant cases has been reported gradually. In this study, we reported the fosfomycin-resistant rate in CRKP strains and further revealed the molecular mechanisms in resistance gene dissemination. Results A total of 294 non-duplicated CRKP strains were collected. And 55 fosfomyin-resistant strains were detected, 94.5% of which were clustered to sequence type (ST) 11 by PCR followed up sequencing. PFGE further revealed two major groups and four singletons. The positive rates of genes responsible to fosfomycin and carbapenem resistance were 81.8% (fosA3), 12.7% (fosA5) and 94.5% (blaKPC-2), respectively. Genomic analysis confirmed insertion sequence (IS) 26 was the predominant structure surrounding fosA3. The fosA3 genes in six isolates were located on plasmids which were able to transfer to E. coli J53 recipient cells by means of conjugation. Conclusions Although the resistant rate of CRKP to fosfomycin is relatively low in our area, considering its gene is located on transferrable plasmid and inserted in IS structure, continuous monitoring is still needed.

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