Molecular characterization of rectal isolates of carbapenemase-negative carbapenem-resistant enterobacterales obtained from ICU patients in Kuwait by whole-genome sequencing

Introduction. Carbapenem-resistant enterobacterales (CRE) are listed among the most urgent antibiotic resistance threats. Hypothesis. Previous studies on the mechanisms of CRE in Kuwait have focused on carbapenemases. There have been no studies on non-carbapenemase-producing CRE in Kuwait. Aim/Gap Statement. The aim of this study was to investigate the genetic characteristics of non-carbapenemase-producing carbapenem-resistant enterobacterales (NCPE) isolates using whole-genome sequencing (WGS). Methodology. Fourteen confirmed NCPE isolates that were negative for genes encoding carbapenemase production by polymerase chain reaction (PCR) assays using rectal swabs from intensive care unit patients were characterized using phenotypic, PCR and WGS methods. Susceptibility testing was performed via Etest and clonality via multi-locus sequence typing (MLST). Results. All of the isolates were resistant to ertapenem; 78.6 % were resistant to imipenem, meropenem and trimethoprim–sulfamethoxazole. Resistance to the other antibiotics was variable, ranging from 28.5 (colistin) through 50 (tigecycline) and 64.3 (amikacin) up to 85.7 % against both amoxicillin–clavulanic acid and ciprofloxacin. WGS detected several resistance genes mediating the production of β-lactamases, genes encoding an outer-membrane porin permeability mutation resulting in reduced susceptibility to β-lactams, including carbapenems, and genes for multidrug-resistant (MDR) efflux pumps. The isolates also possessed global activator protein MarA, which mediated reduced permeability to β-lactams. The existence of β-lactamase genes, overexpression of MDR efflux pumps and reduced permeability mediated by the porin genes were responsible for carbapenem resistance. Conclusions. This finding reflects the superior detection capabilities offered by WGS analysis, which can be used to complement traditional methods and overcome their limited resolution in clinical settings.

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677. Activity of Novel β-Lactamase Inhibitor QPX7728 Combined with β-Lactam Agents When Tested Against Carbapenem-Resistant Enterobacteriaceae (CRE) Isolates

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.745 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S309-S309 ◽

Cited By ~ 3

Author(s):

Mariana Castanheira ◽

Jill Lindley ◽

Holly Huynh ◽

Rodrigo E Mendes ◽

Olga Lomovskaya

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Inhibitory Activity ◽

Multidrug Resistant ◽

Whole Genome ◽

Broth Microdilution ◽

Carbapenem Resistant ◽

Further Development ◽

Lactamase Inhibitor ◽

Carbapenem Resistant Enterobacteriaceae

Abstract Background CREs have been described worldwide and these isolates are often multidrug resistant with few therapeutic options remaining active against them. New β-lactam (BL)/β-lactamase inhibitor (BLI) combinations recently approved are active against KPC and some OXA-48 producers, but not against isolates producing metallo-β-lactamases (MBLs). We evaluated the activity of QPX7728 (QPX), a novel BLI paired with various BLs against a collection of CRE isolates characterized for the presence of carbapenemases. Methods A total of 508 CRE clinical isolates were susceptibility (S) tested by reference broth microdilution methods against meropenem (MER), tebipenem (TEB), cefepime (FEP), ceftolozane (TOL), and ertapenem (ETP), and meropenem (MEM) combined with QPX at fixed 2, 4, and 8 mg/L. Agents were provided by Qpex Biopharma except for FEP, ETP, and MEM. Carbapenemases were detected using PCR/sequencing or whole-genome sequencing. Results All BLs had limited activity against CRE isolates (MIC50/90, ≥32/ >32 mg/L) and QPX lowered the MIC for all agents (figure). Against 157 isolates carrying serine-carbapenemase (SCarb) genes (153 KPC-producers), MEM or ETP plus QPX at fixed 4 or 8 mg/L displayed MIC50 at ≤ 0.03 mg/L and MIC90 ranging from 0.12 to 0.5 mg/L. QPX lowered the FEP or TOL MIC50 to ≤ 0.25 mg/L and MIC90 to 0.25, 0.5 or 1 mg/L depending on the BLI concentration. Over 98.0% of the 150 isolates harboring OXA-48-like genes were inhibited by FEP, TOL, ETP or MEM plus QPX at ≤2 mg/L. Similarly, MEM, FEP, TOL and ETP + QPX inhibited >98.0% of the 51 CREs that did not carry carbapenemases at ≤2 mg/L when using a higher BLI concentration. The activity of FEP (MIC50/90, 0.06/1 mg/L), ETP (MIC50/90, 0.03/4 mg/L), and MEM (MIC50/90, ≤ 0.015/2 mg/L) was mostly restored when 8 mg/L of QPX was combined with these agents and tested against 150 MBL-producing isolates. Conclusion QPX restored the activity of several BLs when tested against 508 CRE isolates that include 157 harboring SCarb, 150 OXA-48-like-producers, and 150 MBL-producing isolates. Further development of this BLI with inhibitory activity against all carbapenemase types seems warranted. Disclosures All authors: No reported disclosures.

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Deciphering the resistome of the widespreadP. aeruginosaST175 international high-risk clone through whole genome sequencing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01720-16 ◽

2016 ◽

pp. AAC.01720-16 ◽

Cited By ~ 14

Author(s):

Gabriel Cabot ◽

Carla López-Causapé ◽

Alain A. Ocampo-Sosa ◽

Lea M. Sommer ◽

María Ángeles Domínguez ◽

...

Keyword(s):

High Risk ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Carbapenem Resistance ◽

Whole Genome ◽

Resistance Mutations ◽

Online Databases ◽

Genes Encoding ◽

Resistance Traits

Whole genome sequencing (WGS) was used for the characterization of the, frequently extensively-drug resistant (XDR),P. aeruginosahigh-risk clone ST175. A total of eighteen ST175 isolates recovered from 8 different Spanish hospitals were analyzed; four isolates from four different French hospitals were included for comparison. The typical resistance profile of ST175 included penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, and fluoroquinolones. In the phylogenetic analysis, the four French isolates clustered together with the two isolates from one of the Spanish regions. Sequence variation was analyzed for 146 chromosomal genes related to antimicrobial resistance and horizontally-acquired genes were explored using online databases. The resistome of ST175 was mainly determined by mutational events, with resistance traits common to all or nearly all of the strains, including specificampRmutations leading toampCoverexpression, specific mutations inoprDconferring carbapenem resistance or amexZmutation leading to MexXY overexpression. All isolates additionally harbored anaadBgene conferring gentamicin and tobramycin resistance. Several other resistance traits were specific to certain geographic areas such as a streptomycin resistanceaadA13gene detected in all four isolates from France and in the 2 isolates from the Cantabria region or aglpTmutation conferring fosfomycin resistance detected in all but these six isolates. Finally, several unique resistance mutations were detected in single isolates; particularly interesting among them were those in genes encoding PBPs (PBP1A, PBP3 and PBP4). Thus, these results provide valuable information for understanding the genetic basis of resistance and the dynamics of dissemination and evolution of high-risk clones.

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2461. Community-Acquired in Name Only: A Cluster of Carbapenem-Resistant Acinetobacter baumannii in a Burn Intensive Care Unit

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.2139 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S852-S852

Author(s):

Erica S Shenoy ◽

Virginia M Pierce ◽

Mohamad Sater ◽

Febriana Pangestu ◽

Ian Herriott ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Whole Genome Sequencing ◽

Acinetobacter Baumannii ◽

Genome Sequencing ◽

Carbapenem Resistance ◽

Environmental Sampling ◽

Whole Genome ◽

Single Source ◽

Carbapenem Resistant

Abstract Background Detection of nosocomial outbreaks often relies on epidemiological definitions of community and nosocomial acquisition. We report a cluster of three carbapenem-resistant Acinetobacter baumannii (CRAB) infections linked to a single source patient with infections occurring within 2 days of admission to a burn intensive care unit (ICU). The epidemiological investigation was supplemented by whole-genome sequencing (WGS) of clinical and environmental isolates. Methods Study participants included burn ICU patients identified with infections caused by CRAB. A detailed review of patient demographic and clinical data was conducted. Clinical A. baumannii isolates were assessed by antimicrobial susceptibility testing and WGS. Review of infection control practices on the affected unit was followed by environmental sampling. A. baumannii isolates obtained through environmental sampling were assessed for carbapenem resistance and then underwent WGS for comparison to the clinical isolates. Results Three cases of CRAB infection in the affected unit spanning a period of 3 months were linked to a preceding source patient, with CRAB isolates from the four patients differing by 5–7 single nucleotide variations. All case patients had been admitted to the same room within 2 days before development of CRAB infection. Environmental sampling performed while the third case patient occupied the room identified highly contaminated areas, and environmental CRAB isolates linked the patient isolates. The contaminated areas were subsequently re-sampled after enhanced terminal cleaning of the room. No additional CRAB was isolated, but other pathogenic organisms were recovered. Conclusion We report a cluster of three infections caused by highly resistant A. baumannii that occurred in a burn intensive care unit over a period of 3 months, linked to a single source patient. Three case patients developed infections classified as community-acquired using standard epidemiological definitions, however, whole-genome sequencing revealed clonality. An extensive investigation identified the role of environmental reservoirs. Burn patients may be particularly vulnerable to early-onset nosocomial infection from environmental contamination. Disclosures All authors: No reported disclosures.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01275-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6625-6628 ◽

Cited By ~ 27

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.

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Genomic characterization of Citrobacter freundii strains coproducing OXA-48 and VIM-1 carbapenemase enzymes isolated in leukemic patient in Spain

Antimicrobial Resistance and Infection Control ◽

10.1186/s13756-019-0630-3 ◽

2019 ◽

Vol 8 (1) ◽

Author(s):

Rym Lalaoui ◽

Ana Djukovic ◽

Sofiane Bakour ◽

Linda Hadjadj ◽

Jaime Sanz ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Mechanism ◽

Carbapenem Resistance ◽

Bioinformatic Analysis ◽

Whole Genome ◽

Citrobacter Freundii ◽

Fecal Samples ◽

Carbapenem Resistant ◽

High Risk Populations

Abstract Background The emergence of carbapenemase-producing (CP) Citrobacter freundii poses a significant threat to public health, especially in high-risk populations. In this study, whole genome sequencing was used to characterize the carbapenem resistance mechanism of three C. freundii clinical isolates recovered from fecal samples of patients with acute leukemia (AL) from Spain. Materials and methods Twelve fecal samples, collected between 2013 and 2015 from 9 patients with AL, were screened for the presence of CP strains by selecting them on MacConkey agar supplemented with ertapenem (0.5 mg/L). Bacteria were identified by MALDI-TOF mass spectrometry and were phenotypically characterized. Whole genome sequencing of C. freundii isolates was performed using the MinION and MiSeq Illumina sequencers. Bioinformatic analysis was performed in order to identify the molecular support of carbapenem resistance and to study the genetic environment of carbapenem resistance encoding genes. Results Three carbapenem-resistant C. freundii strains (imipenem MIC≥32 mg/L) corresponding to three different AL patients were isolated. Positive modified Carba NP test results suggested carbapenemase production. The genomes of each C. freundii tested were assembled into a single chromosomal contig and plasmids contig. In all the strains, the carbapenem resistance was due to the coproduction of OXA-48 and VIM-1 enzymes encoded by genes located on chromosome and on an IncHI2 plasmid, respectively. According to the MLST and the SNPs analysis, all strains belonged to the same clone ST169. Conclusion We report in our study, the intestinal carrying of C. freundii clone ST169 coproducing OXA-48 and VIM-1 identified in leukemic patients.

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mSphere of Influence: Whole-Genome Sequencing, a Vital Tool for the Interruption of Nosocomial Transmission

mSphere ◽

10.1128/msphere.00230-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Ahmed Babiker

Keyword(s):

Health Care ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Multidrug Resistant ◽

Nosocomial Transmission ◽

Whole Genome ◽

Content Type ◽

Genomic Epidemiology ◽

Hospital Outbreak ◽

Carbapenem Resistant

ABSTRACT Ahmed Babiker’s work focuses on the clinical and genomic epidemiology of multidrug-resistant health care-associated pathogens and other high-consequence pathogens. In this mSphere of Influence article, he reflects on how the paper “Tracking a Hospital Outbreak of Carbapenem-Resistant Klebsiella pneumoniae with Whole-Genome Sequencing” by Evan S. Snitkin et al. (Sci Transl Med 4:148ra116, 2012, https://doi.org/10.1126/scitranslmed.3004129) impacted his thinking on the use of whole-genome sequencing for nosocomial transmission investigation.

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Molecular dissection of Carbapenem-resistant Acinetobacter baumannii circulating in Indian hospitals using Whole Genome Sequencing

10.1101/2021.07.30.454432 ◽

2021 ◽

Author(s):

Steffimol Rose ◽

Varun Shamanna ◽

Anthony Underwood ◽

Geetha Nagaraj ◽

Akshatha Prasanna ◽

...

Keyword(s):

Molecular Epidemiology ◽

Whole Genome Sequencing ◽

Acinetobacter Baumannii ◽

Genome Sequencing ◽

Clonal Complex ◽

Carbapenem Resistance ◽

Evolutionary Divergence ◽

Whole Genome ◽

Northern India ◽

Carbapenem Resistant

Objectives: Carbapenem-resistant Acinetobacter baumannii (CRAB) has acquired worldwide recognition as a serious nosocomial infection. It poses a concern to hospitalized patients because of the limited therapeutic options available. Thus, we investigated the molecular epidemiology and antibiotic resistance profiles of A. baumannii isolates in India. Materials and Methods: We characterized 306 retrospective A. baumannii clinical isolates collected from 18 centers across 10 states and 1 Union Territory of India between 2015 and 2019. Molecular epidemiology, and carbapenem resistance were studied by Whole Genome Sequencing. Results: A total of 105 different Sequence Types (STs) were identified including 48 reported STs and 57 Novel STs. 99 isolates were classified into Clonal Complex 451 (CC451) among which ST848 and ST1956 were the common STs. Carbapenemase resistance was confirmed in all the isolates with the presence of intrinsic bla OXA -51-like genes, and the acquired bla OXA-23 and bla NDM-1 genes. Conclusion: Most of the isolates were grouped under clonal complex 451. ST1053 caused an outbreak in Northern India during 2018 and 2019. Novel MLST alleles and STs were also detected, underlining an evolutionary divergence in India. The carbapenem-resistance was dominated by OXA-type carbapenemases and further surveillance of these carbapenem-resistant A. baumannii and antimicrobial stewardship should be strengthened.

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Clinical and Genetic Characteristics of Extended-Spectrum Beta-Lactamase–Producing Enterobacteriaceae Among Canadian Children

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.693 ◽

2020 ◽

Vol 41 (S1) ◽

pp. s167-s168

Author(s):

Nisha Thampi ◽

Jennifer Bowes ◽

Roberto Melano ◽

Nathalie Tijet ◽

Robert Slinger

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Multidrug Resistant ◽

Clinical Isolates ◽

First Episode ◽

Whole Genome ◽

Comorbid Conditions ◽

Genetic Characteristics ◽

E Coli

Background: Infections with extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E) in nonoutbreak settings have not demonstrated the presence of dominant strains. Our objective was to determine the incidence, clinical characteristics, and genetic characteristics of ESBL-E infections among a group of Canadian children. Methods: From 2012 through 2017, patients aged ≤18 years with first-episode ESBL-E infections who presented at a pediatric center were reviewed. All clinical isolates were phenotypically identified in the laboratory as ESBL-producers. Demographic and clinical data were collected, including comorbid conditions, presence of devices, and previous antibacterial exposure. Community-associated infection was defined as a positive culture from a sterile site within the first 48 hours of hospital admission and no healthcare exposure during the preceding year. Isolates were sent to the Public Health Ontario Laboratory for whole-genome sequencing. Multilocus sequence typing was used to determine clonal relationship. Results: During the study period, 102 patients were identified with first-episode ESBL-E infection, and the proportion of ESBL-E isolates among all clinical isolates of E. coli and Klebsiella spp increased from 0.6% to 2.6% between 2012 and 2017, respectively (P = .001). The median age was 1 year (interquartile range, 0.8–5 years). Women comprised 66% of cases. No comorbid conditions were noted among 58 patients (57%), and 24% had previous antibiotic exposure, most frequently a cephalosporin (16%). ESBL-E was most frequently isolated in the urine (91%) and least frequently in the blood (2.2%) and was predominantly Escherichia coli (90%). Infection was most frequently diagnosed in the outpatient setting (61%); there were 11 healthcare-associated infections. Whole-genome sequencing of ESBL-E isolates revealed predominance of blaCTX-M-15 (63 isolates, 62%) and blaCTX-M-27 (16%) genes, and sequence type (ST) 131 (41%). Mutations conferring fluoroquinolone nonsusceptibility were noted among 62 isolates (61%), most frequently associated with ST131 (38 of 62 isolates, 61%) and among all 5 isolates with ST1193, an emerging multidrug-resistant E. coli clone. In addition, 15 patients had recurrence of ESBL-E infection at median of 113 days (IQR, 26–208); blaCTX-M-27 was found in 33% of recurrent infections compared to 12% of primary infections (P = 0.045). Conclusions: This study is the first in Canada to provide whole-genome sequencing data regarding ESBL-E in a pediatric population. The gene blaCTX-M-15 and ST131 clone were predominant. More than 60% of infections were community associated and demonstrated cross resistance to fluoroquinolones. With 76% of infections in antibiotic-naïve children, ESBL-E is a public health concern, and a One Health approach is critical to understanding the epidemiology and curbing the spread of multidrug-resistant Enterobacteriaceae.Funding: NoneDisclosures: None

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Emergence of Carbapenem-Resistant Pseudomonas asiatica Producing NDM-1 and VIM-2 Metallo-β-Lactamases in Myanmar

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00475-19 ◽

2019 ◽

Vol 63 (8) ◽

Cited By ~ 6

Author(s):

Mari Tohya ◽

Tatsuya Tada ◽

Shin Watanabe ◽

Kyoko Kuwahara-Arai ◽

Khwar Nyo Zin ◽

...

Keyword(s):

16S Rrna ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Gene Encoding ◽

Medical Settings ◽

Content Type ◽

Carbapenem Resistant ◽

Genes Encoding ◽

16S Rrna Methylase

ABSTRACT Pseudomonas asiatica is a recently proposed species of the genus Pseudomonas. This study describes eight isolates of carbapenem-resistant P. asiatica harboring blaNDM-1 and blaVIM-2, genes encoding metallo-β-lactamase (MBL). These isolates were obtained from urine samples of patients hospitalized in Myanmar. These isolates were resistant to carbapenems but susceptible to colistin. All eight isolates were positive for a carbapenemase inactivation method, CIMTrisII, and seven were positive on an immunochromatographic assay for NDM-type MBL. One isolate was highly resistant to aminoglycosides. Whole-genome sequencing showed that seven isolates harbored blaNDM-1 and one harbored blaVIM-2, with these genes located on the chromosome. One isolate harbored blaNDM-1 and rmtC, a gene encoding 16S rRNA methylase. Five types of genomic environments surrounding blaNDM-1 and blaVIM-2 were detected in these eight isolates, with four isolates having the same type. These data indicate that P. asiatica isolates harboring genes encoding carbapenemases, including blaNDM-1 and blaVIM-2, are spreading in medical settings in Myanmar.

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Bronchoscope-associated clusters of multidrug-resistant Pseudomonas aeruginosa and carbapenem-resistant Klebsiella pneumoniae

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2018.263 ◽

2018 ◽

Vol 40 (1) ◽

pp. 40-46 ◽

Cited By ~ 11

Author(s):

Alison L. Galdys ◽

Jane W. Marsh ◽

Edgar Delgado ◽

A. William Pasculle ◽

Marissa Pacey ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Relatedness ◽

Multidrug Resistant ◽

Molecular Testing ◽

Whole Genome ◽

Carbapenem Resistant ◽

Clinical Culture

AbstractObjectiveRecovery of multidrug-resistant (MDR) Pseudomonas aeruginosa and Klebsiella pneumoniae from a cluster of patients in the medical intensive care unit (MICU) prompted an epidemiologic investigation for a common exposure.MethodsClinical and microbiologic data from MICU patients were retrospectively reviewed, MICU bronchoscopes underwent culturing and borescopy, and bronchoscope reprocessing procedures were reviewed. Bronchoscope and clinical MDR isolates epidemiologically linked to the cluster underwent molecular typing using pulsed-field gel electrophoresis (PFGE) followed by whole-genome sequencing.ResultsOf the 33 case patients, 23 (70%) were exposed to a common bronchoscope (B1). Both MDR P. aeruginosa and K. pneumonia were recovered from the bronchoscope’s lumen, and borescopy revealed a luminal defect. Molecular testing demonstrated genetic relatedness among case patient and B1 isolates, providing strong evidence for horizontal bacterial transmission. MDR organism (MDRO) recovery in 19 patients was ultimately linked to B1 exposure, and 10 of 19 patients were classified as belonging to an MDRO pseudo-outbreak.ConclusionsSurveillance of bronchoscope-derived clinical culture data was important for early detection of this outbreak, and whole-genome sequencing was important for the confirmation of findings. Visualization of bronchoscope lumens to confirm integrity should be a critical component of device reprocessing.

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