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

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.001409 ◽

2021 ◽

Vol 70 (9) ◽

Author(s):

Amani Al Fadhli ◽

Wafaa Jamal ◽

Vincent O. Rotimi

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Carbapenem Resistance ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Whole Genome ◽

Genetic Characteristics ◽

Carbapenem Resistant ◽

Genes Encoding ◽

Amoxicillin Clavulanic Acid

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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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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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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Whole Genome Sequencing of Peruvian Klebsiella pneumoniae Identifies Novel Plasmid Vectors Bearing Carbapenem Resistance Gene NDM-1

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa266 ◽

2020 ◽

Vol 7 (8) ◽

Author(s):

David Roach ◽

Adam Waalkes ◽

Jorge Abanto ◽

Joseph Zunt ◽

Carolina Cucho ◽

...

Keyword(s):

Developing Countries ◽

Population Structure ◽

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bacterial Pathogen ◽

Carbapenem Resistance ◽

Whole Genome ◽

Carbapenem Resistant ◽

Almost All

Abstract Background Klebsiella pneumoniae is a bacterial pathogen with increasing rates of resistance to carbapenem antibiotics, but the population structure and genetic drivers of carbapenem-resistant K pneumoniae (CRKP) remain underexplored in developing countries. Carbapenem-resistant K pneumoniae were recently introduced into Peru but have grown rapidly in prevalence, enabling study of this pathogen as it expands into an unaffected environment. Methods In this study, using whole genome sequencing, we show that 3 distinct lineages encompass almost all CRKP identified in the hospital where it was first reported in Peru. Results The most prevalent lineage, ST348, has not been described outside of Europe, raising concern for global dissemination. We identified metallo- β -lactamase NDM-1 as the primary carbapenem resistance effector, which was harbored on a novel vector resulting from recombination between 2 different plasmids, pKP1-NDM-1 and pMS7884A. Conclusions This study is the first of its kind performed in Peru, and it furthers our understanding of the landscape of CRKP infections in Latin America.

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Whole-Genome Sequencing for Investigating a Health Care-Associated Outbreak of Carbapenem-Resistant Acinetobacter baumannii

Diagnostics ◽

10.3390/diagnostics11020201 ◽

2021 ◽

Vol 11 (2) ◽

pp. 201

Author(s):

Sang Mee Hwang ◽

Hee Won Cho ◽

Tae Yeul Kim ◽

Jeong Su Park ◽

Jongtak Jung ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Acinetobacter Baumannii ◽

Genome Sequencing ◽

Snp Analysis ◽

Whole Genome ◽

Phylogenetic Tree Analysis ◽

Web Based ◽

Hospital Outbreak ◽

Carbapenem Resistant ◽

Bioinformatics Tools

Carbapenem-resistant Acinetobacter baumannii (CRAB) outbreaks in hospital settings challenge the treatment of patients and infection control. Understanding the relatedness of clinical isolates is important in distinguishing outbreak isolates from sporadic cases. This study investigated 11 CRAB isolates from a hospital outbreak by whole-genome sequencing (WGS), utilizing various bioinformatics tools for outbreak analysis. The results of multilocus sequence typing (MLST), single nucleotide polymorphism (SNP) analysis, and phylogenetic tree analysis by WGS through web-based tools were compared, and repetitive element polymerase chain reaction (rep-PCR) typing was performed. Through the WGS of 11 A. baumannii isolates, three clonal lineages were identified from the outbreak. The coexistence of blaOXA-23, blaOXA-66, blaADC-25, and armA with additional aminoglycoside-inactivating enzymes, predicted to confer multidrug resistance, was identified in all isolates. The MLST Oxford scheme identified three types (ST191, ST369, and ST451), and, through whole-genome MLST and whole-genome SNP analyses, different clones were found to exist within the MLST types. wgSNP showed the highest discriminatory power with the lowest similarities among the isolates. Using the various bioinformatics tools for WGS, CRAB outbreak analysis was applicable and identified three discrete clusters differentiating the separate epidemiologic relationships among the isolates.

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Whole Genome Sequencing detects Inter-Facility Transmission of Carbapenem-resistant Klebsiella pneumoniae

Journal of Infection ◽

10.1016/j.jinf.2018.11.003 ◽

2019 ◽

Vol 78 (3) ◽

pp. 187-199 ◽

Cited By ~ 4

Author(s):

Melanie D. Spencer ◽

Kathryn Winglee ◽

Catherine Passaretti ◽

Ashlee M. Earl ◽

Abigail L. Manson ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Carbapenem Resistant

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Piglet Gut and in-Barn Manure from Farms on a Raised without Antibiotics Program Display Reduced Antimicrobial Resistance but an Increased Prevalence of Pathogens

Antibiotics ◽

10.3390/antibiotics10101152 ◽

2021 ◽

Vol 10 (10) ◽

pp. 1152

Author(s):

Samuel M. Chekabab ◽

John R. Lawrence ◽

Alvin C. Alvarado ◽

Bernardo Z. Predicala ◽

Darren R. Korber

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Fecal Samples ◽

Time Points ◽

Conventional Farms ◽

Production Practices ◽

On Farm ◽

The Impact

In response to new stringent regulations in Canada regarding the use of antibiotics in animal production, many farms have implemented practices to produce animals that are raised without antibiotics (RWA) from birth to slaughter. This study aims to assess the impact of RWA production practices on reducing the actual total on-farm use of antibiotics, the occurrence of pathogens, and the prevalence of antimicrobial resistance (AMR). A 28-month longitudinal surveillance of farms that adopted the RWA program and conventional farms using antibiotics in accordance with the new regulations (non-RWA) was conducted by collecting fecal samples from 6-week-old pigs and composite manure from the barn over six time points and applying whole-genome sequencing (WGS) to assess the prevalence of AMR genes as well as the abundance of pathogens. Analysis of in-barn drug use records confirmed the decreased consumption of antibiotics in RWA barns compared to non-RWA barns. WGS analyses revealed that RWA barns had reduced the frequency of AMR genes in piglet feces and in-barn manure. However, metagenomic analyses showed that RWA barns had a significant increase in the frequency of pathogenic Firmicutes in fecal samples and pathogenic Proteobacteria in barn manure samples.

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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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