44 research outputs found
Transcriptional Landscape of a blaKPC-2 Plasmid and Response to Imipenem Exposure in Escherichia coli TOP10
The diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFIIK plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFIIK-IncFIB blaKPCâ2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance (blaKPCâ2, blaTEM and aph(3âČ)-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of E. coli (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the SoxRS/OxyR oxydative stress regulons, the Fur regulon (for iron uptake machinery), and IscR regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase mepM and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on E. coli (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of E. coli to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments
Cross-border spread of blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae: a European collaborative analysis of whole genome sequencing and epidemiological data, 2014 to 2019
An alert regarding an outbreak of carbapenem-resistant Klebsiella pneumoniae carrying bla NDM-1 and bla OXA-48 carbapenemase-encoding genes was sent by Germany to European Union (EU)/European Economic Area (EEA) countries in October 2019. Since only limited whole genome sequencing (WGS) data on bla NDM-1- and bla OXA-48-positive K. pneumoniae were available in the public domain, national public health reference or equivalent expert laboratories from EU/EEA countries were invited to share WGS data from their national collections with the European Centre for Disease Prevention and Control (ECDC) to investigate the international dissemination of this epidemic strain. The analysis identified a Finnish case with an isolate closely related to the German outbreak strain and with an epidemiological link to St. Petersburg, Russia. In addition, several other clusters of genetically related bla NDM-1- and bla OXA-48-positive K. pneumoniae unrelated to the German outbreak strain but affecting numerous EU/EEA countries were identified. The aim of this follow-up investigation was to characterise these clusters based on the integrated analysis of the WGS dataset on bla NDM-1 - and bla OXA-48-positive K. pneumoniae submitted from 13 EU/EEA countries and additional epidemiological data
genomic and transcriptomic analysis of carbapenemase-producing bacteria
Le combat contre les infections bactĂ©riennes reste un enjeu majeur de santĂ© publique notamment avec la dissĂ©mination des EntĂ©robactĂ©ries productrices de carbapĂ©nĂšmases, capables dâhydrolyser lâensemble des ÎČ-lactamines. On assiste Ă lâĂ©mergence de certains clones Ă©pidĂ©miques, se distinguant par leur distribution mondiale, leur forte transmissibilitĂ© et leur capacitĂ© Ă persister chez les patients. Lâexemple le plus parlant est le cas du clone de Klebsiella pneumoniae appartenant au « sequence type » (ST) 258 et responsable de la diffusion mondiale de la carbapĂ©nĂšmase KPC (Klebsiella Pneumoniae Carbapenemase) portĂ©e majoritairement par des plasmides de la famille InFIIk. Les raisons du succĂšs de ce clone et de lâassociation KPC/IncFIIk/ST258 ne sont pas encore totalement Ă©lucidĂ©es. Par ailleurs, il nâexiste pas de corrĂ©lation entre le niveau dâexpression dâune carbapĂ©nĂšmase, la sensibilitĂ© in vitro de la souche vis Ă vis des carbapĂ©nĂšmes et lâefficacitĂ© clinique dâun traitement par ces molĂ©cules. Les phĂ©nomĂšnes dâhĂ©terorĂ©sistance aux carbapĂ©nĂšmes sont frĂ©quents chez les souches produisant KPC, mais lâimpact clinique est inconnu. Les mĂ©canismes de rĂ©gulation de lâexpression des carbapĂ©nĂšmases ne sont pas Ă©lucidĂ©s.Les objectifs de cette thĂšse rĂ©sident dans lâanalyse des facteurs gĂ©nĂ©tiques associĂ©s Ă la diffusion et la persistance de clones multi-rĂ©sistants ainsi que lâanalyse de lâexpression des ÎČ-lactamases associĂ©es.La premiĂšre partie de ce travail porte sur lâanalyse de lâĂ©volution in vivo dâune souche de K. pneumoniae ST258 produisant KPC ayant persistĂ© chez un patient pendant prĂšs de 5 ans. Lâanalyse comparative des gĂ©nomes provenant de 17 isolats a permis de mettre en Ă©vidence une diversification gĂ©nĂ©tique importante ainsi que la sĂ©lection de mutations modifiant la virulence de la souche et sa sensibilitĂ© aux antibiotiques. Afin de caractĂ©riser les raisons du succĂšs de certains plasmides portant KPC, une analyse transcriptomique dâune souche de Escherichia coli TOP10 transformĂ©e par un plasmide multi-rĂ©plicon IncFIIk-IncFIB exprimant KPC-2, a Ă©tĂ© rĂ©alisĂ©e en prĂ©sence ou non dâimipĂ©nĂšme. Les gĂšnes les plus exprimĂ©s dans ces conditions sont les gĂšnes de rĂ©sistance aux antibiotiques et certains gĂšnes essentiels Ă la rĂ©plication du plasmide. La prĂ©sence dâimipĂ©nĂšme affecte peu la transcription des gĂšnes plasmidiques mais induit un stress oxydatif important dans lâensemble de la souche. Par ailleurs, lâanalyse de lâexpression du gĂšne blaKPC-2 dans diffĂ©rentes espĂšces par 5â-RACE a permis de rĂ©vĂ©ler que ce gĂšne de rĂ©sistance est sous la dĂ©pendance de plusieurs promoteurs, dont la force diffĂšre selon le fond gĂ©nĂ©tique. Cette caractĂ©ristique pourrait expliquer le succĂšs de certains isoformes du transposon Tn4401 permettant une meilleure expression du gĂšne blaKPC-2, dans certaines espĂšces. Les outils dĂ©veloppĂ©s dans cette thĂšse ont Ă©galement Ă©tĂ© appliquĂ©s Ă lâanalyse dâun clone dâEnterobacter kobei ST125 dont la cĂ©phalosporinase naturelle ACT-28 possĂšde une activitĂ© dâhydrolyse accrue vis Ă vis de lâimipĂ©nĂšme. Enfin, lâanalyse du gĂ©nome de la premiĂšre souche Shewanella sp. produisant une BLSE de type CTX-M-15 a permis de rĂ©vĂ©ler la prĂ©sence dâun nouveau variant oxacillinase chromosomique avec activitĂ© carbapĂ©nĂšmase, appelĂ© OXA-535. OXA-535 est proche dâOXA-436, un autre variant carbapĂ©nĂšmase portĂ© par un plasmide ayant dĂ©jĂ dissĂ©minĂ© chez les EntĂ©robactĂ©ries. Lâanalyse de lâenvironnement gĂ©nĂ©tique des gĂšnes blaOXA-535 et blaOXA-436 confirme le rĂŽle du genre Shewanella comme progĂ©niteur des carbapĂ©nĂšmases de classe D. Ce travail contribue Ă une meilleure comprĂ©hension de la diffusion de certains clones multi-rĂ©sistants et des mĂ©canismes contrĂŽlant lâexpression des gĂšnes de rĂ©sistance aux ÎČ-lactamines.Multidrug resistant bacteria and in particular carbapenemase-producing Enterobacteriaceae remain a major health public challenge. Some successful clones are emerging globally, due to their high transmissibility and their ability to colonize and persist in patients over time. Genomic analyses revealed that the dissemination of KPC carbapenemase is closely related to the spread of Klebsiella pneumoniae of the sequence-type (ST) 258 and to few successful plasmids linked to IncFIIk family. However, the reasons of the association between K. pneumoniae ST258, IncFIIk plasmids and KPC that led to the rapid spread of this clone are currently unknown.Furthermore, there is no correlation between expression level of a carbapenemase-encoding gene, in vitro susceptibility to carbapenems and efficiency of a carbapenem-based treatment. Most of the time, KPC-producing K. pneumoniae exhibit a heteroresistant phenotype with carbapenems, but its clinical impact remains unknown. The mechanisms underlying the regulation of carbapenemases expression remain to be explored.The objectives of the thesis are to obtain deeper insights into genomic plasticity of carbapenemaseâproducing clones, and into the expression of their ÎČ-lactamases.The first part of this work was dedicated to the in vivo evolution of a single strain of KPC-producing K. pneumoniae ST258 that colonized a patient for almost 5 years. Genomic comparison of 17 isolates revealed a remarkable diversification with occurrence of several mutations with impact on bacterial virulence and susceptibility to antibiotics.Several studies extensively described the genetic structures of blaKPC-carrying plasmids, but information regarding gene expression at the whole plasmid level are lacking. Accordingly, we performed RNA-seq on Escherichia coli TOP10 transformed with an IncFIIk-IncFI blaKPC-2-carrying plasmid, with or without imipenem exposure. In both conditions, plasmid-encoded genes related to antimicrobial resistance and involved in plasmid replication were the most expressed. Imipenem exposure led to a more general response with overexpression of E. coli numerous chromosome-encoded genes involved in oxidative stress response. In addition, analysis of blaKPC-2 gene expression in several species using 5âRACE revealed the presence of several promoters whose strength depends on the bacterial genetic background. This could promote higher expression of blaKPC-2 gene and explain the association of some isoforms of Tn4401 in different species. The tools developed in the frame of this work were also applied to study a single Enterobacter kobei ST125 clone whose natural cephalosporinase (ACT-28) has increased hydrolytic activity towards imipenem. Finally, genomic analysis of the first ESBL-producing Shewanella sp. was performed. It revealed the presence of blaCTX-M-15 and blaSHV-2 genes carried on an IncA/C plasmid and a new chromosomally-encoded oxacillinase variant of OXA-48 with carbapenemase activity, called OXA-535. OXA-535 was found to be closely related to OXA-436, another carbapenemase which has recently spread in Enterobacteriaceae. Analysis of the genetic environment of both blaOXA-48-like genes confirmed the role of Shewanella spp. as progenitors of class D carbapenemases.Overall, this work contributes to a better comprehension of the diffusion of multi-drug resistant clones and of the mechanisms implicated in ÎČ-lactamase expression
analyse génomique et transcriptomique de bactéries productrices de carbapénÚmases
Multidrug resistant bacteria and in particular carbapenemase-producing Enterobacteriaceae remain a major health public challenge. Some successful clones are emerging globally, due to their high transmissibility and their ability to colonize and persist in patients over time. Genomic analyses revealed that the dissemination of KPC carbapenemase is closely related to the spread of Klebsiella pneumoniae of the sequence-type (ST) 258 and to few successful plasmids linked to IncFIIk family. However, the reasons of the association between K. pneumoniae ST258, IncFIIk plasmids and KPC that led to the rapid spread of this clone are currently unknown.Furthermore, there is no correlation between expression level of a carbapenemase-encoding gene, in vitro susceptibility to carbapenems and efficiency of a carbapenem-based treatment. Most of the time, KPC-producing K. pneumoniae exhibit a heteroresistant phenotype with carbapenems, but its clinical impact remains unknown. The mechanisms underlying the regulation of carbapenemases expression remain to be explored.The objectives of the thesis are to obtain deeper insights into genomic plasticity of carbapenemaseâproducing clones, and into the expression of their ÎČ-lactamases.The first part of this work was dedicated to the in vivo evolution of a single strain of KPC-producing K. pneumoniae ST258 that colonized a patient for almost 5 years. Genomic comparison of 17 isolates revealed a remarkable diversification with occurrence of several mutations with impact on bacterial virulence and susceptibility to antibiotics.Several studies extensively described the genetic structures of blaKPC-carrying plasmids, but information regarding gene expression at the whole plasmid level are lacking. Accordingly, we performed RNA-seq on Escherichia coli TOP10 transformed with an IncFIIk-IncFI blaKPC-2-carrying plasmid, with or without imipenem exposure. In both conditions, plasmid-encoded genes related to antimicrobial resistance and involved in plasmid replication were the most expressed. Imipenem exposure led to a more general response with overexpression of E. coli numerous chromosome-encoded genes involved in oxidative stress response. In addition, analysis of blaKPC-2 gene expression in several species using 5âRACE revealed the presence of several promoters whose strength depends on the bacterial genetic background. This could promote higher expression of blaKPC-2 gene and explain the association of some isoforms of Tn4401 in different species. The tools developed in the frame of this work were also applied to study a single Enterobacter kobei ST125 clone whose natural cephalosporinase (ACT-28) has increased hydrolytic activity towards imipenem. Finally, genomic analysis of the first ESBL-producing Shewanella sp. was performed. It revealed the presence of blaCTX-M-15 and blaSHV-2 genes carried on an IncA/C plasmid and a new chromosomally-encoded oxacillinase variant of OXA-48 with carbapenemase activity, called OXA-535. OXA-535 was found to be closely related to OXA-436, another carbapenemase which has recently spread in Enterobacteriaceae. Analysis of the genetic environment of both blaOXA-48-like genes confirmed the role of Shewanella spp. as progenitors of class D carbapenemases.Overall, this work contributes to a better comprehension of the diffusion of multi-drug resistant clones and of the mechanisms implicated in ÎČ-lactamase expression.Le combat contre les infections bactĂ©riennes reste un enjeu majeur de santĂ© publique notamment avec la dissĂ©mination des EntĂ©robactĂ©ries productrices de carbapĂ©nĂšmases, capables dâhydrolyser lâensemble des ÎČ-lactamines. On assiste Ă lâĂ©mergence de certains clones Ă©pidĂ©miques, se distinguant par leur distribution mondiale, leur forte transmissibilitĂ© et leur capacitĂ© Ă persister chez les patients. Lâexemple le plus parlant est le cas du clone de Klebsiella pneumoniae appartenant au « sequence type » (ST) 258 et responsable de la diffusion mondiale de la carbapĂ©nĂšmase KPC (Klebsiella Pneumoniae Carbapenemase) portĂ©e majoritairement par des plasmides de la famille InFIIk. Les raisons du succĂšs de ce clone et de lâassociation KPC/IncFIIk/ST258 ne sont pas encore totalement Ă©lucidĂ©es. Par ailleurs, il nâexiste pas de corrĂ©lation entre le niveau dâexpression dâune carbapĂ©nĂšmase, la sensibilitĂ© in vitro de la souche vis Ă vis des carbapĂ©nĂšmes et lâefficacitĂ© clinique dâun traitement par ces molĂ©cules. Les phĂ©nomĂšnes dâhĂ©terorĂ©sistance aux carbapĂ©nĂšmes sont frĂ©quents chez les souches produisant KPC, mais lâimpact clinique est inconnu. Les mĂ©canismes de rĂ©gulation de lâexpression des carbapĂ©nĂšmases ne sont pas Ă©lucidĂ©s.Les objectifs de cette thĂšse rĂ©sident dans lâanalyse des facteurs gĂ©nĂ©tiques associĂ©s Ă la diffusion et la persistance de clones multi-rĂ©sistants ainsi que lâanalyse de lâexpression des ÎČ-lactamases associĂ©es.La premiĂšre partie de ce travail porte sur lâanalyse de lâĂ©volution in vivo dâune souche de K. pneumoniae ST258 produisant KPC ayant persistĂ© chez un patient pendant prĂšs de 5 ans. Lâanalyse comparative des gĂ©nomes provenant de 17 isolats a permis de mettre en Ă©vidence une diversification gĂ©nĂ©tique importante ainsi que la sĂ©lection de mutations modifiant la virulence de la souche et sa sensibilitĂ© aux antibiotiques. Afin de caractĂ©riser les raisons du succĂšs de certains plasmides portant KPC, une analyse transcriptomique dâune souche de Escherichia coli TOP10 transformĂ©e par un plasmide multi-rĂ©plicon IncFIIk-IncFIB exprimant KPC-2, a Ă©tĂ© rĂ©alisĂ©e en prĂ©sence ou non dâimipĂ©nĂšme. Les gĂšnes les plus exprimĂ©s dans ces conditions sont les gĂšnes de rĂ©sistance aux antibiotiques et certains gĂšnes essentiels Ă la rĂ©plication du plasmide. La prĂ©sence dâimipĂ©nĂšme affecte peu la transcription des gĂšnes plasmidiques mais induit un stress oxydatif important dans lâensemble de la souche.Par ailleurs, lâanalyse de lâexpression du gĂšne blaKPC-2 dans diffĂ©rentes espĂšces par 5â-RACE a permis de rĂ©vĂ©ler que ce gĂšne de rĂ©sistance est sous la dĂ©pendance de plusieurs promoteurs, dont la force diffĂšre selon le fond gĂ©nĂ©tique. Cette caractĂ©ristique pourrait expliquer le succĂšs de certains isoformes du transposon Tn4401 permettant une meilleure expression du gĂšne blaKPC-2, dans certaines espĂšces.Les outils dĂ©veloppĂ©s dans cette thĂšse ont Ă©galement Ă©tĂ© appliquĂ©s Ă lâanalyse dâun clone dâEnterobacter kobei ST125 dont la cĂ©phalosporinase naturelle ACT-28 possĂšde une activitĂ© dâhydrolyse accrue vis Ă vis de lâimipĂ©nĂšme. Enfin, lâanalyse du gĂ©nome de la premiĂšre souche Shewanella sp. produisant une BLSE de type CTX-M-15 a permis de rĂ©vĂ©ler la prĂ©sence dâun nouveau variant oxacillinase chromosomique avec activitĂ© carbapĂ©nĂšmase, appelĂ© OXA-535. OXA-535 est proche dâOXA-436, un autre variant carbapĂ©nĂšmase portĂ© par un plasmide ayant dĂ©jĂ dissĂ©minĂ© chez les EntĂ©robactĂ©ries. Lâanalyse de lâenvironnement gĂ©nĂ©tique des gĂšnes blaOXA-535 et blaOXA-436 confirme le rĂŽle du genre Shewanella comme progĂ©niteur des carbapĂ©nĂšmases de classe D.Ce travail contribue Ă une meilleure comprĂ©hension de la diffusion de certains clones multi-rĂ©sistants et des mĂ©canismes contrĂŽlant lâexpression des gĂšnes de rĂ©sistance aux ÎČ-lactamines
Transcriptional Landscape of a bla KPC-2 Plasmid and Response to Imipenem Exposure in Escherichia coli TOP10
International audienceThe diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFII K plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFII K-IncFIB bla KPCâ2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance (bla KPCâ2 , bla TEM and aph(3 âČ)-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of E. coli (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the SoxRS/OxyR oxydative stress regulons, the Fur regulon (for iron uptake machinery), and IscR regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase mepM and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on E. coli (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of E. coli to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments
KPC-39-mediated resistance to Ceftazidime-Avibactam in a Klebsiella pneumoniae ST307 clinical isolate
International audienceResistance to ceftazidimeâavibactam (CAZ-AVI) combination is being increasingly reported. Here, we report a CAZ-AVI resistant Klebsiella pneumoniae belonging to the high-risk ST307 clone and producing KPC-39, a single amino-acid variant of KPC-3 (A172T). Cloning experiments, steady state kinetic parameters and molecular dynamics simulations revealed a loss of carbapenemase activity and an increased affinity for ceftazidime. KPC-39 was identified in a patient without prior exposure to CAZ-AVI, suggesting silent dissemination in European healthcare settings
Emergence of New NonâClonal Group 258 High-Risk Clones among Klebsiella pneumoniae CarbapenemaseâProducing K. pneumoniae Isolates, France
International audienceThe worldwide spread of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) isolates was reported to be caused by dissemination of 1 clonal complex (i.e., clonal group [CG] 258, which includes sequence types [STs] 258 and 512). We conducted whole-genome sequencing and epidemiologic analysis of all KPC-Kp isolates in France in 2018 and found that new successful high-risk clones of ST147, ST307, ST231, and ST383 are now the main drivers of blaKPC genes. The blaKPC genes were mostly carried by Tn4401a and Tn4401d structures and a new non-Tn4401 element. Our epidemiologic investigations showed that the emergence of these non-CG258 KPC-Kp isolates in France was linked to dissemination of these clones from Portugal. Thus, KPC-Kp epidemiology has changed in Europe, at least in several non-KPC-endemic countries of western Europe, such as France and Portugal, where CG258 is not the most prevalent clone
In Vitro Activity of Imipenem-Relebactam, Meropenem-Vaborbactam, Ceftazidime-Avibactam and Comparators on Carbapenem-Resistant Non-Carbapenemase-Producing Enterobacterales
Background: Avibactam, relebactam and vaborbactam are ÎČ-lactamase inhibitors that proved their efficiency against KPC-producing Enterobacterales. Regarding their inhibitor activity towards Amblerâs class A extended spectrum ÎČ-lactamases (ESBL) and Amblerâs class C cephalosporinase (AmpC), they should be active on most of the carbapenem-resistant non-carbapenemase-producing Enterobacterales (CR non-CPE). Objectives: Determine the in vitro activity of ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam and comparators against CR non-CPE. Methods: MICs to ceftazidime/avibactam, imipenem/relebactam, meropenem/vaborbactam, but also temocillin, ceftolozane/tazobactam, ertapenem, colistin, eravacycline and tigecycline were determined by broth microdilution (ThermoFisher) on a collection of 284 CR non-CPE (inhibition zone diameter < 22 mm to meropenem). Whole genome sequencing was performed on 90 isolates to assess the genetic diversity as well as resistome. Results: According to EUCAST breakpoints, susceptibility rates of ceftazidime, imipenem, meropenem and ertapenem used at standard dose were 0.7%, 45.1%, 14.8% and 2.5%, respectively. Increased exposure of ceftazidime, imipenem and meropenem led to reach 3.5%, 68.3% and 67.7% susceptibility, respectively. Using the EUCAST clinical breakpoints, susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam were 88.4%, 81.0% and 80.6%, respectively. Susceptibility rates of temocillin, ceftolozane/tazobactam, tigecycline, eravacycline, and colistin were 0%, 4.6%, 27.8%, 54.9% and 90.1%. MICs distributions with and without the presence of the inhibitor demonstrated a better ability of avibactam and relebactam compared to vaborbactam to restore susceptibility to the associated ÎČ-lactam. Conclusions: This study demonstrated the in vitro efficacy of ceftazidime/avibactam, imipenem/relebactam and to a lesser extent meropenem/vaborbactam against CR non-CPE. Moreover, to test all ÎČ-lactams/ÎČ-lactamases inhibitors combinations without a priori for CRE, non-CPE is crucial since resistance to one of the ÎČ-lactam/ÎČ-lactamase inhibitor combinations does not predict resistance to another molecule, depending on the resistance mechanisms involved
CTX-M-15-Producing Shewanella sp Clinical Isolate Expressing OXA-535, a Chromosome-Encoded OXA-48 Variant, Putative Progenitor of the Plasmid-Encoded OXA-436
International audienceShewanella spp. constitute a reservoir of antibiotic resistance determinants. In a bile sample, we identified three extended-spectrum-ÎČ-lactamase (ESBL)-producing bacteria (Escherichia coli, Klebsiella pneumoniae, and Shewanella sp. strain JAB-1) isolated from a child suffering from cholangitis. Our objectives were to characterize the genome and the resistome of the first ESBL-producing isolate of the genus Shewanella and determine whether plasmidic exchange occurred between the three bacterial species. Bacterial isolates were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), standard biochemical tools, and antimicrobial susceptibility testing. Shewanella sp. JAB-1 and ESBL gene-encoding plasmids were characterized using PacBio and Illumina whole-genome sequencing, respectively. The Shewanella sp. JAB-1 chromosome-encoded OXA-48 variant was cloned and functionally characterized. Whole-genome sequencing (WGS) of the Shewanella sp. clinical isolate JAB-1 revealed the presence of a 193-kb plasmid belonging to the IncA/C incompatibility group and harboring two ESBL genes, blaCTX-M-15 and blaSHV-2ablaCTX-M-15 gene-carrying plasmids belonging to the IncY and IncR incompatibility groups were also found in the E. coli and K. pneumoniae isolates from the same patient, respectively. A comparison of the blaCTX-M-15 genetic environment indicated the independent origin of these plasmids and dismissed in vivo transfers. Furthermore, characterization of the resistome of Shewanella sp. JAB-1 revealed the presence of a chromosome-carried blaOXA-535 gene, likely the progenitor of the plasmid-carried blaOXA-436 gene, a novel blaOXA-48-like gene. The expression of blaOXA-535 in E. coli showed the carbapenem-hydrolyzing activity of OXA-535. The production of OXA-535 in Shewanella sp. JAB-1 could be evidenced using molecular and immunoenzymatic tests, but not with biochemical tests that monitor carbapenem hydrolysis. In this study, we have identified a CTX-M-15-producing Shewanella species that was responsible for a hepatobiliary infection and that is likely the progenitor of OXA-436, a novel plasmid-encoded OXA-48-like class D carbapenemase