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 blaOXA-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 blaOXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M blaOXA-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

Horizontal gene transfer mediated bacterial antibiotic resistance

Bacterial antibiotic resistance, especially multidrug resistance (MDR), has become a global challenge, threatening human and animal health, food and environment safety. The number of human deaths accounted for by multidrug resistance (MDR) was estimated to increase to 10 million by 2050, exceeding the number of deaths arising from cancer (WHO, 2014). Although a bacterium is able to establish antibiotic resistance through spontaneous mutation (Salverda et al., 2017), development of MDR in the bacterium would take a long time if it only relies on self-adaptive mutation. Horizontal gene transfer (HGT) allows bacteria to exchange their genetic materials (including antibiotic resistance genes, ARGs) among diverse species (Le Roux and Blokesch, 2018), greatly fostering collaboration among bacterial population in MDR development. Recent studies reveal emergence of ‘superbugs’ that carry a number of HGT-transferred ARGs on plasmids and tolerate almost all antibiotics (Mathers et al., 2015; Wang and Sun, 2015; Malhotra-Kumar et al., 2016). These MDR plasmids are able to be further transferred to different bacterial species, creating new ‘superbugs’ that grow in different environments. Global emergence of ‘superbugs’ carrying MDR plasmids (e. g. NDM-1 and MCR-1) in various environmental niches (e. g. patients, animals and soil) indicates rapid propagation of MDR among bacterial populations. Although HGT and MDR were found to be tightly linked in ‘superbugs’, as revealed by surveillance studies, our knowledge about how and to what extend HGT propels development of MDR under different environmental conditions remain inadequate. The 22 publications collected in the topic “Horizontal Gene Transfer Mediated Bacterial Antibiotic Resistance” show new discoveries and recent advances concerning this issue in a wide range of fields, providing a basis for collaboratively controlling MDR in the future

Identification of Diverse Integron and Plasmid Structures Carrying a Novel Carbapenemase Among Pseudomonas Species

A novel carbapenem-hydrolyzing beta-lactamase, called IMP-63, was identified in three clonally distinct strains of Pseudomonas aeruginosa and two strains of Pseudomonas putida isolated within a 4 year timeframe in three French hospitals. The blaIMP–63 gene that encodes this carbapenemase turned out to be located in the variable region of four integrons (In1297, In1574, In1573, and In1572) and to coexist with novel or rare gene cassettes (fosM, gcu170, gcuF1) and insertion elements (ISPsp7v, ISPa16v). All these integrons except one (In1574) were flanked by a copy of insertion sequence ISPa17 next to the orf6 putative gene, and were carried by non-conjugative plasmids (pNECK1, pROUSS1, pROUSS2, pROUE1). These plasmids exhibit unique modular structures and partial sequence homologies with plasmids previously identified in various non-fermenting environmental Gram-negative species. Lines of evidence suggest that ISPa17 promoted en bloc the transposition of IMP-63-encoding integrons on these different plasmids. As demonstrated by genotyping experiments, isolates of P. aeruginosa harboring the 28.9-kb plasmid pNECK1 and belonging to international “high-risk” clone ST308 were responsible for an outbreak in one hospital. Collectively, these data provide an insight into the complex and unpredictable routes of diffusion of some resistance determinants, here blaIMP–63, among Pseudomonas species

Genomic characterization of an NDM-9-producing Acinetobacter baumannii clinical isolate and role of Glu152Lys substitution in the enhanced cefiderocol hydrolysis of NDM-9

Here, we characterized the first French NDM-9-producing Acinetobacter baumannii isolate. A. baumannii 13A297, which belonged to the STPas25 (international clone IC7), was highly resistant to β-lactams including cefiderocol (MIC >32 mg/L). Whole genome sequencing (WGS) using both Illumina and Oxford Nanopore technologies revealed a 166-kb non-conjugative plasmid harboring a blaNDM-9 gene embedded in a Tn125 composite transposon. Complementation of E. coli DH5α and A. baumannii CIP70.10 strains with the pABEC plasmid carrying the blaNDM-1 or blaNDM-9 gene, respectively, resulted in a significant increase in cefiderocol MIC values (16 to >256-fold), particularly in the NDM-9 transformants. Interestingly, steady-state kinetic parameters, measured using purified NDM-1 and NDM-9 (Glu152Lys) enzymes, revealed that the affinity for cefiderocol was 3-fold higher for NDM-9 (Km = 53 μM) than for NDM-1 (Km = 161 μM), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069 μM−1.s−1, for NDM-9 and NDM-1, respectively). Finally, we showed by molecular docking experiments that the residue 152 of NDM-like enzymes plays a key role in cefiderocol binding and resistance, by allowing a strong ionic interaction between the Lys152 residue of NDM-9 with both the Asp223 residue of NDM-9 and the carboxylate group of the R1 substituent of cefiderocol

Drug susceptibility testing by dilution methods.

International audienceSerial twofold dilution methods are widely used to assess the bacteriostatic activities of antibiotics. This can be achieved by dilution of considered drugs in agar medium or in culture broth, and inoculation by calibrated inoculums. Although seemingly simple, these methods are greatly influenced by the experimental conditions used and may lead to discrepant results, in particular with untrained investigators. The present step-by-step protocol has been validated for Pseudomonas species, including P. aeruginosa. Introduction of appropriate control strains is crucial to ascertain minimal inhibitory concentration values and compare the results of independent experiments

Mutations in gene fusA1 as a novel mechanism of aminoglycoside resistance in clinical strains of Pseudomonas aeruginosa.

International audienceResistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). The present study reports on characterization of a new type of mutants 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, tobramycin), as compared with wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1, and found to result in single amino acid substitutions in domains II, III and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) was found to partly contribute to the resistance conferred by the mutated EF-G1A variants, and to have additive effects on aminoglycosides MICs when mutationally up-regulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides, via a new mechanism involving mutations in elongation factor EF-G1A

A Two-Component Regulatory System Interconnects Resistance to Polymyxins, Aminoglycosides, Fluoroquinolones, and β-Lactams in Pseudomonas aeruginosa▿

Constitutive overexpression of the active efflux system MexXY/OprM is a major cause of resistance to aminoglycosides, fluoroquinolones, and cefepime in clinical strains of Pseudomonas aeruginosa. Upregulation of this pump often results from mutations occurring in mexZ, the local repressor gene of the mexXY operon. In this study, analysis of MexXY-overproducing mutants selected in vitro from reference strain PAO1Bes on amikacin (at a concentration 1.5-fold higher than the MIC) led to identification of a new class of mutants harboring an intact mexZ gene and exhibiting increased resistance to colistin and imipenem in addition to aminoglycosides, fluoroquinolones, and cefepime. Reverse transcription-quantitative PCR (RT-qPCR) experiments on a selected clone named PAOW2 demonstrated that mexXY overexpression was independent of mexZ and the PA5471 gene, which is required for drug-dependent induction of mexXY. Furthermore, the transcript levels of the oprD gene, which encodes the carbapenem-selective porin OprD, were found to be reduced drastically in PAOW2. Whole-genome sequencing revealed a single mutation resulting in an M59I substitution in the ParR protein, the response regulator of the ParRS two-component regulatory system (with ParS being the sensor kinase), which is required for adaptive resistance of P. aeruginosa to polycationic peptides such as colistin. The multidrug resistance phenotype was suppressed in PAOW2 by deletion of the parS and parRS genes and conferred to PAO1Bes by chromosomal insertion of the mutated parRS locus from PAOW2. As shown by transcriptomic analysis, only a very small number of genes were expressed differentially between PAOW2 and PAO1Bes, including the lipopolysaccharide (LPS) modification operon arnBCADTEF-ugd, responsible for resistance to polycationic agents. Exposure of wild-type PAO1Bes to different polycationic peptides, including colistin, was shown to result in increased mexY and repressed oprD expression via ParRS, independent of PA5471. In agreement with these results, colistin antagonized activity of the MexXY/OprM substrates in PAO1Bes but not in a ΔparRS derivative. Finally, screening of clinical strains exhibiting the PAOW2 resistance phenotype allowed the identification of additional alterations in ParRS. Collectively, our data indicate that ParRS may promote either induced or constitutive multidrug resistance to four different classes of antibiotics through the activation of three distinct mechanisms (efflux, porin loss, and LPS modification)

Novel Synthesis and Characterization of Doxycycline-Loaded Gold Nanoparticles: The Golden Doxycycline for Antibacterial Applications

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Effet du nouvel activateur transcriptionnel CmrA sur la résistance et la virulence des souches de Pseudomonas aeruginosa

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Editorial: Horizontal Gene Transfer Mediated Bacterial Antibiotic Resistance

International audienceFour sequence type 664 (ST664) (serotype O:5) strains of Pseudomonas aeruginosa that were highly resistant to antibiotics, including ceftolozane-tazobactam and ceftazidime-avibactam, but were susceptible to colistin were found to harbor the gene encoding the rare class C β-lactamase PAC-1 on a chromosomally located Tn 1721 -like transposon. The bla PAC-1 gene was associated with the 16S rRNA methylase determinant rmtF2 , which confers pan-aminoglycoside resistance