Emergence of resistance to polymyxins in Pseudomonas aeruginosa is mainly due to mutations in two-component systems that promote the addition of 4-amino-4-deoxy-l-arabinose to the lipopolysaccharide (LPS) through upregulation of operon arnBCADTEF-ugd (arn) expression. Here, we demonstrate that mutations occurring in different domains of histidine kinase PmrB or in response regulator PmrA result in coresistance to aminoglycosides and colistin. All seventeen clinical strains tested exhibiting such a cross-resistance phenotype were found to be pmrAB mutants. As shown by gene deletion experiments, the decreased susceptibility of the mutants to aminoglycosides was independent from operon arn but required the efflux system MexXY-OprM and the products of three genes, PA4773-PA4774-PA4775, that are cotranscribed and activated with genes pmrAB. Gene PA4773 (annotated as speD2 in the PAO1 genome) and PA4774 (speE2) are predicted to encode enzymes involved in biosynthesis of polyamines. Comparative analysis of cell surface extracts of an in vitro selected pmrAB mutant, called AB16.2, and derivatives lacking PA4773, PA4774, and PA4775 revealed that these genes were needed for norspermidine production via a pathway that likely uses 1,3-diaminopropane, a precursor of polyamines. Altogether, our results suggest that norspermidine decreases the self-promoted uptake pathway of aminoglycosides across the outer membrane and, thereby, potentiates the activity of efflux pump MexXY-OprM.
The immunochromatographic assay NG-Test Carba 5 (NG-Biotech) was evaluated with a collection of 107 carbapenemase-producing nonfermenters (CP-NF) (55 Pseudomonas spp., 51 Acinetobacter spp., and 1 Achromobacter xylosoxidans isolate) and 61 carbapenemase-negative isolates. All KPC, VIM, and NDM carbapenemase producers tested were accurately detected. Of the 16 IMP variants tested, 6 (37.5%) variants were not detected. Considering the epidemiology of CP-NFs in France, the NG-Test Carba 5 would detect 89.4% of CP Pseudomonas spp. but only 12.9% of CP Acinetobacter spp.
Resistance mechanisms of Pseudomonas aeruginosa to ceftolozane/tazobactam (C/T) were assessed on a collection of 420 nonredundant strains non-susceptible to ceftazidime (MIC > 8 μg/ml) and/or imipenem (> 4 μg/ml), collected by 36 French hospital laboratories over a one month period (GERPA study). Rates of C/T resistance (MIC > 4/4 μg/ml) were equal to 10% in this population (42/420 strains), and 23.2% among the isolates resistant to both ceftazidime and imipenem (26/112). A first group of 21 strains (50%) was found to harbor various extended-spectrum β-lactamases (1 OXA-14; 2 OXA-19; 1 OXA-35; 1 GES-9; 3 PER-1), carbapenemases (2 GES-5; 1 IMP-8; 8 VIM-2), or both (1 VIM-2/OXA-35; 1 VIM-4/SHV-2a). All the strains of this group belonged to widely distributed epidemic clones (ST111, ST175, CC235, ST244, ST348, ST654), and were highly resistant to almost all the antibiotics tested except colistin. A second group was composed of 16 (38%) isolates moderately resistant to C/T (MIC from 8/4 to 16/4 μg/ml), of which 7 were related to international clones (ST111, ST253, CC274, ST352, ST386). As demonstrated by targeted mass spectrometry, cloxacillin-based inhibition tests and gene blaPDC deletion experiments, this resistance phenotype was correlated to an extremely high production of cephalosporinase PDC. In part accounting for this strong PDC upregulation, genomic analyses revealed the presence of mutations in regulator AmpR (D135N/G in 6 strains) and enzymes of the peptidoglycan recycling pathway such as AmpD, PBP4, and Mpl (9 strains). Finally, all the 5 (12%) remaining C/T resistant strains (group 3) appeared to encode PDC variants with mutations known to improve the hydrolytic activity of the β-lactamase to ceftazidime and C/T (F147L, ΔL223-Y226, E247K, N373I). Collectively, our results highlight the importance of both intrinsic and transferable mechanisms in C/T resistant P. aeruginosa. Which mutational events lead some clinical strains to massively produce the natural cephalosporinase PDC remains incompletely understood.
Background Colistin resistance in Acinetobacter baumannii often results from mutational activation of the two-component system PmrAB and subsequent addition of phospho-ethanolamine (pEtN) to lipooligosaccharide by up-regulated pEtN transferase PmrC. Objectives To characterize mechanisms of colistin resistance independent of PmrCAB in A. baumannii. Methods Twenty-seven colistin-resistant A. baumannii were collected from 2012 to 2018. Analysis of operon pmrCAB was performed by PCR and sequencing. Seven strains were investigated further by WGS and whole-genome MLST (wgMLST). Results Seven out of the 27 selected isolates were found to overexpress eptA, a gene homologous to pmrC, likely as a consequence of upstream insertion of an ISAba1 element. Insertion sites of ISAba1 were mapped 13, 18 and 156 bp ahead of the start codon of eptA in five strains, one strain and one strain, respectively. The finding that the isolates did not cluster together when compared by wgMLST analysis supports the notion that distinct insertion events occurred in close, but different, genetic backgrounds. Conclusions Activation of eptA and subsequent addition of pEtN to the cell surface represents a novel mechanism of resistance to colistin in A. baumannii.
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 bla IMP–63 gene that encodes this carbapenemase turned out to be located in the variable region of four integrons (In 1297 , In 1574 , In 1573 , and In 1572 ) and to coexist with novel or rare gene cassettes ( fosM , gcu170 , gcuF1 ) and insertion elements (IS Psp7v , IS Pa16v ). All these integrons except one (In 1574 ) were flanked by a copy of insertion sequence IS Pa17 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 IS Pa17 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 bla IMP–63 , among Pseudomonas species.
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