Derepressed transfer properties leading to the efficient spread of the plasmid encoding carbapenemase OXA-48

The current emergence of the carbapenemase OXA-48 among Enterobacteriaceae is related to the spread of a single IncL/M-type plasmid, pOXA-48a. This plasmid harbors the blaOXA-48 gene within a composite transposon, Tn1999, which is inserted into the tir gene, encoding a transfer inhibition protein. We showed that the insertion of Tn1999 into the tir gene was involved in a higher transfer frequency of plasmid pOXA-48a. This may likely be the key factor for the successful dissemination of this plasmid

Emerging broad-spectrum resistance in Pseudomonas aeruginosa and Acinetobacter baumannii: Mechanisms and epidemiology

Multidrug resistance is quite common among non-fermenting Gram-negative rods, in particular among clinically relevant species including Pseudomonas aeruginosa and Acinetobacter baumannii. These bacterial species, which are mainly nosocomial pathogens, possess a diversity of resistance mechanisms that may lead to multidrug or even pandrug resistance. Extended-spectrum β-lactamases (ESBLs) conferring resistance to broad-spectrum cephalosporins, carbapenemases conferring resistance to carbapenems, and 16S rRNA methylases conferring resistance to all clinically relevant aminoglycosides are the most important causes of concern. Concomitant resistance to fluoroquinolones, polymyxins (colistin) and tigecycline may lead to pandrug resistance. The most important mechanisms of resistance in P. aeruginosa and A. baumannii and their most recent dissemination worldwide are detailed here

Characterisation of OXA-244, a chromosomally-encoded OXA-48-like β-lactamase from Escherichia coli

International audienceABSTRACT Shewanella spp. constitute a reservoir of antibiotic resistance determinants. In a bile sample, we identified three extended-spectrum-β-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, bla CTX-M-15 and bla SHV-2a . bla CTX-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 bla CTX-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 bla OXA-535 gene, likely the progenitor of the plasmid-carried bla OXA-436 gene, a novel bla OXA-48 -like gene. The expression of bla OXA-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

OXA β-lactamases

The OXA β-lactamases were among the earliest β-lactamases detected; however, these molecular class D β-lactamases were originally relatively rare and always plasmid mediated. They had a substrate profile limited to the penicillins, but some became able to confer resistance to cephalosporins. From the 1980s onwards, isolates of Acinetobacter baumannii that were resistant to the carbapenems emerged, manifested by plasmid-encoded β-lactamases (OXA-23, OXA-40, and OXA-58) categorized as OXA enzymes because of their sequence similarity to earlier OXA β-lactamases. It was soon found that every A. baumannii strain possessed a chromosomally encoded OXA β-lactamase (OXA-51-like), some of which could confer resistance to carbapenems when the genetic environment around the gene promoted its expression. Similarly, Acinetobacter species closely related to A. baumannii also possessed their own chromosomally encoded OXA β-lactamases; some could be transferred to A. baumannii, and they formed the basis of transferable carbapenem resistance in this species. In some cases, the carbapenem-resistant OXA β-lactamases (OXA-48) have migrated into the Enterobacteriaceae and are becoming a significant cause of carbapenem resistance. The emergence of OXA enzymes that can confer resistance to carbapenems, particularly in A. baumannii, has transformed these β-lactamases from a minor hindrance into a major problem set to demote the clinical efficacy of the carbapenems

Environmental Shewanella xiamenensis strains that carry bla OXA-48 or blaOXA-204 genes: additional proof for bla OXA-48-Like gene origin

This work was supported by Fundação para a Ciência e a Tecnologia (FCT) through grants SFRH/BD/43468/2008 (M.T.) and SFRH/BPD/63487/2009 (I.H.) and by project Phytomarsh (PTDC/AAC-AMB/118873/2010).publishe

Horizontal transfer of antibiotic resistance genes on abiotic touch surfaces: implications for public health

Horizontal gene transfer (HGT) is largely responsible for increasing incidence of antibiotic-resistant infections worldwide. Whilst studies have focussed on HGT in vivo, this work investigates whether the ability of pathogens to persist in the environment, particularly on touch surfaces, may also play an important role. Escherichia coli, virulent clone ST131, and Klebsiella pneumoniae encoding extended-spectrum ?-lactamase (ESBL) bla CTX-M-15 and metallo-?-lactamase bla NDM-1, respectively, exhibited prolonged survival on stainless steel with approximately 104 viable cells remaining from an inoculum of 107cfu per cm2 after one month at 21oC. HGT of bla to an antibiotic-sensitive but azide-resistant recipient E. coli occurred on stainless steel dry touch surfaces and in suspension, but not on dry copper. Conjugation frequency was approximately 10-50 times greater, occurred immediately and resulting transconjugants were more stable with ESBL E. coli as donor cell compared to K. pneumoniae but bla NDM-1 transfer increased with time. Transconjugants also exhibited the same resistance profile as donor suggesting multiple gene transfer. Rapid death, inhibition of respiration and destruction of genomic and plasmid DNA of both pathogens occurred on copper alloys accompanied by a reduction in bla copy number. Naked E. coli DNA degraded on copper at 21oC and 37oC but slowly at 4oC suggesting a direct role for the metal. Persistence of viable pathogenic bacteria on touch surfaces may not only increase risk of infection transmission but also contribute to spread of antibiotic resistance by HGT. The use of copper alloys as antimicrobial touch surfaces may help reduce infection and HGT

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

Analysis of OXA-204 carbapenemase-producing <i>Enterobacteriaceae</i> reveals possible endoscopy-associated transmission, France, 2012 to 2014.

OXA-48-like beta-lactamase producing bacteria are now endemic in several European and Mediterranean countries. Among this carbapenemase family, the OXA-48 and OXA-181 variants predominate, whereas other variants such as OXA-204 are rarely reported. Here, we report the molecular epidemiology of a collection of OXA-204-positive enterobacterial isolates (n = 29) recovered in France between October 2012 and May 2014. This study describes the first outbreak of OXA-204-producing &lt;i&gt;Enterobacteriaceae&lt;/i&gt; in Europe, involving 12 isolates of an ST90 &lt;i&gt;Escherichia coli&lt;/i&gt; clone and nine isolates of an ST147 &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; clone. All isolates co-produced the cephalosporinase CMY-4, and 60% of them co-produced the extended-spectrum beta-lactamase CTX-M-15. The &lt;i&gt;bla&lt;/i&gt; &lt;sub&gt;OXA-204&lt;/sub&gt; gene was located on a 150-kb IncA/C plasmid, isolated from various enterobacterial species in the same patient, indicating a high conjugative ability of this genetic vehicle

Mechanistic and phenotypic studies of bicarinalin, BP100 and colistin action on Acinetobacter baumannii

Acinetobacter baumannii has been identified by the WHO as a high priority pathogen. It can be resistant to multiple antibiotics and colistin sulphate is often used as a last-resort treatment. However, the potentially severe side-effects of colistin are well documented and this study compared the bactericidal and anti-biofilm activity of two synthetic nature-inspired antimicrobial peptides, bicarinalin and BP100, with colistin. The minimum bactericidal concentration (MBC) against planktonic A. baumannii was approximately 0.5 μg/ml for colistin sulphate and ∼4 μg/ml for bicarinalin and BP100. A. baumannii commonly occurs as a biofilm and biofilm removal assay results highlighted that both bicarinalin and BP100 had significantly greater potential than colistin. Atomic force microscopy (AFM) showed dramatic changes in A. baumannii cell size and surface conformity when treated with peptide concentrations at and above the MBC. Scanning electron microscopy (SEM) visualised the reduction of biofilm coverage and cell surface changes as peptide concentration increased. Liposome assays revealed that these peptides most likely act as pore-forming agents in the membrane. Bicarinalin and BP100 may be effective therapeutic alternatives to colistin against A. baumannii infections but further research is required to assess if they elicit cytotoxicity issues in patients