Rémy A. Bonnin

Analysis of the Resistome of a Multidrug-Resistant NDM-1-Producing Escherichia coli Strain by High-Throughput Genome Sequencing

ABSTRACT The resistome of the multidrug-resistant Escherichia coli strain 271 carrying the plasmid-mediated blaNDM-1 carbapenemase gene was analyzed by high-throughput genome sequencing. The p271A plasmid carrying the blaNDM-1 gene was 35.9 kb in size and possessed an IncN-type backbone that harbored a novel replicase gene. Acquisition of the blaNDM-1 gene on plasmid p271A had been likely the result of a cointegration event involving the transposase of Tn5403. The expression of blaNDM-1 was associated with the insertion sequence ISAba125 likely originating from Acinetobacter baumannii. E. coli 271 accumulated multiple resistance determinants, including five β-lactamase genes (comprising the extended-spectrum β-lactamase CTX-M-15), two 16S RNA methylase ArmA- and RmtB-encoding genes, and the qepA gene encoding an efflux pump involved in resistance to fluoroquinolones. These resistance genes were located on three additional plasmids, of 160 kb (IncA/C), 130 kb (IncF), and 110 kb (IncI1). In addition, several chromosomally encoded resistance determinants were identified, such as topoisomerase mutations, porin modifications and truncations, and the intrinsic ampC gene of E. coli that was weakly expressed. The multidrug resistance pattern observed for E. coli 271 was therefore the result of combined chromosome- and plasmid-encoded mechanisms.

Genetic basis of antibiotic resistance in pathogenic Acinetobacter species

Antibiotic resistance in Acinetobacter spp., particularly Acinetobacter baumannii, is increasing rapidly. A. baumannii possesses two intrinsic β‐lactamase genes, in addition to weak permeability and efflux systems, that together confer a natural reduced susceptibility to antibiotics. In addition, numerous acquired mechanisms of resistance have been identified in A. baumannii. The very high genetic plasticity of A. baumannii allows an accumulation of resistance determinants that give rise to multidrug resistance at an alarming rate. The role of novel genetic elements, such as resistance islands, in concentrating antibiotic resistance genes in A. baumannii requires detailed investigation in the near future.

Outbreak of NDM-1-producing Acinetobacter baumannii in France, January to May 2013

We report the first outbreak of carbapenem-resistant NDM-1-producing Acinetobacter baumannii in Europe, in a French intensive-care unit in January to May 2013. The index patient was transferred from Algeria and led to the infection/colonisation of five additional patients. Concurrently, another imported case from Algeria was identified. The seven isolates were genetically indistinguishable, belonging to ST85. The bla(NDM-1) carbapenemase gene was part of the chromosomally located composite transposon Tn125. This report underscores the growing concern about the spread of NDM-1-producing A. baumannii in Europe.

Identification of the naturally occurring genes encoding carbapenem-hydrolysing oxacillinases from Acinetobacter haemolyticus, Acinetobacter johnsonii, and Acinetobacter calcoaceticus

Carbapenem resistance is increasingly being reported among Acinetobacter species, and results mostly from the expression of acquired carbapenem-hydrolysing oxacillinases (CHDLs). Several Acinetobacter species intrinsically possess chromosomal CHDL genes: Acinetobacter baumannii (bla(OXA-51) ), Acinetobacter radioresistens (bla(OXA-23) ), and Acinetobacter lwoffii (bla(OXA-134) ). We aimed to identify the progenitors of novel CHDL-encoding genes for identification of potential reservoirs. We performed PCR screening using degenerated internal primers designed from a sequence alignment of the known CHDLs (OXA-23, OXA-40, OXA-51, OXA-58, OXA-134, and OXA-143) applied to a collection of 50 Acinetobacter strains belonging to 23 different species. Two strains of Acinetobacter johnsonii, one strain of Acinetobacter calcoaceticus and two strains of Acinetobacter haemolyticus were found to harbour, respectively, the totally novel bla(OXA-211) -like, bla(OXA-213) -like and bla(OXA-214) -like genes. In addition, the complete genomes of those three species available in GenBank, i.e. one A. johnsonii genome, four A. calcoaceticus genomes, and one A. haemolyticus genome, were analysed and found to be positive for the presence of bla(OXA211) -like, bla(OXA-213) -like and bla(OXA-214) -like genes, respectively. The β-lactamases OXA-211, OXA-213 and OXA-214 are diverse, with amino acid identities ranging from 53% to 76%, as compared with the naturally occurring OXA-51-like CHDL from A. baumannii. These β-lactamases showed a peculiar hydrolysis profile, including mostly penicillins and carbapenems. Regarding bla(OXA-23) in A. radioresistens and bla(OXA-134) in A. lwoffii, these genes were not expressed (or expressed at a non-significant level) in their host. Detection of these β-lactamase genes might be used as a useful tool for accurate identification of these Acinetobacter species.

New Delhi metallo-β-lactamase-producing Acinetobacter baumannii : a novel paradigm for spreading antibiotic resistance genes

The impact of carbapenemase production among clinically significant Gram-negative rods is becoming a major medical issue. To date, Acinetobacter baumannii has been considered as a final recipient of carbapenemase genes (imipenemase, Verona metallo-β-lactamase, Guiana extended-spectrum β-lactamase and Klebsiella pneumonia carbapenemase types) from Enterobacteriaceae and Pseudomonas aeruginosa. However, recent findings regarding the spread of the blaNDM carbapenemase genes revealed that A. baumannii likely acts as a source of emerging antibiotic resistance genes. The analysis of genetic structure surrounding the blaNDM-1 gene revealed that the genetic structure (Tn125) responsible for its dissemination most probably originates from Acinetobacter. Moreover, analysis of the blaNDM-1 gene itself demonstrated that it might be constructed in Acinetobacter through a recombination event with another resistance gene found in A. baumannii (aphA6). This novel paradigm highlights a novel and unexpected role played by A. baumannii.

Characterization of a multidrug-resistant Acinetobacter baumannii strain carrying the blaNDM-1 and blaOXA-23 carbapenemase genes from the Czech Republic

Sir, Since its discovery in 2008, the metallo-b-lactamase (MBL) NDM-1 has been identified in different Enterobacteriaceae species and recently also in a number of other bacterial species isolated from water supplies in India, such as Vibrio cholerae and Pseudomonas spp. Genes encoding NDM b-lactamases have been additionally identified in multidrugresistant Acinetobacter spp. Here, we report on the identification and characterization of an Acinetobacter baumannii strain carrying the blaNDM-1 gene that was isolated in the Czech Republic in 2011. A. baumannii strain ANC 4097 was isolated in mid-2011 during a prospective, nationwide study in the Czech Republic focused on the Acinetobacter population structure and resistance mechanisms. It was recovered from the sputum of an elderly patient hospitalized in an intensive care unit (ICU) of a hospital located in the north-western part of the Czech Republic. The patient, who had a malignant tumour, died of complications of the underlying disease several days after the isolation of the strain. The MICs of b-lactams for ANC 4097 determined using Etest (bioMérieux, Solna, Sweden) showed high-level resistance to all b-lactams, including carbapenems (MICs of both imipenem and meropenem .32 mg/L) and broad-spectrum cephalosporins (MICs of both ceftazidime and cefepime .256 mg/L), as well as penicillins in combination with inhibitors (MICs of ampicillin/sulbactam and piperacillin/tazobactam .256 mg/L). The strain was also resistant to fluoroquinolones (MIC of ciprofloxacin .32 mg/L), aminoglycosides (MIC of amikacin .64 mg/L and MIC of gentamicin 32 mg/L) and tetracycline (MIC 128 mg/L). An MBL Etest (bioMérieux) revealed a 24-fold reduction of imipenem MIC when combined with EDTA, which suggested production of an MBL. Isolate ANC 4097 remained susceptible only to tigecycline (MIC 2 mg/L), doxycycline (MIC 2 mg/L), netilmicin (MIC 4 mg/L), tobramycin (MIC 0.5 mg/L) and colistin (MIC 0.25 mg/L). Strain ANC 4097 was genotyped by multilocus sequence typing (MLST) using the Pasteur Institute scheme (http://www.pasteur.fr/ recherche/genopole/PF8/mlst/). It belongs to sequence type (ST) 1 (allelic profile 1-1-1-1-5-1-1), which is typical of the European clone I epidemic lineage. PCR screening followed by sequencing of resistance determinants revealed that the strain harboured genes encoding five b-lactamases: NDM-1, class D carbapenemases OXA-23 (acquired) and OXA-69 (intrinsic), acquired narrowspectrum class A b-lactamase TEM-1 and intrinsic AmpC-type b-lactamase ADC. This strain co-harboured the tetA(A) tetracycline resistance gene and the aacC1, aphA1, aphA6 and aadA1 aminoglycoside resistance genes, but no armA 16S RNA methylase gene was identified. Insertion sequence ISAba1 was detected upstream of each of blaOXA-23, blaOXA-69 and blaADC, thus providing strong promoter sequences likely to enhance the expression of these genes in ANC 4097. In addition, the comM gene (encoding a polypeptide containing an ATPase domain) was found to be disrupted, which, together with the presence of the blaTEM-1, aacC1, aphA1, aadA1 and tetA(A) genes, suggested the presence of an AbaR3-like genomic resistance island typical of the European clone I lineage. Genetic structures associated with the blaOXA-23 and blaNDM-1 genes were analysed using PCR mapping and sequencing as described elsewhere. The blaNDM-1 gene was located in the previously characterized composite transposon Tn125 (two copies of ISAba125 bracketing a 7925 bp region), while the blaOXA-23 gene was found to be part of transposon Tn2008. In order to determine the genetic location of the blaOXA-23 and blaNDM-1 genes, plasmid DNA of ANC 4097 was isolated as described previously. Transfer of the ticarcillin resistance marker was attempted by both electroporation of the ANC 4097 plasmid suspension into A. baumannii BM4547 and liquid mating-out assays of the ANC 4097 and BM4547 strains at 378C. Selection was performed on agar plates supplemented with ticarcillin (100 mg/L). Transformants or transconjugants harbouring the blaNDM-1 and blaOXA-23 genes were not obtained, suggesting a chromosomal location of both genes, as described previously. This study reports on the first blaNDM-1-positive A. baumannii strain in the Czech Republic and adds to the body of evidence of the current spread of multidrug-resistant Acinetobacter harbouring this MBL in Europe.

Emergence of OXA-72-producing Acinetobacter pittii clinical isolates.

The genus Acinetobacter comprises 47 characterised genomic species, among which species belonging to the Acinetobacter calcoaceticus–Acinetobacter baumannii complex are the most clinically relevant. Within this complex, A. baumannii, Acinetobacter nosocomialis (formerly genomic species 13TU) and Acinetobacter pittii (formerly genomic species 3) are frequently associated with hospital-acquired infections [1]. Carbapenem resistance is being increasingly reported in Acinetobacter spp. isolates and this resistance trait is often related to the production of acquired carbapenem-hydrolysing class D -lactamases (CHDLs) that are disseminating worldwide [2]. Five groups of acquired CHDLs have been identified to date in A. baumannii, namely OXA-23, OXA24/-40, OXA-58, OXA-143 and OXA-235 [2]. OXA-72 is a point mutant of OXA-40 that was first described in carbapenem-resistant A. baumannii clinical isolates in China [2]. It was then reported in Colombia from a clinical isolate (A. pittii 2688), which has been used here as a reference strain [3]. This study was initiated by the isolation of three imipenemnon-susceptible Acinetobacter spp. isolates recovered in three hospitals in France in 2011–2013. Isolate RA1 was from the sputum of a patient hospitalised in November 2011, isolate RA2 was from pus of an 84-year-old patient in December 2011, and isolate RA3 was recovered after rectal screening of a 56-year-old patient in May 2013. These isolates were resistant to penicillins and penicillin–inhibitor combinations and were of intermediate susceptibility to carbapenems according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (http://www.eucast.org/clinical breakpoints/). By contrast, they remained susceptible to ceftazidime and cefepime as well as to amikacin, rifampicin, colistin, fluoroquinolones, tetracycline and tigecycline according to the EUCAST guidelines. The isolates were identified using the API32GN system (bioMérieux, Marcy l’Étoile, France), partial sequencing of their 16S rDNA genes and matrix-assisted laser desorption/ionisation time-of-flight mass (MALDI-TOF) analysis. Identification results showed that the three Acinetobacter spp. strains belonged to the A. pittii species. Since the resistance phenotype to -lactams suggested the production of a CHDL, corresponding genes were searched by PCR as described previously [2]. Interestingly, PCR followed by sequencing analysis identified the blaOXA-72 gene in the three isolates. To determine the genetic location of the blaOXA-72 gene, transfer of the ticarcillin resistance marker into A. baumannii BM4547 was attempted by liquid mating-out assays at 37 ◦C and by electrotransformation of a plasmid DNA suspension extracted from the three clinical isolates and the reference strain (A. pittii 2688). Conjugation remained unsuccessful; nevertheless, transformants were obtained for the three clinical isolates and the reference strain, revealing Fig. 1. Results of DiversiLab (bioMérieux, La Balme-les-Grottes, France) analysis. The horizontal similarity line showed the cut-off to separate different clones.

GES-type and OXA-23 carbapenemase-producing Acinetobacter baumannii in Turkey

INSERM U914 ‘Emerging Resistance to Antibiotics’, Le KremlinBicetre, France; Department of Infectious Diseases and Clinical Microbiology, Ege University Medical Faculty, Izmir, Turkey; Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland; Department of Medical Microbiology, Ege University Medical Faculty, Izmir, Turkey; Intensive Care and Reanimation Unit, Ege University Medical Faculty, Izmir, Turkey

Carbapenem resistance in a human clinical isolate identified to be closely related to Acinetobacter indicus

Here we report a case of carbapenem resistance in a human clinical isolate that was found to be closely related to the newly described environmental species Acinetobacter indicus. This strain harboured the blaOXA-23 carbapenemase gene located on a conjugative plasmid. Partial sequencing of 16S rDNA and rpoB genes, together with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) analysis, showed that this strain was distantly related to the Acinetobacter baumannii-calcoaceticus complex and was closely related to A. indicus.