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International Journal of Antimicrobial Agents 44 (2014) 345–350

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents

j o ur nal ho me pag e: ht tp : / /www.e lsev ier .com/ locate / i jant imicag

hort Communication

arbapenem resistance in a human clinical isolate identified to belosely related to Acinetobacter indicus

émy A. Bonnina, Laurent Poirela,b,∗, Tanny J.K. van der Reijdenc, Lenie Dijkshoornc,athilde Lescatd, Patrice Nordmanna,b

INSERM U914 ‘Emerging Resistance to Antibiotics’, K.-Bicêtre, FranceMedical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, SwitzerlandDepartment of Infectious Diseases, Leiden University Medical Center, Leiden, The NetherlandsService de Bactériologie-Virologie, Hôpital Avicenne, Bobigny, France

r t i c l e i n f o

rticle history:eceived 28 February 2014ccepted 21 May 2014

a b s t r a c t

Here we report a case of carbapenem resistance in a human clinical isolate that was found to be closelyrelated to the newly described environmental species Acinetobacter indicus. This strain harboured theblaOXA-23 carbapenemase gene located on a conjugative plasmid. Partial sequencing of 16S rDNA and rpoB

eywords:-LactamasecinetobacterXA-23arbapenemase

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 andwas closely related to A. indicus.

© 2014 Published by Elsevier B.V.

axonomy

. Introduction

The genus Acinetobacter currently comprises 30 validlyamed species (http://www.bacterio.net/). Acinetobacter spp. areidespread in nature and have been found in soil, wild rice, sewage,

nd human and animal samples. Acinetobacter baumannii, Acine-obacter nosocomialis (Acinetobacter genomic species 13TU) andcinetobacter pittii (Acinetobacter genomic species 3) are foundredominantly in clinical specimens [1–4]. They are responsi-le for nosocomial infections in critically ill patients and includeultidrug-resistant (MDR) epidemic strains [5]. One recently

escribed Acinetobacter spp., namely Acinetobacter indicus, haseen identified from a cyclohexane-containing dumpsite [3]. Car-apenem resistance has been increasingly reported in Acinetobacterpp. isolates, mainly in A. baumannii and closely related species (A.ittii and A. nosocomialis). This resistance trait is often related tohe biosynthesis of carbapenem-hydrolysing class D �-lactamases

CHDLs) that are disseminated worldwide [6]. OXA-23 is the mostommonly identified CHDL worldwide, and the corresponding geneay be found located either on plasmids or chromosomes and

∗ Corresponding author. Present address: Medical and Molecular Microbiologynit, Department of Medicine, Faculty of Science, University of Fribourg, Rue Albertockel 3, Fribourg, Switzerland. Tel.: +41 26 300 9580; fax: +41 33670023872.

E-mail address: laurent.poirel@unifr.ch (L. Poirel).

ttp://dx.doi.org/10.1016/j.ijantimicag.2014.05.022924-8579/© 2014 Published by Elsevier B.V.

may be associated either with ISAba1 or more rarely with ISAba4insertion sequences at the origin of its acquisition but also of itsexpression [6].

This study was initiated after the isolation of a carbapenem-resistant non-baumannii Acinetobacter sp. from a human clinicalsample.

2. Materials and methods

2.1. Bacterial strains

Isolate RAB1 was recovered from a patient previously hospi-talised in Algeria for a road traffic accident in August 2011. Hesuffered from a head trauma with meningeal haematoma plushead fracture. He was febrile and was transferred to a Frenchhospital. Empirical treatment with amoxicillin/clavulanic acid wasinitiated, but since the patient still had fever, treatment waschanged to cefotaxime 300 mg/kg/day after which the patientbecame afebrile. During hospitalisation, a carbapenem-resistantAcinetobacter sp. was detected from rectal swabs by routinelyimplemented screening. Other MDR Gram-negative isolates wererecovered from the same patient, including an OXA-48-producing

Escherichia coli and an OXA-48-producing Serratia marcescens, butno other Acinetobacter spp. was identified. Rifampicin-resistant A.baumannii BM4547 was used as recipient strain for conjugationexperiments.

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46 R.A. Bonnin et al. / International Journa

Isolate RAB1 was compared with strains from the collectionf Leiden University Medical Center (Leiden, The Netherlands),amely strains LUH05041, LUH05836, LUH08511, LUH08556 andUH10523.

.2. Susceptibility testing

The resistance phenotype was determined by the disc diffusionethod and was interpreted according to Clinical and Labora-

ory Standards Institute (CLSI) guidelines. Minimum inhibitoryoncentrations (MICs) of �-lactams were determined by EtestbioMérieux, La Balme-les-Grottes, France).

.3. Strain typing and clonal relationship

Identification of the isolates was performed using the API 20End API 32GN systems (bioMérieux, Marcy l’Étoile, France) accord-ng to the manufacturer’s recommendations. Further classification

as realised using molecular techniques. Sequence analysis ofhe 16S rDNA and rpoB (zones 1 and 2) genes was performeds described previously [8]. Characterisation of the strains wasompleted by matrix-assisted laser desorption/ionisation time-of-ight (MALDI-TOF) using an Axima apparatus (Shimadzu Biotech,arne-la-Vallée, France). The clonal relationship between A. indicus

solates was determined using DiversiLab® (bioMérieux) analysisccording to the manufacturer’s recommendations.

.4. PCR experiments and conjugation assays

Whole-cell DNA of Acinetobacter RAB1 was extracted asescribed previously [8] and was used as a template under standardCR conditions [8] with a series of primers designed for the detec-ion of class D carbapenemase genes, including blaOXA-23, blaOXA-40,laOXA-58 and blaOXA-143 [9]. Direct transfer of the ticarcillin resis-ance marker into A. baumannii BM4547 was attempted by liquid

ating-out assays at 37 ◦C and by electroporation of a plasmid DNAuspension as described previously [10]. Selection was performedn agar plates supplemented with 100 mg/L ticarcillin and 60 mg/Lifampicin.

.5. Nucleotide sequence accession nos.

The partial rpoB nucleotide sequences reported in thisork have been deposited in the GenBank nucleotide database

RAB1, KJ847272; LUH05041, KJ847273; LUH05836, KJ847274;UH08511, KJ847275; LUH08556, KJ847276; and LUH10523,J847277).

. Results and discussion

.1. Susceptibility testing

Isolate RAB1 was resistant to penicillins, penicillin/inhibitorombinations and carbapenems but remained susceptible to broad-pectrum cephalosporins, suggesting the production of a CHDL [9].ICs determined by Etest are summarised in Table 1. In addition,

solate RAB1 was resistant to gentamicin, tobramycin, chloram-henicol, fosfomycin and nitrofurantoin. Other A. indicus strains

ere susceptible to all �-lactams, fluoroquinolones, chlorampheni-

ol and aminoglycosides. All strains, including reference strain648T, were resistant to fosfomycin and nitrofurantoin, which isn intrinsic property of the Acinetobacter genus. Ta

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R.A. Bonnin et al. / International Journal of Antimicrobial Agents 44 (2014) 345–350 347

Fig. 1. Phylogenetic tree based on the two concatenated variable zones of the rpoB gene sequence of Acinetobacter indicus strains and representative members of the genusA ar, 0.0i

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cinetobacter. The tree was constructed using the maximum likelihood method. Bndicated in brackets.

.2. Phylogenetic analysis

Isolate RAB1 was initially identified using API 20NE and API2GN as Acinetobacter lwoffii with respective identification scores

f 99% and 75%. Assimilation testing of 32 carbohydrate substratesAPI 32GN) revealed that the organism could grow on sodiumcetate, lactate, l-alanine, valeric acid, 3-hydroxybutyrique acidnd l-proline. According to the manufacturer, isolates of the A.

2 nucleotide substitutions per site. GenBank accession nos. of rpoB sequences are

baumannii–calcoaceticus complex are supposed to use the samesubstrates, but they also grow on itaconic acid, malonate, d-glucose,arabinose, propionic acid, capric acid, citrate, histidine and 4-hydroxybenzoic acid. Noteworthy, isolate RAB1 grew at 37 ◦C but

also at 41 ◦C, similarly to A. baumannii, A. pittii and A. nosocomialisstrains [4].

To further classify isolate RAB1 at the species level, the rpoBsequence was obtained [7] and was compared with that of strains

348 R.A. Bonnin et al. / International Journal of Antimicrobial Agents 44 (2014) 345–350

Fig. 2. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis. Mass spectra of Acinetobacter indicus RAB1, A. indicus LUH5836, the referencestrain A. indicus A648T and two other spectrograms from distant Acinetobacter spp. are indicated.

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R.A. Bonnin et al. / International Journa

rom the collection of Leiden University Medical Center, some ofhem originating from animal samples. This revealed that iso-ate RAB1 was closely linked to a cluster of strains that alsoncluded the type strain of the A. indicus species (Fig. 1). Next,ve additional strains belonging to this cluster were comparedith this type strain using molecular methods. The concatenated

ones of rpoB showed intraspecies similarity values for the seventrains, ranging from 96.3% to 98.6%, and were at 100% for the6S rDNA sequence. Similarity between the concatenated rpoBequences of isolate RAB1 and other species ranged from <78.3%ith Acinetobacter haemolyticus to 86.1% with the closest Acineto-

acter genomic species 15TU. These data correspond to interspeciesnd intraspecies similarity values and highlight the close relation-hip of the six clinical and veterinary strains with A. indicus species,nd their distinctiveness from other described species of the Acine-obacter genus.

MALDI-TOF mass spectrometry analysis has been shown to be useful tool for identification of Acinetobacter spp. [11]. The pro-ein signatures of the six A. indicus were therefore compared withhe reference strain A. indicus A648T [3], but also with representa-ive Acinetobacter spp. strains belonging to different species. Peaknalysis of individual spectra from A. indicus strains and of severalcinetobacter spp. including A. baumannii, A. lwoffii, Acinetobac-er bereziniae and Acinetobacter junii indeed showed significantifferences between the protein signatures (Fig. 2). Whilst iso-

ate RAB1 and the five clinical/veterinary isolates from Leidenhared an identical spectrogram, those strains were different fromhe A. indicus type strain A648T and isolates of other speciesFig. 2). Thus, despite the close phylogenetic relatedness of thoseeven isolates (including RAB1) with strain A648T, significant dif-erences in the cellular make-up were revealed by MALDI-TOFnalysis.

We may conclude that the six human/veterinary isolates belongo a species closely related but distinct from A. indicus, whichas provisionally been named ‘A. indicus-like’. Of note, the def-

nition of A. indicus species is so far based on a single strain,hich makes the present interpretation rather difficult. Description

f single-strain species is increasingly common but also con-roversial in taxonomy since the intraspecies natural diversityemains unknown, complicating taxonomy issues as illustratedere [12].

.3. Identification of the ˇ-lactamases-encoding genes andenetic environment of the blaOXA-23 gene

PCR experiments followed by sequencing analysis identifiedhe blaOXA-23 carbapenemase gene in A. indicus-like RAB1. PCR foretection of ISAba1 upstream of this gene failed. Therefore, shotgunloning was performed to identify the genetic context of this car-apenemase gene. A copy of ISAba4 was identified upstream of thelaOXA-23 gene, corresponding to the previously identified transpo-on Tn2007 [13]. This transposon has been rarely described, beingound in A. baumannii species only, respectively, in Belgium andlgeria [13,14]. Downstream of the blaOXA-23 gene, a fragment of

hypothetical ATPase encoding gene followed by the mobA geneere identified, as previously described [11].

Mating-out assays and electrotransformation were used toetermine the genetic support of the blaOXA-23 gene [10]. Thesexperiments revealed that the blaOXA-23 gene was located on aonjugative plasmid that did not carry any additional resistanceenes.

.4. Clonal relationship of the A. indicus-like isolates

Results showed that the carbapenem-resistant isolate RAB1as not related to the five human/veterinary Dutch isolates

[

timicrobial Agents 44 (2014) 345–350 349

(Supplementary Fig. S1) or to the environmental Indian (type)strain (Table 1).

4. Conclusion

This report constitutes the first description of carbapenemresistance in a group of A. indicus-like strains originating fromdiverse geographical locations. A. indicus has been first describedas an environmental species isolated from a toxic site, the otherisolates being associated with human and veterinary specimens,including a blood culture (Table 1). The difficulty in discriminat-ing several species of Acinetobacter leads to misinterpretationin their identification. Although the A. baumannii–calcoaceticuscomplex remains the main complex associated with humaninfection/colonisation, use of MALDI-TOF and genetic tools foridentification permits a better discrimination at the species leveland reveals that non-baumannii Acinetobacter spp. are morefrequently associated with infections than expected, as previouslyhighlighted [11]. Finally, this study further underlines the spreadof the blaOXA-23 gene, originating from the genome of Acinetobacterradioresistens [15], among other Acinetobacter spp.

Funding: This work was partially funded by a grant from theINSERM (UMR914) (K.-Bicêtre, France), by the Université Paris-Sud (Orsay, France) and by grants from the European Community[R-GNOSIS, FP7/HEALTH-F3-2011-282512 and MAGIC-BULLET,FP7/HEALTH-F3-2001-278232].

Competing interests: None declared.Ethical approval: Not required.

Appendix A. Supplementary data

Supplementary material related to this article can be found,in the online version, at http://dx.doi.org/10.1016/j.ijantimicag.2014.05.022.

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