Journal of Global Antimicrobial Resistance 2 (2014) 327–329

Short communication

Emergence of plasmid mediated carbapenemase OXA-48 in a Klebsiellapneumoniae strain in Algeria

Nadjet Aggoune a,*, Hassiba Tali-Maamar b, Farida Assaous b, Nabila Benamrouche b,Malek Naim a, Kheira Rahal b

a Laboratoire de Microbiologie, Hopital central de l’armee, Universite d’Alger, Faculte de medecine, laboratoire de microbiologie, Algiers, Algeriab Laboratoire de Bacteriologie medicale, Institut Pasteur d’Algerie, Universite d’Alger, Faculte de medecine, laboratoire de microbiologie, Algiers, Algeria

A R T I C L E I N F O

Article history:

Received 26 November 2013

Received in revised form 19 March 2014

Accepted 11 June 2014

Keywords:

Carbapenemase

OXA-48

Klebsiella pneumoniae

ST-307

Algeria

A B S T R A C T

A carbapenem resistant Klebsiella pneumoniae strain was isolated in October 2011 in the pediatric unit of

the Hopital central de l’armee in Algeria, this strain have been confirmed for the production of OXA-48

carbapenemase. The blaOXA-48 gene was located on a self conjugative plasmid of 70 kb. Multilocus

sequence typing indicated the presence of the sequence type ST-307. This is the first isolation of OXA-48-

producing K. pneumoniae in Algeria.

� 2014 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All

rights reserved.

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Journal of Global Antimicrobial Resistance

jo u rn al h om ep age: w ww.els evier .c o m/lo c ate / jg ar

1. Introduction

Carbapenems are usually the last resort to treat severeinfections caused by Gram negative bacteria including multi drugresistant strains. While still rare, Carbapenem resistance inEnterobacteriaceae is increasingly reported worldwide and is amatter of major clinical concern since it seriously compromises theuse of this class of antibiotics. Acquired carbapenemases inenterobacteriaceae represent an important resistance mechanism;these b-lactamases can belong to either Ambler class A, B or D [1].The Ambler class D b-lactamase OXA-48 hydrolyses penicillins andimipenem, but spares extended spectrum cephalosporins [2]. Thebla OXA-48 gene is part of the Tn1999 composite transposon carriedby a self-conjugative plasmid of 70-kb [2].

Since the first identification of the carbapenem hydrolyzingoxacillinase OXA-48 in a Klebsiella pneumoniae from Turkey in2004 [3], several other OXA-48 producing isolates have beenreported worldwide mainly throughout the Mediterranean regionthat became the most important reservoir. Many other strains have

* Corresponding author at: Laboratoire de Microbiologie, Hopital central de

l’armee, BP 244 Kouba, Algiers, Algeria. Tel.: +213 21 54 53 62;

fax: +213 21 54 52 38.

E-mail address: nadjetaggoune@yahoo.fr (N. Aggoune).

http://dx.doi.org/10.1016/j.jgar.2014.06.001

2213-7165/� 2014 International Society for Chemotherapy of Infection and Cancer. Pu

also been recently reported in several countries from Europe, Asia,Africa and further in America [4]. In Europe, the spread of OXA-48carbapenemase in K. pneumoniae is the fact of a clonal dissemina-tion involving the sequence type ST-395 [5]. Many other cloneshave been reported sporadically in several regions of the world [6].In this work, we describe the first detection of K. pneumoniae

producing OXA-48 in Algeria.

2. Materials and methods

2.1. Clinical isolate

K. pneumoniae S103/11 was isolated in October 2011 from 02blood cultures belonging to a male patient aged 18 months oldhospitalized in the pediatric unit of Hopital central de l’armee ofAlgiers. The patient had a myeloblastic acute leukemia and wastransferred from Constantine university hospital where he stayedfor 10 days and presented several episodes of fever. He was febrileat admission and his state worsened despite treatment withimipenem. Susceptibility testing revealed the presence of carba-penem resistant K. pneumoniae but the initial treatment wasmaintained and the patient died few days later. Because ofpatient’s isolation and barrier precautions were respected, no otherisolate with the same resistance was detected from the hospitalduring the same period.

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Table 1Minimum inhibitory concentrations (MICs) of b-lactams for K. pneumoniae S103/11

it is transconjugant T.C E. coli J53-S103/11.

Drug MIC (mg/ml)

K. pneumoniae S103/11 T.C E. coli J53 S103/11 E. coli J53

Amoxicillin >256 >256 6

Amoxicillin + CLAa >256 >256 6

Ticarcillin >256 >256 ND

Ticarcillin + CLAa >256 >256 6

Piperacillin >256 64 2

Piperacillin + TAZb >256 64 ND

Cefalothin >256 >256 ND

Cefoxitin >256 1.5 3

Cefotaxime 4 0.38 0.19

Ceftazidime 1 0.19 0.19

Aztreonam 0.032 0.016 0.047

Cefepime 3 0.032 0.064

Imipenem >32 1 0.5

Ertapenem >32 2 0.016

ND not determined.a Clavulanic acid at a fixed concentration of 2 mg/l.b Tazobactam at fixed concentration of 4 mg/l.

N. Aggoune et al. / Journal of Global Antimicrobial Resistance 2 (2014) 327–329328

2.2. Antibiotic susceptibility testing

Antibiotic susceptibility of the isolate was determined by thedisk diffusion method and minimum inhibitory concentrations(MICs) of b-lactams using E test strips (bioMerieux). The resultswere interpreted according to Clinical and Laboratory StandardsInstitute (CLSI) guidelines [7]. Phenotypic detection of carbaba-penemase included modified Hodge test (MHT) [7] and EDTA assay[8].

2.3. Isoelectric focusing

Enzyme extraction was performed by sonication and followedby an Isoelectric focusing (IEF) of different b-lactamases using apolyacrylamide gel containing ampholines (Amersham, Bios-ciences, Sweden) with a pH range of 3.5–9.5, with TEM-1 (pI5.4), SHV-3 (pI 7.1), SHV-1 (pI 7.6) and Amp C (pI 8.2) as standards.

2.4. Conjugation assays and plasmid analysis

Transferability of the b-lactamase gene was studied byconjugation experiments in broth medium between K. pneumoniae

S103/11 and Escherichia coli J53 Azi R as a recipient strain.Transconjugants were selected on Muller Hinton agar platessupplemented with 200 mg/ml of Ampicillin and 1000 mg/ml ofsodium azide. Plasmid analysis for the clinical strain and it istransconjugant was performed after plasmid DNA extraction bythe method of Kado and Liu [9]. Plasmid size was determined bycomparison with that of plasmids R112 (100.5 kb), R55 (160 kb)and R135/1 (70.4 kb).

2.5. PCR assays

Confirmation of presence of OXA-48 gene was studied byPCR analysis using a specific primer of blaOXA-48: OXA-48A(50-TGGTGGCATCGATTATCGG-30) and OXA-48B (50-GAG-CACTTCTTTTGTGATGGC-30) and performed as described previous-ly [10]. Sequences were analyzed after the amplification of the bla

gene using another specific primer pre-OXA-48 A (50-TATATTG-CATTAAGCAAGGG-30) and pre-OXA-48 B (50-CACAATACGCGC-TAACC-30). Other PCR assays were performed in order to detectplasmidic resistance to quinolones by investigating the presence ofqnr genes A, B and S using the following primers: qnrA (+) 50-TTCTCACGCCAGGATTTGAG-30, qnrA (�) 50-TGCCAGGCACA-GATCTTGAC-3, qnrB (+) 50-TGGCGA AAAAATTGAACAGAA-30, qnrB(�) 50-AGCAACGA(TC)GCCTGGTAG-30, qnrS (+) 50-GACGTGC-TAACTTGCGTGAT-30), qnrS (�) 50-AACACCTCGACTTAAGTCTGA-30.

2.6. Typing

Multilocus sequence typing (MLST) of K. pneumoniae S103/11

with seven housekeeping genes (rpoB, gapA, mdh, pgi, phoE, infB,

and tonB) was performed according to the method of Diancourtet al. [11]. Allele sequences and sequence type (ST) was verified byusing MLST Database (http://www.pasteur.fr/recherche/genopole/PF8/mlst/kpneumoniae.html).

3. Results and discussion

K. pneumoniae S103/11 showed high level resistance toimipenem, ertapenem and meropenem but remains susceptibleto ceftazidime and aztreonam (Table 1). For cefotaxime, the strainwas resistant but a zone diameter of 21 mm was noticed. Onanother hand, it was resistant to nalidixic acid, fluoroquinolones,tetracycline, rifampicine and nitofurantoine, but susceptible to co-trimoxazole, all aminoglycosides, chloramphenicol, colistine and

tigecycline. Modified Hodge test was positive while the isolateyielded a negative EDTA assay. IEF showed the presence of a singleb-lactamase with of pI value of 7.2.

Once the transfer achieved by conjugation, the recipient strainshowed a b-lactam resistance pattern with reduced susceptibilityto carbapenems, also significant differences in carbapenem CMIwere observed between the strain and it is transconjugant(Table 1). Plasmid analysis showed that the bla gene was carriedby a self-conjugative plasmid of 70 kb. This plasmid size hasalready been described in OXA-48 producers]. PCR analysis usingthe specific primer of blaOXA-48: OXA-48A (50-TGGTGGCATCGAT-TATCGG-30) and OXA-48B (50-GAGCACTTCTTTTGTGATGGC-30)confirmed the production of an OXA-48-type carbapenemase withan amplicon size of 432 pb. Other PCR assays studying quinoloneresistance yielded negative results. Sequences analysis followingthe amplification of the bla gene using the specific primer showed100% homology with OXA-48 gene. K. pneumoniae S103/11

displayed a sequence type ST-307(allelic profile 4-1-2-52-1-1-7),this sequence type was more often found in KpC producers [12], buthave recently been described in OXA-48 producers in Morocco [13].

Emergence of carbapenem resistant enterobacterial strains inAlgerian hospitals is a cause for great apprehension. Carbapenemresistance in enterobacteriaceae is poorly documented in ourcountry; molecular studies on resistance enzymes are scarce andfragmented. This is the first time that carbapenem resistancemediated by an OXA-48-type b-lactamase is characterized inAlgeria.

Funding

None declared.

Competing interests

None declared.

Ethical approval

Not required.

Acknowledgement

The authors would like to thank Professor Jean Marc Rolain forhis help in sequences reading and interpretation.

N. Aggoune et al. / Journal of Global Antimicrobial Resistance 2 (2014) 327–329 329

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