Chromosome-mediated OXA-48 carbapenemase in highly virulent
Escherichia coli
Racha Beyrouthy
1–3, Fre´de´ric Robin
1,2,4, Antony Cougnoux
1,2, Guillaume Dalmasso
1,2, Arlette Darfeuille-Michaud
1,2,
Hassan Mallat
3, Fouad Dabboussi
3, Monzer Hamze´
3and Richard Bonnet
1,2,4*
1Clermont Universite´, UMR 1071 Inserm/Universite´ d’Auvergne, 63000 Clermont-Ferrand, France;2INRA, USC 2018, 63000
Clermont-Ferrand, France;3Health and Environment Microbiology Laboratory, AZM Research Center of Biotechnology, Doctoral School of Sciences and Technology, Lebanese University, Tripoli, Lebanon;4Centre Hospitalier Universitaire, 63000 Clermont-Ferrand, France
*Corresponding author. CHU, Laboratoire de Bacteriologie, 58 rue Montalembert, 63000 Clermont-Ferrand, France. Tel:+33-(0)4–73-754-920; Fax:+33-(0)4-73-754-922; E-mail: rbonnet@chu-clermontferrand.fr
Received 13 November 2012; returned 11 December 2012; revised 21 January 2013; accepted 25 January 2013
Objectives: Bacteria multiresistant to antibiotics are widely supposed to be weakly virulent. However, the viru-lence traits of carbapenem-resistant Enterobacteriaceae have not been investigated. In this work, we investi-gated the virulence and resistance mechanism of an extraintestinal pathogenic Escherichia coli (ExPEC) strain (LEB15) that exhibited decreased susceptibility to carbapenems.
Methods: The MICs were determined by a microdilution method. The b-lactamase-encoding gene was identi-fied by PCR and sequencing, and the genetic environment was analysed by PFGE and PCR mapping. The genetic background was investigated by multilocus sequence typing (MLST). Virulence-factor-encoding genes and pathogenic islands (PAIs) were detected by multiplex PCR. Virulence was assessed in a mouse sepsis model. Results: Strain LEB15 produced a chromosomal OXA-48 carbapenemase. The complete blaOXA-48-encoding
Tn1999.2 transposon was inserted in the LEB15 chromosome. The strain belonged to an MLST cluster of emer-ging ExPEC strains (ST-127/ST-22). It had a high pathogenic score and eight PAIs (I536, II536, III536, IV536, VI536,
ICFT073, IICFT073and IIJ96) and induced an unusually high lethality in the mouse sepsis model.
Conclusions: Strain LEB15 combines both an atypical broad accumulation of virulence factors, which confers a strong killer phenotype, and a decrease in susceptibility to carbapenems following the chromosomal acquisition of blaOXA-48. This association of virulence and carbapenemase in E. coli strains might pose major problems in
the future for E. coli infection management.
Keywords: E. coli, virulence, pathogenic islands
Introduction
Carbapenems are used as a last resort to combat multidrug-resist-ant Enterobacteriaceae.1The recent emergence of
Enterobacter-iaceae producing carbapenemases is therefore a major clinical concern.2Carbapenemase OXA-48 was initially reported in Kleb-siella pneumoniae strains isolated in Turkey in 2001.3Since then, carbapenemase-producing Enterobacteriaceae have been in-creasingly observed due to the widespread emergence of OXA-48 producers in many countries.4 OXA-48 is mostly produced by K. pneumoniae and, to a lesser extent, by Escherichia coli and Enterobacter isolates harbouring plasmids encoding blaOXA-48as
part of Tn1999-based composite transposons.5
As a commensal bacterium, E. coli coexists with its mamma-lian host in harmony. However, extraintestinal pathogenic E. coli
(ExPEC) strains have acquired virulence factors (VFs) and can induce infectious diseases such as urinary tract infections, sepsis and meningitis.6 These virulent bacteria are frequently
susceptible to antibiotics.7However, the expression of virulence
traits in OXA-48-producing bacteria has not been investigated. In this work, we describe an ExPEC combining an unusually broad accumulation of VFs and the production of a chromo-some-mediated OXA-48.
Methods
MICs were determined using a microdilution method and interpreted according to the recommendations of EUCAST (http://www.eucast.org/). b-Lactamase content was investigated by analytical isoelectric focus-ing as previously reported.8The blaOXA-48gene was detected by PCR using
#The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
J Antimicrob Chemother 2013; 68: 1558 – 1561
doi:10.1093/jac/dkt051 Advance Access publication 26 February 2013
1558
specific primers (OXA-48A, 5′-GGGGACGTTATGCGTGTATT-3′; and OXA-48B, 5′-GAGCACTTCTTTTGTGATGGC-3′) and sequenced on both strands. Trans-ferability of the blaOXA-48-harbouring plasmid was studied by liquid and solid mating-out assays or by electroporation, as previously reported.8
Plasmids were extracted by an alkaline method, and the chromosome was investigated with I-CeuI-generated chromosome fragments fol-lowed by PFGE as previously reported.8 Hybridizations were performed
with a PCR-generated probe labelled using a digoxigenin kit (Roche Applied Science, Mannheim, Germany).
The phylogroup of the strain was determined by pentaplex PCR.9
Molecular typing was performed by multilocus sequence typing accord-ing to the University College Cork (http://mlst.ucc.ie/) and Pasteur Insti-tute (http://www.pasteur.fr/mlst) schemes. The presence of 62 extraintestinal virulence genes and eight pathogenic islands (PAIs) was assessed by multiplex PCR, and a pathogenicity score was calculated as previously described.10,11The killer phenotype of LEB15 was
investi-gated in a mouse sepsis model. The experiments were conducted in outbred female OF1 mice (4– 5 weeks old, 14–18 g), challenged by intra-peritoneal injection of E. coli (10 mice/group). All animal experiments
were performed in accordance with protocols approved by the local animal Ethics Committee following the institutional guidelines of the European Community (Council Directive 86/609/EEC).
Results and discussion
In January 2011, a 1-month-old premature male newborn suffering from respiratory distress was admitted to the Paediatric Medical Unit of Nini Hospital, Tripoli, Lebanon. Bacteriological ana-lysis of a bronchial aspiration sample revealed the presence of an E. coli strain (.106cfu/mL) designated LEB15. The strain was resistant to all penicillins and their combination with b-lactamase inhibitors. Susceptibility to carbapenems was decreased (the MICs of ertapenem, imipenem, meropenem and doripenem being 0.50, 0.50, 0.25 and 0.25 mg/L, respectively). However, the strain remained susceptible to cefotaxime, ceftazidime, aztreonam and cefepime (MICs≤0.06 mg/L). This resistance pattern was suggest-ive of the presence of a class D carbapenemase.
1.00 (a) (b) 0.75 0.50 0.25 0.00 1.00 0.75 0.50 0.25 0.00 0 12 24 36 48 60 72 84 96 0 12 24 36 48 Time (h post-infection) Time (h post-infection) Surviv al r ate (f ra ction) Surviv al r ate (f ra ction) 60 72 84 96 LEB15 536 ED1a LEB15 536 ED1a
Figure 1. Kaplan– Meier survival curves of mice intraperitoneally challenged with E. coli strains LEB15, 536 and ED1a. Mice were injected with 106cfu (a) and 105cfu (b) and monitored for 72 h.
Virulent OXA-48-producing E. coli
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Analysis of b-lactamase content revealed an enzyme at pI 7.1, consistent with OXA-48. The presence of the blaOXA-48
gene was confirmed by PCR and sequencing. We failed to trans-fer a blaOXA-48-encoding plasmid from LEB15 into an E. coli
recipi-ent. The analysis of the plasmid content revealed two plasmids of 85 and 100 kb. However, their hybridization with a PCR-generated probe specific to blaOXA-48was negative. In
con-trast, a chromosomal fragment of900 kb hybridized with the blaOXA-48probe. PCR mapping and sequencing revealed the
pres-ence of the complete blaOXA-48-encoding Tn1999.2 transposon.12
These results show that E. coli LEB15 harboured an OXA-48-encoding gene located in the chromosome, as the result of Tn1999.2 transposition or chromosomal insertion of a Tn1999.2-encoding plasmid.
E. coli LEB15 belonged to phylogroup B2 and was genotyped as ST-127 and ST-22 according to the University College Cork and Pasteur Institute schemes, respectively. Hence, E. coli LEB15 belongs to the same sequence type (ST-127) as the uro-pathogenic reference strain E. coli 53613,14according to the
Uni-versity College Cork scheme. These two strains are clustered in the same clonal complex (CCIII) according to the Pasteur Insti-tute scheme.15 LEB15 and the ExPEC reference strain E. coli 53614therefore possess a similar genetic background, which is
usually observed in strains isolated from animals.14The strains belonging to this background are considered in humans as a re-cently emerging clone14possessing a high number of VFs and susceptibility to antibiotics.14,16 In contrast, the LEB15 strain
has acquired a chromosome-mediated carbapenemase, which confers a decreased susceptibility to b-lactams, including carbapenems.
PAIs are large chromosomal regions where VFs are accumu-lated. They are acquired by horizontal genetic transfer and con-tribute to the pathogenic evolution of bacterial populations. To analyse the pathogenicity of strain LEB15, we investigated the presence of PAIs and calculated a pathogenic score as previous-ly reported.10,11 The strain had a high pathogenic score [15 versus a median (range) of 10 (6 –13) for ST-131 strains;11
VFs in LEB15: pap, sfa, foc, fim, hra, hly, cnf1, vat, clb, fyu, iron, kpsMT II, uspA, ompT and malX] and eight extra-intestinal PAIs; five previously observed in E. coli 536 (I536, II536, III536,
IV536and VI536)13and three additional ones previously reported
in CFT073 and J96 (ICFT073, IICFT073and IIJ96). To the best of our
knowledge, this is the first report of such a broad accumulation of PAIs in E. coli.
To confirm the virulence of the strain, the lethality induced by LEB15 was investigated in a mouse sepsis model in compari-son with that of two reference strains, killer strain 53617and the
B2 non-virulent strain E. coli ED1a.17 Mice injected with 108
E. coli LEB15 or E. coli 536 died in ,24 h, in contrast to mice inoculated with ED1a. However, when mice were inoculated with 106and 105cfu, we observed a significant increase in le-thality with strain LEB15 in comparison with strain 536 (Figure 1; Gehan –Breslow–Wilcoxon test, P value 0.0027 for 106cfu and 0.0035 for 105cfu). A total of 105cfu of LEB15 killed 85% of the injected mice in 10 h, whereas 536 killed no mouse during the same period (Figure1b). To eliminate the pos-sibility that the difference in virulence observed in vivo was due to a difference in growth rate, strains LEB15, 536 and ED1a were cultured in LB liquid medium and their growth rates assessed by optical density analysis at 600 nm. The strains
exhibited similar growth behaviour (data not shown), suggesting that the lethality induced by LEB15 was due to the unusual accumulation of PAIs.
Tourret et al.18have demonstrated that all PAIs cooperate in the virulence of E. coli 536 in killing mice during sepsis, but have also highlighted the key role of PAIs II536 and III536 in this
model. Likewise, Le Gall et al.19reported that the mouse killer
phenotype was associated with PAIs ICFT073, IIJ96, III536 and
IV536. Strain LEB15 harbours an unusually high number of
PAIs and all high-pathogenic PAIs observed in ExPEC. These PAIs encode numerous VFs linked to virulence such as haemo-lysin, siderophores, P fimbriae, vacuolating toxin, colibactin, group 2 capsule, haemagglutinin and haemoglobin pro-teases.17,18,20 Multiple copies of the corresponding genes are present in LEB15, which could explain the strain’s enhanced mouse killer phenotype.
In conclusion, LEB15 belongs to a highly virulent emerging cluster of ExPECs. Another facet of this cluster was revealed by characterization of the strain, which combines both an atypical broad accumulation of VFs conferring a strong killer phenotype and a decrease in susceptibility to carbapenems following the chromosomal insertion of the carbapenemase OXA-48. This association of virulence and carbapenemase might pose major problems in the future for E. coli infection management.
Acknowledgements
We thank Marle`ne Jan and Laurent Guillouard for their technical assistance.
Funding
This work was supported by AZM Research Center of Biotechnology, Leba-nese University, Lebanon, the Ministe`re de la Recherche et de la Techno-logie and Institut national de la sante´ et de la recherche me´dicale (UMR Inserm U1071), l’Institut National de la Recherche Agronomique (USC-2018) and the Centre Hospitalier Re´gional Universitaire de Clermont-Ferrand, France.
Transparency declarations
None to declare.
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