Carbapenemase and virulence factors of Enterobacteriaceae in
North Lebanon between 2008 and 2012: evolution via endemic
spread of OXA-48
R. Beyrouthy
1–4, F. Robin
1,2,4, F. Dabboussi
3, H. Mallat
3, M. Hamze´
3and R. Bonnet
1,2,4*
1
Clermont 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 Centre of Biotechnology,
Doctoral School of Sciences and Technology, and Faculty of Public Health, Lebanese University, Tripoli, Lebanon;
4
Centre 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 4 January 2014; returned 21 February 2014; revised 24 April 2014; accepted 30 April 2014
Objectives: To investigate the resistance to carbapenems in Enterobacteriaceae and the underlying resistance
mechanisms in North Lebanon between 2008 and 2012.
Methods: A total of 2767 Enterobacteriaceae isolates recovered from clinical samples collected in Nini Hospital
(North Lebanon) were screened for a decrease in susceptibility or resistance to ertapenem (MIC .0.25 mg/L).
Enterobacteriaceae were similarly screened from 183 faecal samples obtained from non-hospitalized patients.
The bacterial isolates were assigned to clonal lineages by PFGE and multilocus sequence typing. Carbapenemase
genes, their genetic environment and virulence genes were characterized by molecular approaches.
Results: The rate of Enterobacteriaceae exhibiting a decrease in susceptibility or resistance to ertapenem
increased from 0.4% in 2008 –10 to 1.6% in 2012 for the clinical isolates recovered from hospitalized patients.
Of these isolates, scattered among seven enterobacterial species, 88% produced OXA-48 carbapenemase.
However, Escherichia coli represented 73% of the OXA-48-producing Enterobacteriaceae collected in 2012 at
this hospital. During the faecal carriage study performed in non-hospitalized patients, E. coli was the only
species producing OXA-48. The bla
OXA-48gene was mainly found within Tn1999.2-type transposons inserted
into E. coli chromosomes or in
50, 62 or 85 kb plasmids. The plasmids and chromosomal inserts were related
to pOXA-48a. Molecular typing of the isolates revealed clonal diversity of E. coli and Klebsiella pneumoniae
pro-ducing OXA-48. OXA-48 was observed in all major E. coli phylogroups, including D and B2, and isolates harbouring
virulence genes of extra-intestinal pathogenic E. coli. Although not belonging to highly virulent capsular
geno-types, the OXA-48-producing K. pneumoniae harboured genes associated with virulence or host colonization.
Conclusions: Horizontal transfer of related plasmids has facilitated the spread of the bla
OXA-48gene into several
Enterobacteriaceae species, including virulent E. coli. Their clonal diversity and the presence of faecal carriers in
the community suggest an endemic spread of OXA-48.
Keywords: OXA-48, Escherichia coli, Klebsiella pneumoniae
Introduction
Carbapenems are broad-spectrum antibiotics that constitute the
last-line therapeutic option available to treat infections caused
by multidrug-resistant Enterobacteriaceae.
1However, due to
the emerging resistance to carbapenems worldwide, the
anti-microbial activity of these drugs is no longer guaranteed.
2The
most concerning mechanism of resistance is the acquisition of
carbapenem-hydrolysing b-lactamases. The most prevalent
carbapenemases are Ambler class A enzymes such as KPC-type
b
-lactamases, class B enzymes (such as VIM-, IMP- and
NDM-type metallo-b-lactamases) and class D enzymes (such as
OXA-48).
3OXA-48 was initially identified from a Klebsiella pneumoniae
isolate in Turkey
4and then spread to various Enterobacteriaceae
species, especially throughout the southern Mediterranean area
and in Europe.
5,6OXA-48-encoding genes are primarily found in
K. pneumoniae and, to a lesser extent, in Escherichia coli and
#The Author 2014. 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
Enterobacter spp.
6Their emergence is mediated by the rapid
spread of broad host-range conjugative plasmids harbouring
the bla
OXA-48gene located within a Tn1999-type composite
transposon.
7–9The bla
OXA-48gene was essentially observed
in the IncL/M plasmids pOXA-48a and derivatives (62 kb),
7,8pKPoxa-48N2 (160 kb) characterized from a K. pneumoniae strain
isolated in France
8and pJEG011 (72 kb) characterized from a
K. pneumoniae strain isolated in Australia.
9In addition to often being multiresistant, K. pneumoniae is an
opportunistic pathogen and can give rise to severe diseases
responsible for community- and hospital-acquired infections. Its
virulence trait is due to the presence of the K1 and K2 capsular
ser-otypes, lipopolysaccharide synthesis, iron-scavenging systems
and adhesins and its ability to form biofilms.
10E. coli organisms
include commensal isolates belonging to the E. coli phylogroups
A and B1 and virulent isolates, including intestinal pathogenic
E. coli and extra-intestinal pathogenic E. coli (ExPEC).
11The latter
belong to the B2 and D E. coli phylogroups and have acquired
pathogenicity islands encoding virulence factors.
12Although
the association of virulence with resistance was observed in
an OXA-48-producing E. coli isolate,
13the virulence traits of
carbapenemase-producing bacteria remain largely unknown.
The aim of this work was to investigate the evolution of
the resistance to carbapenems in Enterobacteriaceae isolated
in North Lebanon between 2008 and 2012, the underlying
resistance mechanisms and the genetic background and
viru-lence factors of these bacteria.
Materials and methods
Bacteria
A total of 2767 Enterobacteriaceae strains isolated from clinical samples in Nini Hospital, Lebanon, between January 2008 and December 2012 were screened for reduced susceptibility or resistance to ertapenem (MIC .0.25 mg/L). Nini Hospital has 125 general-care beds and 12 intensive-care beds. The annual number of hospital admissions is18000 and the hospital serves an area population of1000000. Twenty-four non-redundant clinical strains were recovered from hospitalized patients. None of the patients included in the study had travelled recently.
In parallel, Enterobacteriaceae exhibiting decreased susceptibility or resistance to ertapenem (MIC .0.25 mg/L) were collected from 183 faecal samples obtained in December 2012 from healthy children who had not been admitted to hospital and had not taken antibiotics in the previous 6 months. Non-redundant intestinal Enterobacteriaceae were recovered by spreading the corresponding faecal samples on MacConkey agar plates (bioMe´rieux, Marcy-l’E´toile, France) supplemented with ertapenem (0.5 mg/L) or cefotaxime (4 mg/L).
The strains were identified using the MALDI-TOF mass spectrometry system VITEK MS (bioMe´rieux). Rifampicin-resistant E. coli C600 was used for mating-out assays and E. coli DH5a for electroporation.
Determination of genetic relatedness and the
E. coli phylogroup
PFGE was performed using a CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s recommendations. Pulsotypes were interpreted according to criteria previously defined by Tenover et al.14Allelic profiles and sequence types (STs) were assigned using the multilocus sequence typing (MLST) scheme described by Diancourt et al.15 (http://www.pasteur.fr/recherche/genopole/PF8/mlst/ Kpneumoniae.html) and Achtman (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli)
for K. pneumoniae and E. coli isolates, respectively. E. coli phylogenetic groups were assigned by pentaplex PCR.16
Antibiogram and MIC determination
Antibiotic susceptibility was assessed by the disc diffusion method and the determination of MICs by a broth microdilution method according to the guidelines of EUCAST (http://www.eucast.org/). Antimicrobial agents were obtained from Sigma Chemical Co. (St Louis, MO, USA) or directly from manufacturing companies. Carbapenemase production was detected by a modified Hodge test using combined disc tests of ertapenem with and without cloxacillin and the Mastdiscs ID inhibitor combination disc method (Mast Diagnostics, Bootle, UK).17,18Extended-spectrum b-lactamase (ESBL) production was detected by both the combination disc test and the double-disc synergy test according to the guidelines of EUCAST (http://www.eucast.org/).
Molecular identification of b-lactamases and additional
associated resistance genes
b-Lactamase-encoding genes (blaTEM, blaCTX-MblaKPC, blaNDM, blaVIMand
blaOXA-48) were detected by PCR amplification (Table S1, available as
Supplementary data at JAC Online). The genetic environment of the blaOXA-48gene was further investigated by PCR and sequencing using
primers specific for the insertion sequence IS1999 and the blaOXA-48
gene to map the composite transposon Tn1999 and its derivatives Tn1999.2, Tn1999.3 and Tn1999.4.
Analysis of plasmids and chromosome
Transferability of the blaOXA-48 gene was studied by a mating-out
assay. When these experiments failed, plasmid DNA was extracted with NucleoBondwXtra (Macherey-Nagel, Germany) and transferred by
electro-poration into a bacterial recipient. Selection was performed on agar plates supplemented with ticarcillin (32 mg/L) and rifampicin (300 mg/L) for the mating-out assay and ticarcillin (32 mg/L) for electroporation. The plasmid content of the bacteria and the size of plasmids were determined using plas-mid DNA extracted by the method of Kado and Liu19with the plasmids Rsa (39 kb), TP114 (61 kb), pCFF04 (85 kb) and pCFF14 (180 kb) as standards.
PCR-based replicon typing (PBRT) was used to identify plasmid incom-patibility groups in transconjugants or transformants.20The repA, traU and parA genes of the pOXA-48a plasmid were detected by PCR, as previously described.8
Chromosome analysis
The location of the blaOXA-48gene was investigated by PFGE using the
endonuclease I-CeuI and hybridizations with probes specific for the 16S rRNA gene, the blaOXA-48gene and IS1999, as previously described.21
The chromosomal insertion of the blaOXA-48gene was further mapped
by PCR using blaOXA-48-containing DNA fragments purified from PFGE
gels and primers specific for pOXA-48a, as previously described.7
Virulence factor-encoding genes and
pathogenicity islands
K. pneumoniae virulence genes (allS, cf29a, kfu, magA, mrkD, rmpA, uge, ureA, wabG, wzy and wzx) and the polyketide synthetase (pks) gen-omic island were screened by PCR, as previously described.10,22,23 Similarly, virulence factors encoded by the E. coli isolates were detected by PCR targeting the genes encoding adhesins (papC, papG alleles, sfa and hra), siderophores (fyA, iroN and ear), cyclomodulins (pks, cnf and hlyA), malx and pathogenicity islands (IJ96, IIJ96, ICFT073, IICFT073,
I536, II536, III536, IV536and VI536), as previously described.24,25
Beyrouthy et al.
Biofilm formation
Biofilm production was assessed using the microtitre plate assay, as described by the modified protocol of O’Toole and Kolter.26The F plasmid-bearing Escherichia coli TG1 strain27was used as a positive control.
Results
Decrease in susceptibility to ertapenem
in hospitalized patients
In total, 2767 Enterobacteriaceae isolates were recovered from
clinical samples at Nini Hospital, Tripoli, Lebanon, between 2008
and 2012. These isolates mainly belonged to the species E. coli
(n ¼ 2284, 82.6%), K. pneumoniae (n ¼ 230, 8.3%), Enterobacter
cloacae (n¼ 10, 0.3%), Enterobacter aerogenes (n ¼ 6, 0.2%) and
Proteus mirabilis (n ¼ 106, 3.8%). Twenty-four non-redundant
isolates exhibiting resistance or reduced susceptibility to
ertape-nem (MIC .0.25 mg/L) were recovered between 2008 and
2012. The isolates belonged to seven bacterial species and were
predominantly collected from urine (n ¼ 14) and pus (n ¼ 6)
(Table
1
). The rate of isolates exhibiting reduced susceptibility or
resistance to ertapenem increased from 0.4% (n ¼ 6/1385) in
2008 – 10 to 0.9% (n ¼ 6/650) in 2011 and 1.6% (n ¼ 12/732)
in 2012.
Intestinal carriage in the community
In parallel, the faecal carriage of Enterobacteriaceae
exhibit-ing resistance or reduced susceptibility to ertapenem (MIC
Table 1. Phenotypic and genotypic characteristics of OXA-48-producing isolatesIsolate
STa(E. coli
phylogroup)
Isolation site (year)
OXA-48 genetic environment
Other b-lactamases
MIC (mg/L)
transposon parAb repAb traUb
genetic
support ETP IPM MEM CTX CAZ
E. coli
EC8 131 (B2) urine (2012) DTn1999.2c + + + chromd 2 1.0 0.5 0.12 0.25 0.5
EC37 88 (A) stoole(2012) DTn1999.2c + 2 2 chromd 2 2 2 0.5 0.5 0.5
EC49 38 (D) stoole(2012) NTf + 2 2 chromd CTX-M-24, TEM-1 1 0.5 0.12 256 1
EC119 3877 (A) stoole(2012) Tn1999.2 + + + 62 kb 2 1 0.5 0.25 0.06 0.12
EC253 617 (A) urine (2012) Tn1999.2 + + + 62 kb CTX-M-15, OXA-1, TEM-1 2 1 0.25 128 16
EC254 38 (D) urine (2012) DTn1999.2c + 2 2 chromd CTX-M-24, TEM-1 1 0.5 0.25 16 1
EC260 1711 (B1) urine (2012) Tn1999.2 + + + 62 kbg TEM-1 0.5 0.25 0.25 0.06 0.06
EC264 227 (A) urine (2012) DTn1999.2c + 2 2 chromd TEM-1 0.5 0.25 0.12 0.06 0.06
EC265 38 (D) pus (2012) DTn1999.2c + 2 2 chromd CTX-M-24, TEM-1 1 0.5 0.12 128 1
EC267 88 (A) sputum (2012) DTn1999.2c + 2 2 chromd CTX-M-15, TEM-1 2 1 0.25 16 16
EC269 617 (A) urine (2012) Tn1999.2 + + + 62 kbg CTX-M-15, TEM-1 2 1 0.25 512 128
EC15 127 (B2) sputum (2011) DTn1999.2c + 2 2 chromd 2 0.5 0.25 0.25 0.06 0.06
K. pneumoniae KP78 1157 blood (2012) Tn1999.2 + + + 62 kbg SHV-1 0.5 1 0.25 2 2 KP87 1156 pus (2012) Tn1999 + + + 62 kbg CTX-M-15, SHV-1, TEM-1 0.5 1 0.25 64 32 KP34 45 urine (2011) Tn1999.2 + + + 62 kbg SHV-1, TEM-1 1 1 0.25 0.06 0.06 KP9 866 urine (2009) Tn1999.2 + + + 62 kbg SHV-1 8 8 4 32 16 KP26 866 urine (2008) Tn1999.2 + + + 62 kbg SHV-1 32 64 8 16 1 KP112 866 pus (2008) Tn1999.2 + + + 85 kbh SHV-1 8 8 4 8 0.5
E. aerogenes EA2 — pus (2012) Tn1999.2 + + + 62 kbg 2 1 0.5 0.25 32 64
C. koseri CK8 — urine (2011) Tn1999.2 + + + 62 kbg CTX-M-15, TEM-1 2 1 0.25 256 32
R. planticola RA35 — pus (2011) Tn1999.2 + + + 62 kbg 2 2 1 2 0.5 1
C. freundii CF29 — urine (2010) Tn1999.2 + + + 50 kbi TEM-1 0.5 0.5 0.5 0.25 0.25
E. cloacae ECL6 — urine (2010) Tn1999.2 + + + 62 kbg 2 2 0.5 0.25 1 0.5
E. cloacae ECL8 — urine (2009) Tn1999.2 + + + 62 kbg 2 8 8 4 128 32
ETP, ertapenem; IPM, imipenem; MEM, meropenem; CTX, cefotaxime; CAZ, ceftazidime.
aSTs for E. coli and K. pneumoniae isolates. bPCR for genes specific for the pOXA-48a backbone. c
Incomplete structure of Tn1999.2.
dChromosome-mediated OXA-48. e
Faecal carriage in children having no known contact with the hospital.
fNon-typeable Tn1999-type environment. g62 kb plasmid encoding OXA-48. h
85 kb plasmid encoding OXA-48.
i50 kb plasmid encoding OXA-48.
OXA-48 carbapenemase in North Lebanon
JAC
.0.25 mg/L) was screened in 2012 from samples obtained from
183 healthy children in the community. The children were enrolled
in two schools with different socio-economic levels. School 1
(n ¼ 120) was a welfare school catering for children of poor
families. School 2 (n ¼ 87) was a private institution, with pupils
that were drawn from upper-class families. Three E. coli isolates
exhibiting resistance to ertapenem (n ¼ 3/183, 1.5%) were
recovered from children (9, 11 and 12 years old) enrolled in
three different classes at school 1.
Phenotypic and molecular identification of b-lactamases
PCR and sequencing confirmed the presence of the bla
OXA-48gene
in 88% of the clinical isolates (n ¼21/24) and in the three faecal
E. coli isolates from healthy children (Table
1
). No other gene
encoding carbapenemase was detected in the isolates, including
those devoid of the bla
OXA-48gene (Enterobacter spp., n¼ 3/24). In
addition, the ESBL synergy test was positive for eight
OXA-48-producing isolates. PCR and sequencing showed the presence of
the ESBL-encoding genes bla
CTX-M-15and bla
CTX-M-24in five and
three isolates, respectively (Table
1
).
MICs of b-lactams for OXA-48-producing bacteria
The OXA-48-producing isolates were homogeneously resistant to
penicillins and their combinations with b-lactamase inhibitors.
The MIC of temocillin, which has been proposed as a screening
test for OXA-48 detection, was .32 mg/L for all isolates except
E. aerogenes EA2. The isolates exhibited various levels of
resist-ance to carbapenems (Table
1
). The MICs of oxyimino
cephalos-porins were in the resistance range for isolates producing
both OXA-48 and an ESBL. The MIC values of cefotaxime and
ceftazidime were in the susceptible range (0.06 –2 mg/L) for the
isolates devoid of an ESBL, except for three K. pneumoniae isolates
(KP9, KP26 and KP112) and E. cloacae ECL8.
Plasmidic genetic support of the bla
OXA-48gene
Except for eight E. coli strains, transconjugants (n ¼ 15) and
transformants (n¼ 2) were obtained from the OXA-48-encoding
strains by a mating-out assay and electroporation. All of the
trans-conjugants and transformants exhibited a b-lactam resistance
phenotype compatible with OXA-48 production, and the presence
of the corresponding gene was confirmed by PCR experiments.
Plasmids from natural isolates and their transformants or
trans-conjugants were extracted and hybridized with a probe specific
for the bla
OXA-48gene. The results showed that the bla
OXA-48gene was predominantly located on
62 kb conjugative plasmids
(Table
1
). Further analysis of these plasmids from the
transconju-gants revealed similar restriction profiles (data not shown).
However, the bla
OXA-48gene was also harboured by plasmids of
50 and 85 kb in Citrobacter freundii CF29 and K. pneumoniae
KP112 (Figure
1
and Table
1
). Resistance to non-b-lactam
antibio-tics was not observed in the transconjugants and transformants,
except for that containing the
85 kb plasmid, which exhibited
resistance to sulphonamides and spectinomycin. No plasmids
could be assigned to an incompatibility group by the PBRT method.
We detected the repA, traU and parA genes of the pOXA-48a
plas-mid in all of the transformants or transconjugants (Table
1
). These
results suggested that the plasmids had an IncL/M-pOXA-48a-like
backbone, including the
50 and 85 kb plasmids.
Chromosomal genetic support of the bla
OXA-48gene
Despite many attempts, no transconjugants or transformants
were obtained from six clinical and two faecal E. coli isolates.
The insertion of the bla
OXA-48gene into the bacterial chromosome
was therefore investigated by PFGE after I-CeuI nuclease
treat-ment and hybridization. Different DNA fragtreat-ments (size range
75 to 0.88 Mb) hybridized with the bla
OXA-48probe (Figure
2
).
These results show that the insertion of the bla
OXA-48gene into
the E. coli chromosome is not a rare event (33%, n ¼ 8/24) and
may occur at different sites. The parA gene of pOXA-48a (but
not repA and traU) was detected by PCR and sequenced in the
bla
OXA-48-harbouring DNA fragments, which were eluted from
PFGE. These results suggest that the strains contain a large
frag-ment inserted into the chromosome and that the DNA fragfrag-ments
harbouring the bla
OXA-48gene originated in a pOXA-48a-type
plasmid.
Genetic environment of the bla
OXA-48gene
To characterize the genetic environment of the bla
OXA-48gene in
our strains, we mapped Tn1999-type transposons harbouring
the bla
OXA-48gene by PCR and sequencing. The bla
OXA-48gene
was predominantly associated with Tn1999.2 (63%, n ¼ 15/24).
In addition, seven bla
OXA-48genes (29%, n ¼ 7/24) were located
in incomplete Tn1999.2 elements. These incomplete Tn1999.2
85kb 62kb 50kb A B B 1 2 1 2 3 4 3 4 5 6 5 6 A A BFigure 1. Plasmid DNA content of representative strains and their transconjugants and transformants. A, DNA on a 0.7% agarose gel; B, hybridization using a probe specific for blaOXA-48. 1 and 2, plasmid content of KP112 and the corresponding transformant, respectively; 3 and 4, plasmid content of
RA35 and the corresponding transconjugant, respectively; 5 and 6, plasmid content of CF29 and its transformant, respectively.
Beyrouthy et al.
elements were observed only in the isolates containing a
chromosome-mediated bla
OXA-48gene.
Genetic background and virulence factors
of K. pneumoniae and E. coli
The genetic background of K. pneumoniae and E. coli isolates
pro-ducing OXA-48 was investigated by PFGE and MLST analyses
(Table
2
). Most of the OXA-48-producing K. pneumoniae and
E. coli isolates were different according to Tenover’s
14criteria for
interpreting PFGE patterns (data not shown). Only K. pneumoniae
isolates KP9, KP26 and KP112 were closely related. Two E. coli
isolates presented identical pulsotypes (EC254 and EC265) and
one was closely related (EC49).
Four STs were represented among the six K. pneumoniae
isolates (Table
1
). No K. pneumoniae isolate exhibited the virulent
capsular serotype K1 or K2. Among the genes encoding adhesins,
only mrkD was detected. However, all isolates harboured the
genes wabG and uge, which favour host colonization and
viru-lence (Table
2
). The ferric iron uptake system encoded by kfu
was detected in four isolates. All strains produced biofilms; four
isolates in particular showed biofilm production that was not
sig-nificantly different from that observed for the reference strain TG1
(Table
2
; ANOVA, P. 0.05).
Seven STs were represented among the E. coli isolates
(Table
1
), belonging predominantly to the commensal
phy-logroups A (n ¼ 6) and B1 (n ¼ 1). Five isolates were assigned to
phylogroup D or B2. These latter isolates harboured virulence
genes and pathogenicity islands previously observed in ExPEC
strains (Table
3
). Strains EC8 and EC15, which belonged to
phy-logroup B2, contained a higher number of pathogenicity islands
(five or more) and virulence genes (six or more) than the E. coli
isolates of phylogroup D.
Discussion
In this study, we showed that the rate of Enterobacteriaceae
exhibiting a decrease in susceptibility to ertapenem in Nini
Hospital, North Lebanon, increased from 0.4% in 2008 – 10 to
1.6% in 2012. This rise was associated with the emergence of
carbapenemase OXA-48, which had been previously reported
in Lebanon in 2008 – 10.
28–30E. coli was the predominant
species producing OXA-48, in contrast to previous studies, which
generally cite K. pneumoniae as the main OXA-48-producing
Enterobacteriaceae.
4,6,7,31–35E. coli isolates represented 10% of
the OXA-48 clinical producers in 2008 – 11 and 73% in 2012. In
addition, intestinal carriage of OXA-48-producing E. coli was
observed in the community and was marked by a diversity of
A B C A B C A B C A B C A B C
600kb_
840kb_ 870kb_
740kb_
880kb_
Figure 2. Chromosomal location of blaOXA-48in five representative E. coli isolates. A, I-CeuI fragments of chromosomes analysed by PFGE; B, hybridization
after transfer to a nylon membrane using probes specific for the 16S RNA gene; C, hybridization after transfer to a nylon membrane using probes specific for blaOXA-48.
Table 2. STs, virulence factor-encoding genes and biofilm formation in OXA-48-producing K. pneumoniae isolates
Isolate Source ST
Virulence factor-encoding genes
Biofilm formation relative to E. coli strain TG1
ureA wabG uge cf29a kfu mrkD rmpA allS magA pks
KP9 urine 866 + + + 2 + + 2 2 2 2 0.99+0.28 KP26 urine 866 + + + 2 + + 2 2 2 2 1.11+0.17 KP34 urine 45 + + + 2 + + 2 2 2 2 0.15+0.01 KP78 blood 1157 + + + 2 2 + 2 2 2 2 0.71+0.18 KP87 pus 1156 + + + 2 2 + 2 + 2 2 0.23+0.07 KP112 pus 866 + + + 2 + + 2 2 2 2 0.84+0.20
OXA-48 carbapenemase in North Lebanon
JAC
strains, suggesting that OXA-48 has become endemic in North
Lebanon.
OXA-48 was observed in all major phylogroups of E. coli. Most
of the isolates belonged to phylogroup A, which predominantly
comprises commensal strains. However, D and B2
OXA-48-producing E. coli strains harboured virulence genes of
extra-intestinal pathogenic E. coli. No highly virulent OXA-48-producing
K. pneumoniae isolate was found. However, OXA-48-producing
K. pneumoniae isolates produced a significant amount of biofilm
and harboured genes previously associated with virulence and
favouring host colonization.
A recent study showed that pOXA-48a is highly transferable
in vitro.
36Our findings showed that the emergence of OXA-48
car-bapenemase was facilitated by the horizontal transfer of plasmids
related to pOXA-48a-like plasmids.
32A recent study reported the
presence of the bla
OXA-48gene in the bacterial chromosome of a
sporadic E. coli isolate.
13In this study, the bla
OXA-48
gene was
located in the chromosome in 33% of the OXA-48-producing
Enterobacteriaceae and in 67% of the OXA-48-producing E. coli.
A chromosomal location of the bla
OXA-48gene is therefore not
rare and is observed in unrelated E. coli isolates. The Tn1999.2
elem-ent was predominant in the plasmids and the E. coli chromosomes
harbouring the bla
OXA-48gene. However, chromosome-mediated
Tn1999.2 was truncated, most likely as a result of its insertion
into the E. coli chromosome. Furthermore, the parA gene, which
is located
13 kb from the bla
OXA-48gene on pOXA-48a, was
observed in a chromosomal DNA fragment harbouring the
bla
OXA-48gene, suggesting that Tn1999.2 is most likely not the
only DNA fragment inserted. These results suggest that multiple
acquisitions of the bla
OXA-48gene in E. coli chromosomes involve
a Tn1999.2-containing fragment of a pOXA-48a-like plasmid.
In conclusion, the distribution and diversity of Enterobacteriaceae
producing OXA-48 carbapenemase demonstrate that this
resist-ance mechanism is becoming widespread in hospitals and
in the community in North Lebanon. The occurrence of the
bla
OXA-48gene is worrying due to the spread of both highly
diffusing plasmids and their presence in E. coli producers. This
situation resembles the ecological success story of CTX-M-type
ESBLs, the worldwide emergence of which is most likely due to
their association with E. coli.
Acknowledgements
We thank Laurent Guillouard and Alexis Pontvianne for their technical assistance. We also thank the two schools for their helpful collaboration.
Funding
This research project was supported by the National Council for Scientific Research, Lebanon, the AZM Research Centre of Biotechnology, Lebanese University, Lebanon, the Ministe`re de la Recherche et de la Technologie, 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. Table 3. ST s, pa thog enicity isla nds and other virulence fa ctor-encoding gene s in O XA-4 8-pr oducing E. co li st ra in s belo ngin g to th e D and B2 ph ylogr ou ps Isola te Sour ce ST (p h ylogr oup) Pa thogen icity islands Virulence fa ctor-encod ing genes I536 II536 III 536 IV 536 V536 VI536 ICF073 IICFT073 IJ96 IIJ96 hr a fuyA ear ir oN mal X pks sfa hly cnf pap C pap GI papGII papGIII EC8 urine 131 (B2) + 22 + 2 ++ + 22 ++ + 2 + 22 2 2 + 2 + 2 EC15 sputu m 127 (B2) ++ + + + + + + 2 ++ + + + + + 2 ++ + 22 + EC49 stool 38 (D) 22 ++ 2 + 22 2 2 ++ 22 2 2 2 2 2 2 2 2 2 EC254 urine 38 (D) 22 ++ 2 + 22 2 2 ++ 22 2 2 2 2 2 2 2 2 2 EC265 pus 38 (D) 22 ++ 2 + 22 2 2 ++ 22 2 2 2 2 2 2 2 2 2Beyrouthy et al.
Supplementary data
Table S1 is available as Supplementary data at JAC Online (http://jac. oxfordjournals.org/).
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