CC BY-NC-ND
0Contents lists available at ScienceDirect
International Journal of Infectious Diseases
journal homepage www.elsevier.com/locate/ijid
Countrywide spread of OXA-48 carbapenemase in Lebanon: surveillance and genetic characterization of carbapenem-non-susceptible Enterobacteriaceae in 10 hospitals over a one-year period
D. Hammoudiab*, C. Ayoub Moubareck abc, J. Aires d, A. Adaime abe, A. Barakatf, N. Fayad g, N. Hakime h, M. Houmanii, T. Itania,b,j, Z. Najjark, M. Suleimanl, R. Sarrafm, D. Karam Sarkis a,b
a Microbiology Laboratory, School of Pharmacy, Saint-Joseph University, Beirut, Lebanon b Rodolphe Merieux Laboratory, Beirut, Lebanon
c Department of Natural Science and Public Health, College of Sustainability Sciences and Humanities, Zayed University, Dubai, United Arab Emirates
d EA4065, Sorbonne Paris Cite, Universite Paris Descartes, Paris, France
e Clinique du Levant, Beirut, Lebanon
fBellevue Medical Center, Beirut, Lebanon
g Secours Populaire Libanais, Nabatieh, South Lebanon
h Saint George Hospital and University of Balamand, Beirut, Lebanon
i Labib Medical Center, Saida, South Lebanon
j Arz Hospital, Beirut, Lebanon
k Chtoura Hospital, Bekaa, Lebanon
' Farhat Hospital, Bekaa, Lebanon
m Monla Hospital, Tripoli, North Lebanon
CrossMark
ARTICLE INFO
SUMMARY
Article history: Received 26 January 2014 Received in revised form 26 May 2014 Accepted 24 July 2014 Corresponding Editor: Ziad Memish, Riyadh, Saudi Arabia
Keywords:
Enterobacteriaceae
OXA-48
Carbapenem resistance AmpC
Objectives: To detect, characterize, and assess the genetic clonality of carbapenem-non-susceptible Enterobacteriaceae in 10 Lebanese hospitals in 2012.
Methods: Selected Enterobacteriaceae isolates with reduced susceptibility to carbapenems were subject to phenotypic study including antibiotic susceptibility, cloxacillin effect, modified Hodge test, and activity of efflux pump inhibitor. Carbapenemase genes were detected using PCR; clonal relatedness was studied by pulsed field gel electrophoresis.
Results: Out of 8717 Enterobacteriaceae isolated in 2012, 102 (1.2%) showed reduced susceptibility to carbapenems. Thirty-one (70%) of the 44 studied clinical isolates harbored blaOXA-48, including 15 Klebsiella pneumoniae, eight Escherichia coli, four Serratia marcescens, three Enterobacter cloacae, and one Morganella morganii. The majority of OXA-48 producers co-secreted an extended-spectrum beta-lactamase, while one had an acquired AmpC of the ACC type. In the non-OXA-48 producers, carbapenem resistance was attributed to the production of acquired AmpC cephalosporinases of MOX or CIT type, outer membrane impermeability, and/or efflux pump overproduction. DNA fingerprints revealed that OXA-48 producers were different, except for clonal relatedness among four K. pneumoniae, two E. coli, two E. cloacae, and three S. marcescens.
Conclusions: Nosocomial carbapenem-non-susceptible Enterobacteriaceae are moderately spread in Lebanon and the predominant mechanism is OXA-48 production.
© 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/3.0/).
* Corresponding author. Tel.: +961 1 614006; fax: +961 1 421022. E-mail addresses: dalal.hammoudi@net.usj.edu.lb, dalal.hammoudi@liu.edu.lb (D. Hammoud).
1. Introduction
Enterobacteriaceae are Gram-negative rod-shaped bacteria that normally colonize the human intestinal tract and are capable of causing opportunistic infections in both community and hospital settings. They are easily spread among individuals and can acquire
http://dx.doi.org/10.1016/j.ijid.2014.07.017
1201-9712/© 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
genetic material through lateral gene transfer, largely mediated by mobile elements like plasmids and transposons.1 The dissemination of Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs) has compromised susceptibility to cephalosporins in many areas of the world, including Lebanon, and has increased the consumption of carbapenems.2,3 Recently developed antibiotics among the class of beta-lactams, carba-penems (imipenem, meropenem, ertapenem, and doripenem) are not hydrolysable by EBSLs and represent the antimicrobial options of last resort.2 In 1993, the first incidence of carbapenem resistance in Enterobacteriaceae was described in Enterobacter cloacae due to production of carbapenemase NmcA.4 Since then, various carbapenemases have been detected in Enterobacteriaceae and the majority belong to three Ambler classes: (1) class A, such as KPC, first described in the USA but now disseminated worldwide; (2) class B, such as IMP, VIM, and NDM metallo-beta-lactamases, encountered in Japan, Taiwan, and Greece, as well as many European countries; and (3) class D oxacillinases, such as OXA-48, originating in Turkey and now detected in Europe and the Mediterranean region.1 Rare acquired cepha-losporinases of Ambler class C may also show low carbapenem-hydrolyzing activity.5 Carbapenem resistance in Enterobacter-iaceae may also arise from non-carbapenemase-mediated mechanisms, like permeability defects or the impact of efflux transporters.6
In Lebanon, carbapenem-resistant Enterobacteriaceae have been detected since 2008, with single case reports of OXA-48, 1MP-1, and NDM-1 carbapenemases.7-10 The aims of the current study were to evaluate the dissemination of carbapenem-resistant Enterobacteriaceae in various Lebanese hospitals, to identify the mechanisms of resistance, to examine the types of carbapene-mases involved in resistance, and to analyze the clonal relationship of such isolates collected during the year 2012. Part of this work was presented at the R1CA1 (Reunion Interdisciplinaire de Chimiotherapie Anti-infectieuse) 2012 congress in Paris, France (November, 22-23, 2012; abstract number 300).
2. Materials and methods
2.1. Bacterial isolates
From January 1, 2012 to December 31, 2012, carbapenem-intermediate/resistant isolates of Enterobacteriaceae were collected from the bacteriology laboratories of 10 Lebanese hospitals located in diverse geographic areas: Beirut (Hotel Dieu de France, Saint-George Hospital, Clinique de Levant); Mount Lebanon (Bellevue Medical Center, Arz Hospital); North Lebanon (Monla Hospital); South Lebanon (Secours Populaire Libanais, Labib Medical Center), and Bekaa (Farhat Hospital, Chtaura Hospital). Using standard disk diffusion testing, and following the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST),11 strains with inhibition zone diameters of imipenem smaller than 22 mm, or of ertapenem smaller than 25 mm, were included. These isolates were delivered to the central microbiology laboratory at the Faculty of Pharmacy, Saint-Joseph University, Beirut.
2.2. Phenotypic analysis
2.2.1. Antimicrobial susceptibility testing and detection of ESBLs
Antimicrobial susceptibility testing was performed by disk diffusion on Mueller-Hinton agar plates, according to EUCAST guidelines.11 Amoxicillin/clavulanic acid (AUG), ceftazidime (CAZ), cefotaxime (CTX), cefepime (CPM), cefoxitin (FOX), aztreonam (ATM), and imipenem (1MP) disks (Mast Diagnostics, Merseyside, UK) were tested. After overnight incubation, an increase in the
inhibition zone of the third- and/or fourth-generation cephalospo-rin disk towards the clavulanate-containing disk by at least 5 mm was considered as indicating synergy and the presence of an ESBL.12 The minimum inhibitory concentration of ertapenem (M1CERT) was determined using the Etest (Liofilchem, Roseto degli Abruzzi, 1taly).
2.2.2. Cloxacillin test for detection of AmpC cephalosporinases
Antibiotic susceptibility testing was performed with cloxacillin
(25-500 mg/l) to inhibit AmpC cephalosporinases. An increase in susceptibility to cephalosporins in the presence of cloxacillin was considered to be due to AmpC production. The cloxacillin test was also used to allow better visualization of synergy between: (1) clavulanic acid and third- and/or fourth-generation cephalospor-ins, indicating the possibility of ESBL production, and (2) clavulanic acid and imipenem, indicating the production of an ESBL with carbapenem-hydrolyzing activity.12
2.2.3. Modified Hodge test
A modification of the Hodge test was applied to screen for carbapenemase production.13 The indicator organism, Escher-ichia coli ATCC 25922 at a turbidity of 0.5 McFarland, was used to inoculate the surface of Mueller-Hinton agar plates, and a 10 mg meropenem disk (Mast Diagnostics, Merseyside, UK) was placed at the center. The test strain was heavily streaked from the disk to the plate periphery. After overnight incubation, the presence of a cloverleaf-shaped indentation of growth of the test strain versus the indicator strain was interpreted as carbape-nemase production, with the highest sensitivity to those belonging to Ambler classes A and D.14 Heavy streaks of carbapenemase-positive and carbapenemase-negative strains were used as internal controls.
2.2.4. Activity of efflux pump inhibitor
Mueller-Hinton plates containing 100 mg/l of efflux pump inhibitor phenylalanine-arginine b-naphthylamide hydrochloride (PAbN; Sigma-Aldrich, USA) were prepared. Selected isolates were swabbed on plates with and without PAbN, and Etest strips of ertapenem, cefixime, and levofloxacin (Liofilchem, Via Scozia, 1taly) were placed on the plates. After overnight incubation at 37 °C, MICs in the presence and the absence of the inhibitor were compared, and a two-fold decrease in MIC in the presence of PAbN was considered as indicative of efflux activity.15,16 E. coli ATCC 25922 was used as internal control.
2.3. Genotypic analysis
The presence of various types of beta-lactamase genes was screened for by PCR using primers designed by the 1nstitut Pasteur, Unite des Agents Antibacteriens, Paris, France.17 When ESBLs were detected by phenotypic tests, PCR experiments to detect blaCTX-M group 1 and blaSHV genes were performed. When hyperproduction of AmpC was suspected by the cloxacillin test, multiplex PCR for plasmid-encoded AmpC genes was performed, including blaACC, blaFOX, blaMOX, blaDHA, blaC1T, and blaEBC. To detect carbapenemases, strains were tested for blaKPC, blaGES, blaOXA-48, blaV1M, bla1MP-1, bla1MP-2, and blaNDM genes. For OXA-48 producers, the presence of insertion sequence 1S1999 of transposon Tn1999 known to carry the blaOXA-48 gene was investigated, as described by Aubert et al.18
2.3.1. Conjugation experiments with Klebsiella pneumoniae
Conjugation experiments were performed with K. pneumoniae Kpd2, using the recipient nalidixic acid-resistant E. coli K12, as described previously,19 but changing the mating broth medium to brain-heart infusion.
Table 1
Numbers and percentages of carbapenem-non-susceptible Enterobacteriaceae (CNE) collected from 10 participating hospitals in different areas of Lebanon during the year 2012
Species Total Number (%) of CNEa
Klebsiella pneumoniae 1826 26 (1.4)
Escherichia coli 6631 51 (0.8)
Enterobacter cloacae 513 12 (2.3)
Serratia sppb 199 11 (5.5)
Morganella morganii 229 2 (0.9)
Salmonella spp 87 1 (1.1)
All Enterobacteriaceae 8717 102 (1.2)
a The percentage for each species indicates the proportion of CNE among total isolated strains within the same species. b Serratia marcescens and Serratia odorifera.
2.3.2. Clonality of the OXA-48-positive strains
Genomic DNA digested with Xbal (Roche, Meylan, France) was subjected to pulsed field gel electrophoresis (PFGE) using a clamped homogeneous electric-field apparatus (CHEF-DRII, BioRad). Bionumerics (Applied Math, Kortrijk, Belgium) was used to establish a similarity matrix for the DNA based on calculation of the Dice coefficient (pairwise comparison of strains). A dendrogram was generated with the unweighted pair group using arithmetic means (UPGMA) hierarchical algorithm. Gels were compared using Staphylococcus aureus NCTC 8325 as a reference strain. PFGE patterns were interpreted according to international recommendations.20
3. Results
Out of 8717 Enterobacteriaceae isolated in 2012,102 (1.2%) non-duplicate carbapenem-non-susceptible strains were reported in the records of the participating hospitals (Table 1). The distribution of the 102 strains among hospitals was as follows: 31 were from Hotel Dieu de France, 32 from Saint-George Hospital, six from Clinique de Levant, two from Bellevue Medical Center, two from Arz Hospital, one from Monla Hospital, 27 from Farhat Hospital, and one from Chtaura Hospital. Of these, only 44 isolates were delivered to the central laboratory. The studied species and corresponding hospital sources are shown in Figure 1. Twenty-two specimens were isolated from urine, 10 from sputum, four from pus, four from wound swabs, two from blood, one from stool, and one from pleural fluid. Eighteen isolates out of 44 were nosocomial.
The results of disk diffusion tests and MICs of ertapenem are shown in Table 2. Isolates of K. pneumoniae and E. coli displayed ertapenem-susceptible (M1CERT <0.5 mg/l), intermediate (M1CERT >0.5-1 mg/l), or resistant (M1CERT >1 mg/l) phenotypes, concurrent either with sensitivity or resistance to tested cephalosporins
and aztreonam. All E. cloacae were resistant to ertapenem, tested cephalosporins, and aztreonam. Serratia marcescens, Serratia odorifera, and Salmonella enterica subsp. arizonae were highly resistant to the beta-lactams tested, while the Morganella morganii isolate was resistant to cephalosporins and aztreonam but intermediate to carbapenems.
Synergy between amoxicillin/clavulanic acid and third/fourth-generation cephalosporins was observed for 31 strains including H K. pneumoniae, six E. coli, nine E. cloacae, four S. marcescens, and one M. morganii. These isolates were candidates for ESBL gene detection. None of the isolates displayed synergy between amoxicillin/clavulanic acid and imipenem, indicating the absence of ESBLs with carbapenem-hydrolyzing activity. Moreover, synergy between amoxicillin/clavulanic acid and third/fourth-generation cephalosporins was better visualized on cloxacillin-containing media for the majority of the 31 strains described above.
Inhibition zones of third/fourth-generation cephalosporins (CAZ, CTX, and CPM) on Mueller-Hinton agar plates with 25500 mg/l of cloxacillin increased for two K. pneumoniae and two E. coli, indicating the possibility of production of an acquired AmpC cephalosporinase. E. cloacae, S. marcescens, and M. morganii naturally produce chromosomal AmpC beta-lactamases and hyperproduction can be induced by exposure to beta-lactams.21 In two out of nine E. cloacae strains, three out of five Serratia strains, and in M. morganii, the cloxacillin test was positive. In the remaining strains, the test was negative, possibly due to a lack of hyperproduction of a chromosomal AmpC, or to masking by coproduction of an ESBL or a carbapenemase.
The cloverleaf-like growth pattern in the modified Hodge test was positive for 35 strains including 15 K. pneumoniae, eight E. coli, seven E. cloacae, four S. marcescens, and one M. morganii.
3.1. OXA-48-producing strains
Out of 44 tested Enterobacteriaceae, 31 (70.4%) harbored OXA-48, including 15 K. pneumoniae, eight E. coli, three E. cloacae, four S. marcescens, and one M. morganii. Of the 31 OXA-48 producers, 28 (90.3%) carried 1S1999. In addition to OXA-48, 20 (64.5%) isolates had an ESBL of CTX-M group 1 type, including 11 K. pneumoniae, five E. coli, three E. cloacae, and one M. morganii. 1n Serratia, the four (12.9%) OXA-48-positive isolates were also positive for an ESBL of the SHV type. In all these strains with both carbapenemase and ESBL enzymes, there was non-susceptibility to third-generation cephalosporins in addition to ertapenem. One K. pneumoniae harbored OXA-48 with an acquired AmpC of the ACC type. 1n conjugation experiments, OXA-48 was not transferable to recipient E. coli K12.
Figure 1. Distribution of the tested carbapenem-non-susceptible Enterobacteriaceae strains collected during the study by species and hospital source (HDF, Hotel Dieu de France; SG, Saint-George Hospital; BMC, Bellevue Medical Center; AH, Arz Hospital; CL, Clinique de Levant; FH, Farhat Hospital; MH, Monla Hospital.).
Table 2
Antimicrobial susceptibility patterns and genotypic profiles of tested carbapenem-non-susceptible strains
Species3 Tested strains Genotype (number of strains) ESBL Acquired AmpC Carbapenemase Hodge test AMC FOX CTX FEP CAZ ATM IMP MICert (mg/l)
K. pneumoniae 17 I(1) Neg MOX Neg Neg R R R S R R I 0.25
II (1) Neg ACC OXA-48 Pos R R I I S S R >32
III (11) CTX-M1 Neg OXA-48 Pos R R R R R R R >32
IV (3) Neg Neg OXA-48 Pos R I/R I S S/I S/I I/R (0.75-32)
V(1) Neg Neg Neg Neg R R R R R R R >32
E. coli 11 I(1) Neg CIT Neg Neg R R R I R R I 0.5
II (1) CTX-M1 CIT Neg Neg R R R I R R I >32
III (5) CTX-M1 Neg OXA-48 Pos R I/R R R I/R R I/R (1->32)
IV (3) Neg Neg OXA48 Pos R I S S S/I S I (0.5-1)
V(1) Neg Neg Neg Neg R R R R R R R >32
E. cloacae 9 I (3) CTX-M1 Neg OXA48 Pos R R R R R R R >32
II (6) 1 CTX-M1 1 SHV 4 Neg Neg Neg 4 Pos 2 Neg R R R R R R I/ R (2->32)
S. marcescens 4 I (4) SHV Neg OXA-48 Pos R R R R R R R >32
S. odorifera 1 I(1) Neg Neg Neg Neg R R R R R R R >32
M. morganii 1 I(1) CTX-M1 Neg OXA-48 Pos R R R R R R I 0.75
S. enterica subsp. 1 I(1) Neg Neg Neg Neg R R R R R R R >32
arizonae
ESBL, extended-spectrum beta-lactamase; AMC, amoxicillin/clavulanic acid; FOX, cefoxitin; CTX, cefotaxime; FEP, cefepime; CAZ, ceftazidime; ATM, aztreonam; IMP, imipenem; M1CERT, minimum inhibitory concentration of ertapenem; S, sensitive; I, intermediate; R, resistant.
a K. pneumoniae = Klebsiella pneumoniae; E. coli = Escherichia coli; E. cloacae = Enterobacter cloacae; S. marcescens = Serratia marcescens; S. odorifera = Serratia odorifera; M. morganii = Morganella morganii; S. enterica = Salmonella enterica.
Figure 2. Dendrogram of DNA macrorestriction patterns of OXA-48-producing Enterobacteriaceae with Xbal. Strains were clustered by the unweighted pair-group method of arithmetic averages (UGPMA).
The Xbal-digestion PFGE patterns of the OXA-48-producing strains are presented in Figure 2. Data show the existence of clonally related strains among K. pneumoniae (4/15 isolates), E. coli (2/8 isolates), E. cloacae (2/3 isolates), and S. marcescens (3/4 isolates). The clonally related K. pneumoniae and E. coli were isolated from different hospitals.
3.2. Acquired AmpC-producing strains
Various acquired AmpC enzymes belonging to the groups MOX, ACC, and CIT were detected. One K. pneumoniae harbored MOX and another harbored ACC along with carbapenemase OXA-48. CIT was detected in two E. coli isolates; one of these also expressed an ESBL of CTX-M group 1 type.
3.3. Ertapenem resistance and efflux activity
Eleven isolates with a high level of resistance to ertapenem were subjected to the efflux inhibition test. These included one K. pneumoniae genotype V, two E. coli genotypes II and V, six E. cloacae genotype II, S. odorifera genotype II, and S. enterica subsp. arizonae. Ertapenem MICs were reduced by two-fold in six isolates that revealed a negative modified Hodge test, indicating the possibility of efflux pump activity in the absence of carbapene-mase. These included one K. pneumoniae genotype V, four E. cloacae genotype II, and S. odorifera genotype II. The efflux test, however, was negative in three isolates (one E. coli genotype V, one E. cloacae genotype II, and S. enterica subsp. arizonae) eliminating efflux as a mechanism of ertapenem resistance in these strains and suggesting the possibility of outer membrane protein mutation. The two remaining isolates, one E. coli genotype II and one E. cloacae genotype II, did not grow in the presence of the inhibitor; the test was inconclusive.
4. Discussion
Although several studies have addressed the issue of the emergence of carbapenem-non-susceptible pathogens worldwide, no epidemiological survey has been carried out in Lebanon,
stressing the need for a nationwide investigation. The present study represents the first broad surveillance of carbapenem-non-susceptible Enterobacteriaceae isolated from Lebanese hospitals. 1t also represents the first report of acquired AmpC enzymes in Lebanon.
The overall prevalence of carbapenem-non-susceptible Enter-obacteriaceae in Lebanon during 2012 was 1.2%. If compared to the 4.2% reported by the Centers for Disease Control and Prevention (CDC) in the year 2011, carbapenem resistance in Enterobacter-iaceae in Lebanon appears modest.22 Concerning the distribution of carbapenem resistance among enterobacterial species, the European network on carbapenemases reported in 2012 that carba-penemase producers in Europe are mainly identified among K. pneumoniae and E. coli; the prevalence for K. pneumoniae was found to vary significantly, from very high in Greece (59.9%) to low in Germany (0.2%).23 In this study, carbapenem resistance was disseminated among different enterobacterial species including K. pneumoniae, E. coli, E. cloacae, S. marcescens, and M. morganii. With the exception of Serratia spp, resistance ranged from 0.8% for E. coli to 2.3% for E. cloacae. With regard to Serratia spp, 10 out of the 11 non-susceptible strains isolated during 2012 originated from a single hospital, likely indicating a limited epidemic.
OXA-48 carbapenemase was the prominent mediator of carbapenem resistance in Lebanese hospitals. The detection of OXA-48 among K. pneumoniae and E. coli is in agreement with other data reported in Lebanon.7,10 Since the first description of OXA-48-producing K. pneumoniae in Turkey, numerous reports have indicated its dissemination to Mediterranean countries including France, Egypt, Tunisia, and Morocco.8,24-27 No other carbapene-mases were detected among Enterobacteriaceae strains in this study. According to our results, the metallo-beta-lactamases NDM-1 and 1MP-1 previously detected in case reports in Lebanon did not propagate to Lebanese patients.9,10 Also, although KPC is now endemic in 1srael, it was not detected in this study.28 This may indicate that the spread of carbapenemases of classes A and B among isolates of Enterobacteriaceae in Lebanon is still restricted, unlike that of OXA-48.
All isolates producing OXA-48 were associated with a positive modified Hodge test. This is consistent with previous data indicating that the test shows excellent sensitivity for the detection of carbapenem resistance mediated via the production of class D carbapenemases.29 However, the test may lack sensitivity in Enterobacter species;25 this may explain the false-positive results in four E. cloacae of genotype 11, which were OXA-48-negative but modified Hodge test-positive. 1n this study, the majority of OXA-48 producers harbored insertion sequence IS 1999 suggesting horizontal transfer of the blaOXA-48 gene. However, conjugation experiments were not efficient, which is indicative of probable blaOXA-48 chromosomal integration, as reported previ-
ously.30,31
To test the genetic relatedness of the strains, PFGE allowed the comparison of the genome of the blaOXA-48-positive Enterobacter-iaceae obtained from different hospitals. For each species, certain strains were clonally related, indicating that clonal dissemination played a role in the overall spread of blaOXA-48. The results of PFGE for OXA-48-producing K. pneumoniae and E. coli strains revealed high genetic diversity. OXA-48-producing E. cloacae and S. marcescens strains demonstrating genetic relatedness originated from the same hospitals, revealing small institutional-level outbreaks. In order to obtain a better analysis of clonality in these two genera, greater sampling from different hospital sources is needed.
1n non-OXA-48 producers, acquired AmpC enzyme alone (K. pneumoniae genotype 1 and E. coli genotype 1) conferred only intermediate resistance to imipenem. Like chromosomal AmpC, the plasmid-encoded AmpC enzymes slightly affect carbapenem susceptibility.5 However, carbapenem resistance can arise in
clinical isolates where AmpC enzymes or ESBLs exist together with lower permeability, like in E. coli genotype 11.32 1ndeed, the emergence of resistance to carbapenems can, in part, be due to the loss or alteration of outer membrane porins.33 This could be the case in samples in which no investigated carbapenems of classes A, B, or D have been detected, like E. coli genotype V. In K. pneumoniae genotype V and S. odorifera, highly resistant to both cephalosporins and carbapenems, no beta-lactamase genes were detected. Efflux pump inhibitor tests were positive, indicating a beta-lactamase-independent resistance mechanism. 1n contrast, in S. enterica subsp. arizonae, neither beta-lactamase genes nor the efflux inhibition test were positive, suggesting the existence of mutant outer membrane proteins.
1n conclusion, carbapenem-non-susceptible Enterobacteriaceae are moderately spread in Lebanese hospitals and resistance depends predominantly on OXA-48 production. A combination of AmpC production, efflux pump overexpression, and/or mutant porin proteins may also play a role in carbapenem resistance. As the reservoirs of OXA-48 producers are growing worldwide, concerted surveillance and infection control measures are needed for the containment of further dissemination in Lebanon.
Acknowledgements
We thank Prof. Patrice Courvalin for the gift of reference strains and reading the manuscript.
Funding: This work was supported by the Saint-Joseph University Research Council (project number FPH37) and by the Lebanese National Council for Research and Development (project number 02-09-12).
Conflict of interest: None declared.
References
1. Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011;17:1791-8.
2. Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol Med 2012;18:263-72.
3. Moubareck C, Daoud Z, Hakime N1, Hamze M, Mangeney N, Matta H, et al. Countrywide spread of community- and hospital-acquired extended-spectrum beta-lactamase (CTX-M-15)-producing Enterobacteriaceae in Lebanon. J Clin Microbiol 2005;43:3309-13.
4. Naas T, Nordmann P. Analysis of a carbapenem-hydrolyzing class A beta-lactamase from Enterobacter cloacae and of its LysR-type regulatory protein. Proc Natl Acad Sci U S A 1994;91:7693-7.
5. Jacoby GA. AmpC beta-lactamases. Clin Microbiol Rev 2009;22:161-82.
6. Fernandez L, Hancock RE. Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance. Clin Microbiol Rev 2012;25:661-81.
7. Matar GM, Dandache 1, Carrer A, Khairallah MT, Nordmann P, Sabra A, et al. Spread of OXA-48-mediated resistance to carbapenems in Lebanese Klebsiella pneumoniae and Escherichia coli that produce extended spectrum beta-lacta-mase. Ann Trop Med Parasitol 2010;104:271-4.
8. Carrer A, Poirel L, Yilmaz M, Akan OA, Feriha C, Cuzon G, et al. Spread of OXA-48-encoding plasmid in Turkey and beyond. Antimicrob Agents Chemother 2010;54:1369-73.
9. Daoud Z, Hobeika E, Choucair A, Rohban R. Isolation of the first metallo-beta-lactamase producing Klebsiella pneumoniae in Lebanon. Rev Esp Quimioter 2008;21:123-6.
10. Baroud M, Dandache 1, Araj GF, Wakim R, Kanj S, Kanafani Z, et al. Underlying mechanisms of carbapenem resistance in extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a tertiary care centre in Lebanon: role of OXA-48 and NDM-1 carbapenemases. IntJAntimicrob Agents 2013;41:75-9.
11. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of M1Cs and zone diameters. Version 2.0. EUCAST; 2012. Available at: http://www.eucast.org (accessed as of 01/01/2012).
12. Thomson KS. Extended-spectrum beta-lactamase, AmpC, and carbapenemase issues. J Clin Microbiol 2010;48:1019-25.
13. Miriagou V, Cornaglia G, Edelstein M, Galani 1, Giske CG, Gniadkowski M, et al. Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues. Clin Microbiol Infect 2010;16:112-22.
14. Girlich D, Poirel L, Nordmann P. Value of the modified Hodge test for detection of emerging carbapenemases in Enterobacteriaceae. J Clin Microbiol 2012;50:477-9.
15. Doumith M, Ellington MJ, Livermore DM, Woodford N. Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J Antimicrob Chemother 2009;63:659-67.
16. Grobner S, Linke D, Schutz W, Fladerer C, Madlung J, Autenrieth IB, et al. Emergence of carbapenem-non-susceptible extended-spectrum beta-lacta-mase-producing Klebsiella pneumoniae isolates at the university hospital of Tubingen, Germany. J Med Microbiol 2009;58(Pt 7):912-22.
17. Antibiotic resistance techniques, Sixth ed., Paris, France: Institut Pasteur Unite des Agent Antibacteriens; 2011.
18. Aubert D, Naas T, Heritier C, Poirel L, Nordmann P. Functional characterization of 1S1999, an 1S4 family element involved in mobilization and expression of beta-lactam resistance genes. J Bacteriol 2006;188:6506-14.
19. Moubareck C, Lecso M, Pinloche E, Butel MJ, Doucet-Populaire F. 1nhibitory impact of bifidobacteria on the transfer of beta-lactam resistance among Enterobacteriaceae in the gnotobiotic mouse digestive tract. Appl Environ Microbiol 2007;73:855-60.
20. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. 1nterpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9.
21. Paterson DL. Resistance in Gram-negative bacteria: Enterobacteriaceae. Am J Infect Control 2006;34(5 Suppl 1):S20-8. discussion S64-73.
22. Centers for Disease Control and Prevention (CDC). Vital signs: carbapenem-resistant Enterobacteriaceae. MMWR Morb Mortal Wkly Rep 2013;62:165-70.
23. Canton R, Akova M, Carmeli Y, Giske CG, Glupczynski Y, Gniadkowski M, et al. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin Microbiol Infect 2012;18:413-31.
24. Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother 2012;67:1597-606.
25. Nordmann P, Poirel L. Strategies for identification of carbapenemase-producing Enterobacteriaceae. J Antimicrob Chemother 2013;68:487-9.
26. Ktari S, Mnif B, Louati F, Rekik S, Mezghani S, Mahjoubi F, et al. Spread of Klebsiella pneumoniae isolates producing OXA-48 beta-lactamase in a Tunisian university hospital. J Antimicrob Chemother 2011;66:1644-6.
27. Hays C, Benouda A, Poirel L, Elouennass M, Nordmann P. Nosocomial occurrence of OXA-48-producing enterobacterial isolates in a Moroccan hospital. Int J Antimicrob Agents 2012;39:545-7.
28. Warburg G, Hidalgo-Grass C, Partridge SR, Tolmasky ME, Temper V, Moses AE, et al. A carbapenem-resistant Klebsiella pneumoniae epidemic clone in Jerusalem: sequence type 512 carrying a plasmid encoding aac(6')-1b. J Antimicrob Chemother 2012;67:898-901.
29. Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH. Modified Hodge and EDTA-disk synergy tests to screen metallo-beta-lactamase-producing strains of Pseudomonas and Acinetobacter species. Clin Microbiol Infect 2001;7:88-91.
30. Poirel L, Abdelaziz MO, Bernabeu S, Nordmann P. Occurrence of OXA-48 and V1M-1 carbapenemase-producing Enterobacteriaceae in Egypt. Int J Antimicrob Agents 2013;41:90-1.
31. Gulmez D, Woodford N, Palepou MF, Mushtaq S, Metan G, Yakupogullari Y, et al. Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss. Int J Antimicrob Agents 2008;31:523-6.
32. Livermore DM, Woodford N. The beta-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol 2006;14:413-20.
33. Dahmen S, Mansour W, Charfi K, Boujaafar N, Arlet G, Bouallegue O. Imipenem resistance in Klebsiella pneumoniae is associated to the combination of plasmid-mediated CMY-4 AmpC beta-lactamase and loss of an outer membrane protein. Microb Drug Resist 2012;18:479-83.
