Carbapenemases and extended-spectrum β-lactamases producing Enterobacteriaceae isolated from Tunisian and Libyan hospitals

Introduction: The aim of the study was to investigate the prevalence of extended-spectrum β-lactamase (ESBL) and carbapenemase production among clinical isolates of Enterobacteriaceae recovered from Tunisian and Libyan hospitals. Methodology: Bacterial isolates were recovered from patients in intensive care units and identified by biochemical tests and MALDI-TOF. Antibiotic susceptibility testing was performed by disk diffusion and the E-test method. ESBL and carbapenemase activities were detected using standard microbiological tests. Antibiotic resistance-encoding genes were screened by PCR and sequencing. Clonal relationships between Klebsiella pneumoniae strains were carried out using multi-locus sequence typing (MLST). Results: A total of 87 isolates were characterized, with 51 and 36, respectively, identified as E. coli and K. pneumoniae. Overall the resistance prevalence was high for aminoglycosides (> 60%), fluoroquinolones (> 80%), and extended-spectrum cephalosporins (> 94%), and was low for imipenem (11.4%). Among this collection, 58 strains (66.6%) were ESBL producers and 10 K. pneumoniae strains (11.4%) were carbapenemase producers. The antibiotic resistance-encoding genes detected were blaCTX-M-15 (51.7%), blaTEM-1 (35.6%), several variants of blaSHV (21.8%), and blaOXA-48 (11.4%). The MLST typing of K. pneumoniae isolates revealed the presence of multiple clones and three novel sequence types. Also, close relationships between the OXA-48-producing strains from Tunisia and Libya were demonstrated. Conclusions: This study is the first paper describing the emergence of carbapenemaseand ESBL-producing Enterobacteriaceae, sensitive to colistin, isolated in Tunisia and Libya. Active surveillance and testing for susceptibility to colistin should be implementing because resistance to colistin, mainly in Klebsiella, has been recently reported worldwide.


Introduction
Enterobacteriaceae are the most common pathogens causing both community-acquired and hospital-acquired infections, including infections of the urinary and gastrointestinal tracts, peritonitis, meningitis, sepsis, and medical device-associated infections [1].The infections caused by these bacteria are associated with significant morbidity and mortality.In fact, a previous report in Israel demonstrated that the mortality rates investigated in clinical studies ranged from 22% to 72% [2].Within this family, E. coli is a frequent cause of urinary tract infections, and K. pneumoniae is an important cause of pneumonia [3].Emerging resistance in Enterobacteriaceae is a significant problem that requires immediate attention.Indeed, resistance phenotype due to the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases is becoming a major public health concern worldwide [1,4].ESBLs include class A βlactamases, namely TEM and SHV, which confer resistance to ampicillin, amoxicillin, and other penicillins, as well as cephalosporins [3].
Enterobacteriaceae may also express ESBLs that are not closely related to TEM-or SHV-related species, including CTX-M-and OXA-type ESBLs.These ESBLs are typically plasmid-mediated rather than chromosomally mediated β-lactamases [5].ESBLs that hydrolyze carbapenems should be distinguished from other β-lactamases [3].Indeed, they have broader-range activity, covering carbapenems as well as extendedspectrum cephalosporins [6].The clinically most important groups are the increasingly emerging NDM-1, KPC, and OXA-48 enzymes; their producers are spread around the world, becoming an alarming public health problem [7].ESBL-and carbapenemaseproducing Enterobacteriaceae strains are being increasingly reported in Europe, South America, Asia, Oceania, and Africa [8].The situation is still worse in low-income countries, where there is a lack of antimicrobial-resistance surveys and an absence of adequate policies on antibiotics use [9,10].The poor state of health has undoubtedly exacerbated the problem of antimicrobial resistance in these countries [10].In Tunisia and Libya, misuse of antimicrobial agents by the public is widespread.Indeed, in both countries, antimicrobials can be purchased from pharmacies without a prescription, which has led to the rapid emergence of many resistant bacteria.In addition, there is a lack of infection prevention policies and simple control measures, such as hand hygiene, to avoid the spread of pathogens in the hospital setting [9,10].
In view of the emergence of ESBLs and carbapenemases in Tunisia [9,[11][12][13] and because there is no detailed information on the occurrence of these enzymes in Libyan hospitals, this study was undertaken to investigate the prevalence of antibiotic resistance and to characterize the molecular mechanisms of these resistances in E. coli and K. pneumoniae clinical isolates collected from Libyan and Tunisian hospitals.Herein, we describe the first multiclonal spread of K. pneumoniae-harbored blaOXA-48 and ESBL-encoding genes in Libyan hospitals.

Bacterial isolates
A total of 87 clinical isolates were collected over 12 months, between March 2014 and March 2015.Among these strains, 47 were recovered from Benghazi Medical Center (BMC), Benghazi, Libya, and 40 strains were isolated from Avicenne Clinic in Tunisia.All strains were isolated from hospitalized patients in intensive care units (ICUs).The isolates were previously identified using biochemical tests, a Phoenix automated microbiology system, and confirmed by matrix-assisted laser desorption and ionization time-offlight mass spectrometry (MALDI-TOF MS).
For all isolates, minimum inhibitory concentrations (MICs) of ceftazidime and imipenem were determined using an E-test strip (AB BioMerieux, Grenoble, France).MICs values were interpreted according to the CA-SFM breakpoints.

Phenotypic detection of ESBLs and carbapenemases
ESBL production was detected by a double-disk synergy test (DDST).Enhancement of the inhibition zone between the disks containing clavulanic acid and cefotaxime, ceftazidime, or aztreonam indicated the presence of ESBL production [14].Imipenem-resistant isolates were screened for carbapenemase production using the modified Hodge test (MHT), the modified Carba NP test (MCNP) and the EDTA test as previously described [15][16][17][18].

DNA sequencing
PCR products were purified and sequenced using the Big Dye terminator chemistry on an ABI 3730 automated sequencer (Applied Biosystems, Foster City, USA).The obtained nucleotide sequences and their deduced amino acids sequences were compared against the NCBI database using, respectively, BlastN and BlastP functionalities.(www.ncbi.nlm.nih.gov).

Molecular strain typing
The clonal relationships between imipenemresistant K. pneumonie isolates were studied by multilocus sequence typing (MLST).Isolates were attributed to a sequence type (ST) number according to the allelic profiles available in the Institute Pasteur's MLST web site (www.pasteur.fr/mlst).

Results
After identification, it was possible to conclude that the collection of 87 isolates included 51 strains of E. coli and 36 of K. pneumoniae.Antibiotic susceptibility testing for the 87 isolates is summarized in Figure 1.In general, a high prevalence of resistance was observed against the greater part of antibiotics, especially thirdgeneration cephalosporins.Eighty percent of the isolates were very highly resistant to ceftazidime, cefotaxime, amoxicillin-clavulanic acid, amoxicillin, and ciprofloxacin.In addition, 10 strains of K. pneumoniae showed a high level of resistance to carbapenems, with MICs for imipenem > 16 mg/L (Table 2).In the present study, only ESBLs and carbapenemase-producing Enterobacteriaceae were presented in Table 2 and Table 3.All isolates were susceptible to colistin.The double-disk synergy test showed that 66.6% of isolates were ESBL positive.Detection of blaCTX-M, blaSHV and blaTEM using PCR amplification showed that 58 strains were positive for the genes encoding these enzymes.As shown in Table 2, the nucleotide sequences of the amplicons revealed the presence of sequences that were 100% similar to that of blaCTX-M-15, blaTEM-1 and many variants of blaSHV (Table 3).None of the isolates harbored either the blaPER, blaVEB or blaGES genes.The results of carbapenemase production tests showed that all imipenem-resistant isolates were positive by MHT and MCNP, suggesting carbapenemase production.However, the activity of β-lactamases was not inhibited by EDTA, an indicated that the imipenem-resistant isolates were not MBL producers.Screening for carbapenemase-encoding genes by PCR showed that all 10 isolates contained a blaOXA-48like gene (Table 2).All of the isolates were negative against blaKPC-like or blaNDM genes.
All 36 K. pneumoniae isolates were analyzed by MLST, and different STs were observed (Table 2).This analysis showed that the 36 isolates of K. pneumoniae belong to 15 different STs.The most frequent clone was ST101, corresponding to imipenem-resistant K. pneumoniae strains (Table 2).
Using MLST analysis, three isolates of K. pneumoniae collected from BMC, Libya, were found to have novel STs.K. pneumoniae 330 was assigned as ST1949, K. pneumoniae 203 was assigned as ST1950, and K. pneumoniae 82 was assigned as ST1951.It is worth mentioning that those isolates were positive for OXA-48.

Discussion
In this study, we characterized carbapenemase-and ESBLs-producing E. coli and K. pneumoniae clinical isolates isolated from patients admitted to Tunisian and Libyan hospitals.Among the 87 isolates, about twothirds produced ESBLs.Several studies have also shown the emerging problem of ESBL-producing E. coli and K. pneumoniae isolates in different geographic regions, including the Mediterranean basin [19] and North Africa [9,[20][21][22][23].In our work, blaCTX-M-15 was the most frequently detected (54%) gene in the ESBLpositive Tunisian and Libyan isolates.These results suggest that blaCTX-M-15 is the most common gene responsible for mediating extended-spectrum cephalosporin resistance in these isolates.These data confirm previous studies showing that this enzyme is widely present in Tunisia [11,21,[24][25][26][27][28][29][30][31] (Table 4).[24] (Table 4).However, in Libya, few articles have been published describing the presence of ESBL-producing Enterobacteriaceae [32] (Table 4).In this study, similar phenotypes and genotypes related to antibiotic resistance were found in Tunisia and Libya.Indeed, in North Africa, many studies also demonstrated the incidence of ESBL-producing E. coli and K. pneumoniae.Agabou et al. demonstrated the frequency and diversity of ESBLs produced by E. coli isolates from patients hospitalized in the Regional Military Hospital of Constantine in Algeria [33].Additionally, in Morocco, Girlich et al. reported the high rate of fecal carriage of ESBL-producing Enterobacteriaceae at a university hospital [34].In Egypt, a neighboring country of Libya, the CTX-M-15 encoding-gene has been found in clinical isolates of E. coli from Cairo [35].Interestingly, we identified the presence of the blaOXA-48 gene in ten imipenem-resistant K. pneumoniae isolates in our study.Indeed, three Tunisian and seven Libyan K. pneumoniae isolates resistant to imipenem were found to produce OXA-48 (Table 3).Several studies concerning the emergence of OXA-48producing K. pneumoniae have been reported in Tunisia (Table 5), but our study is the first that detected OXA-48-positive K. pneumoniae isolated directly from patients hospitalized in Libyan hospitals.Indeed, in Tunisia, two carbapenem-resistant K. pneumoniae clinical isolates carrying the plasmid-harbored OXA-48 carbapenemase gene were reported in 2010 [12].Ktari et al. reported the spread of 21 (13.7%)K. pneumoniae isolates producing the blaOXA-48 encoding-gene in a Tunisian university hospital [36].In 2012, Saidani et al. screened 21 ESBL-producing Enterobacteriaceae with reduced susceptibilities to carbapenems; they found that 5 of the 21 isolates investigated were OXA-48 positive [13].More recently, among enterobacterial clinical isolates recovered in the Center of Maternity and Neonatology of Monastir, Tunisia, Charfi et al. identified one isolate positive for the OXA-48 gene that co-expressed the blaCTX-M-15 gene [37].These data showed the dissemination of imipenem-resistant K. pneumoniae carrying the blaOXA-48 gene in Tunisian hospitals (Table 5).

Indeed, Mamlouk et al. detected the blaCTX-M-15 gene in
Concerning Libya, to the best of our knowledge, our study is the first that detected the emergence of OXA-48-producing K. pneumoniae in this country.However, three studies reported the presence of OXA-48producing K. pneumoniae isolated from Libyan patients transferred to Europe for treatment.Indeed, Kocsis et al. reported that a carbapenem-resistant K. pneumoniae carrying the blaOXA-48 gene was recovered from the blood culture of a Libyan patient hospitalized in the ICU of the Sacro Cuore-Don Calabria Hospital in Negrar, Italy [38].Italy is the country with the highest incidence of OXA-48-producing K. pneumoniae isolates, and has been considered to be the epicenter of the spread of this enzyme in Mediterranean basin [39].Indeed, several isolates of K. pneumoniae producing OXA-48 have been reported, proving that the situation in this country is becoming endemic [39,40].In addition, Pirš et al. reported the first case of OXA-48producing K. pneumoniae in Slovenia, isolated from a rectal swab collectedfrom a patient transferred from Libya.The patient was colonized by both ESBLproducing E. coli and ESBL-and OXA-48-producing K. pneumoniae [41].Hammerum et al. reported that patients transferred from Libya to Denmark carried OXA-48-producing K. pneumoniae [42].These studies underscore the importance of an early warning system at the European level and screening upon admission of patients transferred across countries.These findings may also confirm that North Africa, including Tunisia and Libya, are considered to be reservoirs of oxacillinase producers, particularly OXA-48; to date, this enzyme represents the most common carbapenemase type circulating in this region [43][44][45][46] (Table 5).Recently, in 2014, Agabou et al. reported the first description of OXA-48-producing E. coli and the pandemic clone ST131 from patients hospitalized at the Regional Military Hospital of Constantine [33].In Morocco, Girlich et al. showed a high prevalence of multidrug-resistant Enterobacteriaceae, and particularly OXA-48 producers, at a university hospital [34] (Table 5).MLST analysis of Tunisian and Libyan isolates showed the occurrence of multiple clones, with clones belonging to ST101 being the most frequent.This is the first report using MLST analysis for typing of K. pneumoniae isolates in Libya, but MLST analysis studies have reported that ST101 is the most prevalent ST type in Tunisia.Indeed, the results of this work are consistent with the studies of Charfi et al., who assigned the K. pneumoniae KP51 strain to ST101, a widespread clone harboring various β-lactamases, mostly OXA-48, which had been already reported in Tunisia and which had also accounted for an outbreak in Spain [37,47].In addition, Cuzon et al. reported a plasmid-mediated OXA-48 in K. pneumoniae ST101 from Tunisia [48].Three novel STs were found among the K. pneumoniae isolates from Benghazi, Libya: ST1949, ST1950, and ST1951.These isolates were positive for OXA-48, as were other isolates assigned to previously described STs (20, 100, 101, 414, and 1322).These findings show that BMC, which is a tertiary hospital in Benghazi and covers the eastern part of Libya in terms of providing healthcare services, may suffer from multiclonal spread of nosocomial pathogens; more importantly, the CTX-M-15 and OXA-48-producing K. pneumoniae detected in this study demonstrates the long-standing infection control problems needing urgent attention.

Conclusions
This study described the emergence of ESBL-and carbapenemase-producing Enterobacteriaceae in Tunisian and Libyan hospitals.These findings are of great concern because of the rapid dissemination of multidrug-resistant bacteria, particularly carbapenemresistant strains, which represent a major therapeutic and epidemiological threat.The implementation of strict infection prevention and control precautions in addition to regular surveillance studies is urgently needed to contain the increasing spread of nosocomial pathogens in these countries.

Figure 1 .
Figure 1.Antibiotic susceptibility of Escherichia coli and Klebsiella pneumoniae clinical strains.

Table 1 .
Primers used for PCR and real-time PCR amplification of carbapenemases, ESBLs, and MBLs genes.

Table 2 .
Phenotypic and genotypic characteristics features of the Tunisian and Libyan Klebsiella pneumoniae clinical isolates producing ESBLs and carbapenemases.

Table 3 .
Phenotypic and genotypic characteristics features of the Tunisian and Libyan Escherichia coli clinical isolates producing ESBLs.

Table 4 .
Studies reporting ESBL-producing Enterobacteriaceae in Tunisia and Libya.

Table 5 .
Studies reporting OXA-48 producing Enterobacteriaceae in North Africa.Enterobacteriaceae (35 E. coli and 27 K. pneumoniae) collected from different wards of Charles Nicolle Hospital in Tunis [30].More recently, in 2014, Ferjani et al. reported that 88% of cefotaxime-resistant E. coli strains, isolated from urine of patients in a Tunisian hospital, harbored the blaCTX-M-15 gene [28].These studies confirm the current spread of the CTX-M-15 encoding-gene, which encodes the most prevalent β-lactamase detected among ESBL-positive K. pneumoniae and E. coli strains in Tunisian hospitals (Table 4).The increased consumption of cefotaxime and ceftazidime might have contributed to the emergence of ESBLs, and particularly these CTX-Mtype enzymes.The occurrence of the blaSHV-11 encoding gene among the ESBL-positive Tunisian strains is consistent with the finding of Abbassi et al., who reported the blaSHV-11 encoding gene in K. pneumoniae ESBL-positive isolates recovered in the Centre of Bone Marrow Transplantation of Tunisia