Prevalence and molecular typing of extended-spectrum β-lactamases in Escherichia coli , Enterobacter cloacae and Citrobacter freundii isolates from Laghouat Hospital , Algeria

1 Department of Natural Sciences and Life, Faculty of Science and Technology, Salhi Ahmed University Center, P. B: 66, Naama 45000, Algeria. 2 Laboratory of Food, Biomedical and Environmental Microbiology « LAMAABE », Aboubakr Belkaïd University. P. B: 119, Tlemcen 13000. Algeria. 3 Laboratory of Bacteriology–Mycology-Parasitology, Gabriel-Montpied Hospital; 58 Montalembert Street, 63003, Clermont-Ferrand, Cedex. France.


INTRODUCTION
ESBL-producing bacteria are responsible for many local, national and international outbreaks which originated from the different hospital wards and mostly in intensive care units (Rodriguez-Villalobos and Struelens, 2006).Infections due by these strains have been associated with high mortality in affected patients, and represent an increased risk of therapeutic failure and are associated with longer duration of hospital stay and higher hospital charges (Soraas et al., 2013).Production of extended-spectrum β-lactamases (ESBLs) is the principal mechanism of resistance to oxyimino-cephalosporins evolved by members of the family Enterobacteriaceae (Marco et al., 2013).These enzymes can hydrolyse penicillins, first, second and thirdgeneration cephalosporins, and aztreonam, but do not hydrolyse cephamycins or carbapenems (Chanal et al., 1992).The activity of ESBL can be inhibited by βlactamase inhibitors such as clavulanic acid (Paterson, 2000).This family of plasmid-mediated ESBL belongs to Ambler class A and group 2be of the Bush-Jacoby and Medeiros classification.
In the recent years, a new family of plasmid-mediated ESBLs called CTX-M which preferentially hydrolyze cefotaxime has emerged.CTX-M enzymes are not closely related to TEM or SHV β-lactamases, as they only show approximately 40% similarity in sequence (Shahid et al., 2009).These ESBLs have been classified into five phylogenetic families on the basis of their amino acid identities: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTXM-25 (Bradford, 2001;Bonnet, 2004).
CTX-M genes might be associated with insertionsequence ISEcp1 or related insertion sequences involved in their expression and mobilization (Poirel et al., 2003).It is encoded by transferable plasmids that vary in size from 7 to 160 kb (Bonnet, 2004).Now, CTX-M-15 β-lactamase, which was first described in 2001, it is recognized as the most widely distributed CTX-M enzyme (Livermore et al., 2007).This βlactamase is found mainly in Enterobacteriaceae and was recently named "plasmids of resistance responsible for outbreak" because of their capacity to acquire genes of resistance and to transfer among bacteria (Coque et al., 2008).
The majority of previous studies on ESBL-producing isolates have been reported at the west, centre and east of Algeria (Baba Ahmed-KaziTani et al., 2013;Ramdani-Bouguessa et al., 2006;Gharout-Sait et al., 2012).But no study has been performed at southern regions of Algeria.
In this context, the aim of this study was to determine the prevalence and molecular typing of extendedspectrum β-lactamases (ESBLs) producing clinical isolates of Escherichia coli, Enterobacter cloacae, and Citrobacter freundii isolated in the Laghouat "Ahemida Ben Adjila" hospital, Algeria.

Clinical isolates
During a three years period from January 2010 to December 2012, Lagha et al. 1431 255 non-repetitive clinical isolates of E. coli, E. cloacae and C. freundii were isolated from patients hospitalized at the Laghouat "Ahemida Ben Adjila" Hospital, Algeria.These isolates were obtained from various clinical specimens (including urine, pus, blood-culture, catheters and rectal swabs), and only one isolate per patient was investigated.The samples were performed on hospitalized patients from different wards: General surgery, orthopedics, women medicine, men medicine, pulmonology, obstetrics-gynecology, pediatrics ward and intensive care unit.All clinical isolates of enterobacteria were identified with the API 20E® system (bioMérieux, Marcy l'Etoile, France).

Antimicrobial susceptibility testing and ESBL detection
The susceptibility to 32 antibiotics was determined by the standard disk diffusion method on Mueller-Hinton agar recommended by the Antibiogram Committee of the French Society for Microbiology (CASFM, 2010).
Extended spectrum β-lactamase (ESBL) production was phenotypically confirmed using the double-disk synergy test, described by Jarlier et al. (1988).Synergy was determined between a disc of amoxicillin-clavulanate (20/10 μg) and a 30-μg disc of each third-generation cephalosporin test antibiotic (cefotaxime, ceftriaxone, ceftazidime, and aztreonam) placed at a distance of 20 mm from center to center on a Mueller-Hinton Agar (MHA) plate swabbed with the test isolate.Clear extension of the edge of the inhibition zone of cephalosporin toward the augmentin disc was interpreted as positive for ESBL production (Bradford, 2001;Giriyapur et al., 2011).E. coli ATCC 25922 was used as quality control strains.

Extraction of bacterial DNA
Total DNA of ESBL-producing isolates was extracted by boiling to100°C for 10 min a suspension of the strains in 200 µl of distilled water and centrifugation for 7 min at 13,000 × g, then the supernatant obtained was stored at -20°C.PCR experiments were performed with these crude lysates.
Primers used to amplify β-lactamases genes and annealing temperatures are shown in Table 1, thus the operating conditions and assay methods were performed to all ESBL producing isolates as previously described in detail (Lagha et al., 2014).The PCR products were visualized using UV after migration in agarose gel 1% and staining with ethidium bromide.
DNA sequencing was performed with the dideoxy chain termination method (Sanger et al., 1977), in GATC Biotech AG (European Custom Sequencing Centre, Gottfried-Hagen-Stra ße 20, 51105 Köln).The nucleotide and deduced protein sequences were analyzed using the Codon Code Aligner software and compared to sequences available at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).

Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR)
The all ESBL producing clinical isolates were analyzed by enterobacterial repetitive intergenic consensus PCR (ERIC2-PCR) as previously reported (Lagha et al., 2014).DNA was amplified using the only one primer ERIC-2: 5'AAGTAAGTGACTGGGGTGAGCG3' (Dumarche et al., 2002), as shown in Table 1.Amplified products were visualized on agarose gels 1.5% stained with ethidium bromide.The profiles of tested clinical isolates were considered different when at least one band differed (Khan et al., 2002).

Conjugation experiments
Conjugation experiments were performed as previously described (Sambrook et al., 1989) with E. coli C600 Rif R (Rifampicin resistant) as a recipient strain.A donor strain (clinical isolates) and a recipient strain were grown separately in Brain Heart Infusion broth (Oxoid) at 37°C for overnight.Then the transconjugants were selected on Mueller Hinton agar (Oxoid) containing rifampicin (300 µg/L) and cefotaxime (1 µg/L).All transconjugants were subjected to antimicrobial susceptibility testing and double-disk synergy test.

Plasmid analysis
Plasmids DNA from ESBL strains and their transconjugants were extracted by the method of Kado and Liu (Kado and Liu, 1981).Plasmid profiles were analyzed by electrophoresis in agarose gel at 0.7% and the sizes of the plasmids were determined by comparison with that of plasmids-size standards: Rsa (39 kb), TP114 (61 kb), pCFF04 (85 kb), and (180 kb) as previously described (Robin et al., 2005).
The clinical and genetic characteristics, including isolation date, specimen and ward distribution of the 21 ESBL producing isolates are shown in Table 3.
These ESBL-producing strains were isolated from 21 individual patients, consisting of 10 men (47.61%) and 11 women (52.38%) with a mean age of 45.42 years.The patients have been hospitalized during periods between 9
Moreover, it is worth noting that all 21 strains (100%) were susceptible to imipenem, amikacin, colistin and fosfomycin.Results of antimicrobial susceptibility testing are shown in Table 4.
In this study, all 21 ESBL-producing isolates were analyzed for their β-lactamases content by isoelectric focusing.It showed the presence of one to five βlactamase bands with different pIs.The first band of pI5.4 corresponded to the TEM-1-type chromosomal penicillinase.The following bands were of pI: 7.5 (compatible with the OXA-1-type oxacillinase); and pI:  The genotypic analysis by PCR showed the presence of bla SHV , bla TEM , and bla CTX-M-1 group on the chromosomal DNA of strains (Figure 2).Nucleotide sequence analysis of the bla CTX-M genes showed the presence of CTX-M-15 in the twenty ESBL-producing isolates (95.24%).Whereas TEM-4 was detected in three isolates (14.28%) mainly in E. coli and C. freundii, and SHV-12 was identified in only one strain (04.76%) of E. coli isolated from the women medicine ward.The distribution of the different ESBL types for each strain is shown in Table 3.
ERIC-PCR analysis of genomic DNA from the 21 ESBL-producing clinical isolates revealed that there were 11 different patterns among the 13 E. coli isolates (Figure 3A), whereas all strains of E. cloacae and C. freundii have been different (8 distinct ERIC-PCR patterns were seen in the 8 isolates) (Figure 3B).This indicated clearly heterogeneity in isolates genetic profiles.
Concerning the conjugation assays, 10 ESBLproducing transconjugants were obtained from the 21 ESBL isolates selected, which consisted of six E. coli and four E. cloacae.All transconjugants also expressed resistance to aminoglycosides (gentamicin and tobramycin), fluoroquinolones, sulfamid and trimethoprimsulfamethoxazole.
After gel electrophoresis, comparison of the plasmidic content of isolates E. coli and E. cloacae and their transconjugants showed the presence for each strain, one to four bands, different sizes, between 5 and 180 kb.A common band was observed in all extracts which isolates a high molecular weight ˃ 85 kb (≈130 kb); it indicated that the bla CTX-M-15 gene is carried by this plasmid and transferred between strains.

DISCUSSION
Extended-spectrum β-lactamases producing Enterobacteriaceae (ESBL-E) are emerging worldwide in hospitals and in the community (Bradford, 2001).Their incidence varies according to countries, regions or even hospitals (Arnaud et al., 2015).
In our study, ESBLs were found in 21 (8.23%) of 255 clinical isolates with E. coli being the major ESBLs produce (13/21), following of E. cloacae (6/21) and C. freundii (2/21).This rate of ESBLs isolates is in accordance with those reported in Kingdom of Saudi Arabia (8.9%) (El-Khizzi and Bakheshwain, 2006), but even higher than the rate observed in recent European studies, from 2.4% to 5.1% in France (Toubiana et al., 2016), and 2.9% in Sweden (Kaarme et al., 2013).Studies conducted in Ethiopia (Mulisa et al., 2016) reported the prevalence rate of 25% of ESBLs producers respectively among Enterobacteriaceae family.These prevalence rates are high as compared to our study which may be attributed to variation in drug management policies or follow other control programs.
Additionally, Latin America, the Middle East, Europe, and the South Pacific displayed a prevalence of ESBL of approximately 10 to 35% (Morrissey et al., 2013;Fernandez-Reyes et al., 2014).However, more than 40% of clinical isolates from Asia were ESBL producers in 2011 (Lukac et al., 2015).
At Laghouat hospital, the patients were hospitalized more frequently in orthopedics wards (33.33%), intensive care unit (23.8%) and the women medicine wards (19.04%).As they were previously-shown, that orthopedic surgical site infections are often associated with substantial morbidity and exorbitant costs, and are challenging to treat, especially in case of multi-resistant pathogens or presence of implants (Martinez-Pastor et al., 2010).Additionally, many studies in Intensive care units have established risk factors for the acquisition of infection due to ESBL producing E. coli and even to other bacterial species (Oteo et al., 2013).Moreover, it is worth noting that, the principal source of isolation the ESBL-producing isolates in our study was urine with the proportion 52.38%; our results are in accordance with other recent studies realized on a group of 124 Enterobacteriaceae isolates resistant to third generation cephalosporin, and collected in distinct health care facilities of different Portuguese regions, which have even described a 58.9% clinical strains were isolated from urine (Jones-Dias et al., 2014).However, studies have shown that ESBL producing uropathogens have their reservoir in the digestive tract (Anil Kumar and Babu, 2013).
The major risk factors found in this study were length of hospitalization, the hospital ward where the ESBL-strains was isolated (orthopedics, intensive care unit and the women medicine wards), urinary tract, and antibiotic therapy, as previously reported (Dayan et al., 2013) Our antimicrobial susceptibility analysis of the all ESBLproducing isolates found highly prevalent resistances against to the majority of β-lactams, and even to others tested antibiotics: Gentamicin, tobramycin, ofloxacin, chloramphenicols, trimethoprim, and sulfonamides, which confirmed the presence of multidrug-resistant isolates in this hospital.Unfortunately, comparable results of the susceptibility rate to these molecules were also reported by Rakotonirina et al. (2013).
This correlates with other studies, where many ESBL producers are multi-resistant to non-β-lactam antibiotics, including fluoroquinolones and aminoglycosides (Livermore et al., 2007), trimethoprim, tetracyclines, sulfonamides, and chloramphenicol, which are often encoded by the same plasmids that determine the ESBL (Karisik et al., 2006).
Consequently, effective antibiotic therapy for treating these infections is limited to a small number of drugs, such as carbapenems and thus increasing the chance of resistance to carbapenems among the Enterobacteriaceae (Pitout, 2010).
In this study, among the twenty one ESBL-producing clinical strains isolated, bla CTX-M-15 was the most commonly detected genotype in all clinical isolates (95.24%).
The blaCTX-M-15 ESBL gene is considered to be the most prevalent ESBL worldwide, as it is found in a Tunisian study analyzed 32 ESBL E. coli isolates collected during a 10-month period, which reported the emergence of CTX-M-15 in 97% (31/32) of isolates; and 81% (26/32) also harbored TEM-1 (Réjiba et al., 2011).
According to the previously studies realized, among ESBL-producing Enterobacteriaceae in Algeria County, CTX-M-15 with CTX-M-3 enzymes were most frequently reported in the west (Baba Ahmed-KaziTani et al., 2013), centre (Ramdani-Bouguessa et al., 2006) and east of Algeria (Gharout-Sait et al., 2012).The rapid dissemination of CTX-M-15 producing Enterobacteriaceae were reported in a number of countries is a significant public health concern (Bonnet, 2004;Livermore et al., 2007).
Thus, in our study, 14.28% of the clinical isolates harbored bla TEM-4 , followed bla SHV-12 which is also distributed, but only in a single strain of E. coli with rate of 4.76%.This SHV-12 type extended-spectrum βlactamase is most often found in Asia (Kim et al., 1998) and including Africa (Kasap et al., 2010).It is already found in Algeria, as shown previously in E. cloacae (Iabadene et al., 2008), and K. pneumonia (Berrazeg et al., 2013).
However, the spread of TEM-4 enzyme is very rare in Algeria; their presence is reported only in one study by Kermas et al. (2012) where this enzyme is detected in Salmonella enterica.
The molecular typing by ERIC-PCR revealed that the majority of ESBL-producing isolates from our hospital showed distinct genetic profiles, with the presence of 19 different patterns among the 21 genetic profiles.These isolates were considered genetically unrelated.This property explains the easy horizontal dissemination of bla CTX-M-15 -harboring plasmids, a high molecular weight ˃ 85 kb (≈130 kb); and their emergence between different strains.
This result correlates with previous studies in Algeria, where showed that bla CTX-M-15 , was carried by conjugative plasmid of high molecular weight, vary from 85 kb (Messai et al., 2008), to ≥ 125 kb (Nedjai et al., 2012).Already, Messai et al. (2008) showed that the CTX-M-15 enzyme was developed from CTX-M-3 under Algerian clinical context.
This dissemination of the CTX-M-15-type ESBLs is not restricted to the nosocomial setting but also involves the community.This phenomenon is acting to modify the epidemiology of ESBLs, whereas those enzymes were, previously, mostly restricted to the nosocomial setting (Rossolini et al., 2008).
This study is the first report conducted on ESBLproducing strains of E. coli, E. cloacae and C. freundii, isolated from various clinical specimens in Laghouat hospital, Southern Algeria; that demonstrates such a high prevalence of CTX-M-15 enzymes among clinical strains.Although, TEM-4 and SHV-12 are the less frequent enzymes isolated in some strains of E. coli and C. freundii.All this indicates the dissemination of multidrug resistant isolates in Laghouat, in different hospital wards and probably in the community.

Table 1 .
Primers used in PCR for detection of bla-genes.

Table 2 .
Distribution of extended-spectrum β-lactamase producing strains isolated in Laghouat hospital by species and years.

of isolation Wards Sample origin Sex Age (years) β-lactamase pI β-lactamase gene Conjugaison
The principal source of isolation was urine (52.38%); following of the catheters source (19.04%), then pus, blood and rectal sources with a percentage of isolation 9.52% for each one.However, 33.33% of the patients were