First characterization of CTX-M-15 and DHA-1 β-lactamases among clinical isolates of Klebsiella pneumoniae in Laghouat Hospital , Algeria

1 Laboratoire de microbiologie Appliquée à l’Agroalimentaire Au Biomédical et à l’Environnement « LAMAABE », faculté SNV-STU, université de Abou Bekr Belkaïd, B.P 119, Tlemcen 13000, Algérie. 2 Laboratoire de microbiologie, faculté des sciences biologiques, université Amar Telidji, Laghouat 03000, Algérie. 3 Laboratoire de Bactériologie-Mycologie-Parasitologie, Hôpital Gabriel-Montpied ; 58 rue Montalembert 63003 ClermontFerrand, Cédex. France.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Table 1.Sequences of the primers used to detect bla-genes.ERIC-PCR ERIC2 5' -AAG TAA GTGACT GGG GTG AGC G -3' 64 Dumarche et al. (2002) they have been involved in many nosocomial outbreaks and are associated with increased mortality (Edelstein et al., 2003;Lebessi et al., 2002;Podschun and Ullmann, 1998).CTX-M β-lactamases enzymes belong to class A. They preferentially hydrolyze cefotaxime more than ceftazidime (Bonnet, 2004).Currently, the CTX-M group 1, particularly CTX-M-15, appear to be more widespread and prevalent in many countries in the world (Carrer and Nordmann, 2011;Lavigne et al., 2007), while other ESBLs of the TEM and SHV subgroups seem to decrease.However, resistance to third generation cephalosporins in K. pneumoniae is no longer exclusively due to class A beta-lactamases.The acquisition of AmpC cephalosporinases is associated with a high level of cephalosporin resistance (Hanson, 2003).The earliest report of AmpC beta-lactamases was in 1990, with the identification of MIR-1 beta-lactamase (Papanicolaou et al., 1990).These enzymes are classified in Bush's Group 1 (Ambler's class C).They are generally highly expressed and confer resistance to most β-lactams, except for carbapenems (Philippon et al., 2002).The main objectives of this study were to determine the frequency of resistance to 3rd generation cephalosporins of K. pneumoniae strains isolated from patients hospitalized in Laghouat hospital, Algeria, to characterize the β-lactamases involved and to highlight the genetic diversity of these strains.

Bacterial strains
This is a retrospective study over a period of three years from 1 January 2010 to 31 December 2012.It focused on 112 nonrepetitive Klebsiella pneumoniae strains isolated from patients hospitalized in "Ahemida Ben Adjila" hospital, Laghouat, Algeria.All patients hospitalized for more than 48 hours were included in the study.The samples correspond to different colonization sites (urine, catheters, rectal swabs and pus), and essentially isolated from services: Women Medicine; General Surgery, Orthopedics, Pulmonology and Intensive Care Unit.Some epidemiological data such as age, gender and origin of service were recorded in the first time for each patient.Species' biochemical identification was performed using the API 20E® identification system (bioMérieux, Marcy l'Etoile, France).

β-Lactamase characterization
The DNA extraction was conducted by preparing a suspension of the strains to be studied in 200μl of distilled water.After boiling for 10 min of suspension and centrifugation for 7 min at 13, 000 × g, and the supernatant was collected in a new 1.5 ml Eppendorf tube and stored at -20°C.
The detection of genes encoding the β-lactamase ESBL and plasmid-mediated AmpC cephalosporinases (DHA-1) was performed by Polymerase Chain Reaction (PCR), the operating conditions and primers used were described in Table 1.The PCR reactions mixture consists of: 5 µl of each primer (10 picomoles/µl), 1 µl of dNTP, 10μl of PCR reaction buffer 5X and 0.25 µl of Taq DNA polymerase (Promega).The DNA was amplified in a final volume of 50 µl, in a thermocycler: either Biometra T Personal (Labgene) or the Primus 96 plus (Biotech).The PCR products were separated in 1% agarose gels.When PCR was positive, the amplicon was purified and sequenced in GATC Biotech AG (European Custom Sequencing Centre, Gottfried-Hagen-Straße 20, 51105 Köln), to identify precisely the desired β-lactamases.

Conjugation transfer experiments
Conjugation experience was performed with E. coli C600 Rif R (resistant to rifampicin) as the recipient strain.The strains were inoculated into a brain-heart infusion broth (BHIB) and incubated overnight at 37 C°.The transconjugants were selected on Mueller-Hinton agar containing rifampicin (300 µg/L) and cefotaxime (1 µg/L).After, the transconjugants pushed into the selection boxes were tested vis-à-vis the antibiotics to detect the presence of resistance phenotype towards 3rd generation cephalosporins and cefoxitin.

RESULTS
During the period of our study, 215 patients were included and 112 strains of Klebsiella pneumoniae were isolated.The double synergy test was positive for nine strains of K. pneumoniae, suggesting the probable production of ESBL.These nine strains produced extended-spectrum β-lactamases (ESBLKp) (frequency of 8%).The majority of patients were women (78%) and their ages ranged from 27 to 85 years.Some hospital services appeared more concerned with the problem of resistance due to ESBL production, including the intensive care unit of which 33% of cases were noticed, followed by the orthopedics and women medicine service with 22% for each (Table 2).
The nine isolates tested were resistant to extendedspectrum cephalosporins, amoxicillin, ticarcillin, amoxicillin/clavulanic acid and ticarcilline/clavulanic acid.Only 33% of the strains were susceptible to piperacillintazobactam.In addition, three strains resistant to cefoxitin.For these three strains induction was observed between imipenem and third generation cephalosporins, suggesting the production of an inducible céphalosporinase.All strains remained susceptible to imipenem (Figure 1).
Molecular characterization of β-lactamases by polymerrase chain reaction (PCR) and sequencing revealed that the nine strains of K. pneumoniae producing ESBL from hospitalized patients in different departments in the hospital carried the same gene bla CTX-M-15 .Moreover, we noticed the presence of plasmid-mediated AmpC cephalosporinase by bla DHA-1 gene in three strains Kp18, Kp55.2 and kp73.
Following the conjugation, antimicrobial susceptibility of the nine transconjugants showed resistance towards 3rd generation cephalosporins with the presence of synergy,   aminoglycosides, kanamycin, and fluoroquinolones.Thus, the resistance to céfoxitine was observed in 3 transconjugants.This explained that the gene bla CTX-M-15 and the gene bla DHA-1 gene have been carried by a plasmid, and transferred to the transconjugants by conjugation.Molecular typing by ERIC-PCR showed the presence of seven different ERIC profiles.One profile was common to the 3 strains co-producing CTX-M-15 and DHA-1, suggesting a clonal relatedness (Figure 3).

DISCUSSION
In our study, the prevalence of ESBL producing Klebsiella pneumoniae isolates was 8%.The overall prevalence of ESBL production varied considerably according to geographic areas of countries and the different hospital structures.ESBL prevalence rate in this study is still lower compared to those reported in northern Algeria (19.9%) (Messai et al., 2008) and Tunisia (20.2%) (Ben Haj Khalifa and Khedher, 2009).However, higher prevalence rates of ESBL produced by Klebsiella were detected in South America (45.4% to 51.9%) (Villegas et al., 2008) and Saudi Arabia (55%) (Al-Agamy et al., 2009).In Pakistan, the prevalence of ESBLs produced in K. pneumoniae reached a very high alarming rate with 70% (Shah et al., 2004).
The antibiotics susceptibility profile of the nine strains studied showed resistance to most β-lactams tested except cefoxitin (30% of strains were resistant), and imipenem remained active in all strains studied.Thus, cross-resistance is observed with aminoglycosides and fluoroquinolones, and this could be related to the misuse of broad-spectrum antibiotics (penicillins, cephalosporins, chloramphenicol, tetracyclines, fluoroquinolones, and aminoglycosides).
The proportions found by Ben Haj Khalifa and Khedher in a study of ESBLs produced in uropathogen Klebsiella spp, isolated in Tunisian university hospital, are closer to those we observed (100% to 3rd generation cephalosporins, 92.5% to gentamicins, and 67.5% to fluoroquinolones) (Ben Haj Khalifa and Khedher, 2009).
It is now proven that the use of antibiotics, especially 3rd generation cephalosporins for therapeutic purposes, is the most important risk factor in the development of bacterial resistance (Rubin and Samore, 2002).It has become a major public health problem.
In our study, the phenotype of the DHA-1-producing strains was characterized in three cefoxitin-resistant strains, two strains (Kp18, Kp55.2) were isolated in the women medicine service, and one strain (kp73) was isolated in the intensive care unit (ICU).
These enzymes have been recently detected among Enterobacteriaceae such as E. coli, K. pneumoniae, Klebsiella oxytoca, P. mirabilis and Salmonella spp.These plasmid-mediated cephalosporinases are genetically very close to chromosomal AmpC cephalosporinases, and the transfer of the genes coding these enzymes requires mobile genetic elements (Philippon et al., 2002;Bush et al., 1995).
Our results are comparable with those of other studies in Spain, China, and France, which observed the diffusion of DHA-1 plasmid-mediated cephalosporinases in K. pneumoniae strains (Hennequin et al., 2012;Guo et al., 2012;Tobes et al., 2013).There are few studies concerning the cephalosporinases AmpC diffusion in this species in Algeria.Only one study was conducted in three hospitals of Algiers, where there is the detection of the production of cephalosporinases CMY-2 in K. pneumoniae and DHA-1 was detected in Enterobacter cloacae strains (Iabadene et al., 2009).So, this is the first detection of DHA-1 in K. pneumoniae in Algeria.
Typing ESBL by ERIC-PCR showed a single profile for the three strains producing CTX-M-15 and DHA-1, and a genetic diversity for other strains that produced only CTX-M-15.This suggests that the distribution of DHA-1 is the result of the spread of a single clone among patients in the two services: Women Medicine and Intensive Care Unit, hospital.
The acquisition of the gene blaCTX-M-15 by strains of K. pneumoniae probably occurred by horizontal transfer from strains of E. coli.Several studies have described that the gene blaCTX-M-15 has been carried by transferable plasmids.A study in Spain showed that the gene blaCTX-M-15 is carried by the same plasmid with a size of 180kb (Valverde et al., 2008).On the other hand, in another study, the size of plasmid was 150kb, on the basis of which it is associated with genes encoding for aminoglycosides resistance (Mesko et al., 2009).
Our study is the first report made to "Ahemida Ben Adjila" Laghouat Hospital, Algeria.It allowed us to reveal the distribution of β-lactamase CTX-M-15 and DHA-1 in K. pneumoniae strains which produce the extendedspectrum β-lactamases, with the first detection of plasmid-mediated cephalosporinases DHA-1 produced by K. pneumoniae in Algeria.
Nowadays, controlling antibiotics use is essential to monitor the increasing spread of genes resisting to anti-biotics.