Phenotypic Detection of Extended Spectrum Beta-Lactamase and Carbapenemases Produced by Klebsiella spp Isolated from Three Referrals Hospitals in Yaounde, Cameroon

Aims: The main objective of this study was to determine the resistance phenotype of β-lactamines by Klebsiella in three hospitals in Yaoundé. Study Design: A cross-sectional descriptive study. Methodology: A cross-sectional descriptive study was carried out over a 6 month-period from May to November 2013 in the bacteriology laboratories of three referrals hospitals in Yaounde. 99 strains of Klebsiella spp. were collected for the study. Antimicrobial susceptibility testing was done using the disc diffusion method. The antibiotics tested were the β-lactams, other inhibitors like clavulanic acid, tazobactam, Ethylene-Diamine-Tetra-Acetic Acid (EDTA), cloxacillin and 3-aminophenyl boronic acid hydrochloride. The minimum inhibitory concentration (MIC) was also determined to enable the classification of the different resistance phenotypes. Results: Ninety-nine Klebsiella spp. were identified from urine (52.5%), blood (21.2%), pus (15.2%) and others sites (11.1%). The distribution of the Klebsiella spp . was: Klebsiella pneumoniae pneumoniae (78.7%), Klebsiella oxytoca (12.12%), Klebsiella pneumoniae ozaenae (5.05%), and Klebsiella pneumoniae rhinoscleromatis (4.04%). The isolates were most resistant to piperacillin (76%) and cephalothin, (85%). The most active antibiotics were imipenem (99%), and ertapenem (77%). The phenotypic tests revealed the following resistance phenotypes: extended-spectrum beta-lactamase (30.30%), wild (27.27%), penicillinase resistant to inhibitors (16.16%), carbapenemase (11.11%). Out of these 99 Klebsiella spp . , 5 were carbapenemases producers of class C and 6 of class D. The MIC were variable with different antibiotics tested but the MIC of imipenem were always lower than 1 µg/ml. Conclusion: The interpretation of the antimicrobial susceptibility testing has enabled the establishment of a high prevalence of expanded spectrum β-lactamase and consequently leading to an increase in the presence of carbapenemase producing Klebsiella spp. This could lead to therapeutic failure in case of treatment with beta-lactamines antibiotics. Therefore this trend needs to be monitored.


INTRODUCTION
Bacterial infections acquired in the hospital as well as in the community settings are a serious public health concern. These infections are mostly caused by Gram-negative bacilli which are nowadays increasingly implicated in the development of antibiotic resistance. The resistance of Klebsiella to antibiotics has in recent years experienced an alarming global increase [1].
Since 1980s, the third generation cephalosporins (3GC) belong to the group of antibiotic used for the treatment of severe enterobacterial infection. Quickly the extended-spectrum beta-lactamases (ESBLs), Plasmid transmitted enzymes responsible for resistance to penicillins, cephalosporins and to monobactams began to develop. Due to the broad spectrum activity of the carbapenemes, they have recently become the alternatives to the 3GC [2].
The ESBLs are broad spectrum enzymes conferring resistance to bacteria against most of the β-lactam antibiotics except cephamycins and carbapenemes [2]. The resistance of gram-negative bacilli to carbapenems, particularly by the production of transmissible carbapenemases is a major public health problem [3]. Most of these Carbapenemases have been identified in Klebsiella pneumoniae [4]. The emergence and spread of carbapenemase-producing Enterobacteriaceae in different parts of the world is a major threat especially in nosocomial infections [2]. The isolation of carbapenemaseproducing Klebsiella spp. is increasing worldwide [4]. In Cameroon, some phenotypic studies presumptively reveal the existence of carbapenemase-producing Klebsiella spp. (C Mbakop, University of Yaounde 1, Cameroon, Unpublished results).
Klebsiella spp. are involved in many nosocomial infections and many strains are resistant to antibiotics. Studies carried out at the Yaounde Central Hospital found a resistance rate of 18.8% of ESBL -producing Klebsiella [5]. However, this observation is unknown in many other hospitals. This study aimed at determining the resistance and the phenotypes of Klebsiella spp., isolated from three clinical laboratories in three referrals hospitals in Yaounde.

MATERIALS AND METHODS
Ninety-nine Klebsiella spp. were isolated from all clinical specimens at three bacteriology laboratories from three referrals hospitals in Yaounde from May to November 2013. The identification of the Klebsiella isolates was done using standard bacteriological procedures and biochemically using the API 20E gallery (BioMérieux SA, Lyon, France).
The minimum inhibitory concentration (MIC) was determined using the E-test. The phenotypes were determined from the interpretation of the antimicrobial susceptibility testing as recommended by the CA-SFM. Detection of ESBLs was done using the synergy test by the combination of amoxicillin and clavulanic acid disks, ceftazidime, aztreonam and cefepime.
The detection and classification of carbapenemase were performed by the modified Hodge test and the use of different inhibitors (cloxacillin, boronic acid and EDTA)

RESULTS AND DISCUSSION
This study was conducted in the bacteriology laboratories of three public referrals hospitals in Yaounde: University Teaching Hospital, Yaoundé (UTHY), Yaounde General Hospital (YGH) and Gynaeco-Obstetric and Pediatric Hospital, Yaounde (HGOPY). Among the 99 strains of Klebsiella spp. isolated from 99 patients, most of them were from hospitalized patients (n = 85 or 85.85%), mainly from the pediatrics unit, n = 34 (35.35%) and intensive care unit n = 27 (27, 27%). These results are similar to those obtained in a study carried out in Cameroon which revealed a predominance of Klebsiella in the pediatric unit (48.4%) and medical intensive care unit (21.3%) (C Mbakop, University of Yaounde 1, Cameroon, Unpublished results). This could be partly due to misuse of antibiotics or poor application of hygiene in these units. On the other hand it may be due to a high concentration of patients in these units of intensive care which could enhance the transmission of germs between patients [3]. Table 1 shows that all strains were resistant to amoxicillin (aminopenicillin) and ticarcillin (carboxypenicillin).
The wild type was found in all strains. The isolated bacterial strains expressed high resistance to ureidopenicillins (piperacillin, 76%) and first-generation cephalosporins (1GC: cephalothin, 85%).
Regarding the sensitivity of strains to antibiotics, they expressed a high level of resistance with respect to most of the antibiotics tested. The wild phenotype was confirmed for all of our strains as recommended by CASFM, [6]. Overall, there has been a slight increase in the level of antibiotic resistance as comparison to a 2011 study (C Mbakop, University of Yaounde 1, Cameroon, Unpublished results) with a p-value = 0.001. The resistance of the 1GC (cephalothin), 2GC (cefoxitin), 3GC (ceftazidime), 4GCs (cefepime), monobactam (aztreonam) and imipenem were 85%, 55%, 54%, 56%, 51% and 1% respectively. They also found 84%, 52%, 45%, 50.3%, 38% and 12% respectively. This increased level of resistance to β-lactam antibiotics could probably be as a result of inappropriate prescribing and indiscriminate use of this antibiotic family [7]. The high prevalence of "resistant" phenotypes acquired primarily within hospital strains (strains 66/85 or 77.64%) shows the possibility of an increased antibiotics selection pressure [8,9,10]. In addition, Escherichia coli and Klebsiella spp. have usually been cited as being the Enterobacteriaceae species presenting the most resistant phenotypes [11,12]. The frequency of the "wild type" (27.27%) obtained in our study was low and well below the frequency of the known wild type of Klebsiella spp. (70%) [13,14].
The interpretation of the AST enabled us to highlight the different resistance phenotypes expressed by the isolates (Fig. 1, Table 2, Table  3, Fig. 2).
Nearly a third of the Klebsiella spp. were ESBL producers (30.3%), showing a remarkable increase in the frequency of these strains in Cameroon, as previous studies in the Yaounde Central Hospital and UTHY had revealed frequencies of 18.8% in 2005 [15] and 29% in 2012 respectively (N Kouya, School of Health Sciences / Catholic University of Central Africa, Cameroon, Unpublished results). However, the Alert, Investigation and Surveillance of Nosocomial Infections Network in France indicated in 2008 that 17% of K. pneumoniae responsible for bacteremia were ESBLproducing. The high frequency of ESBLproducing Klebsiella in our context would be the consequence of regular use of β-lactam antibiotics or prolonged hospitalization of patients [16]. Resistance related to the production of carbapenemase by E. coli and Klebsiella spp. have been described [17,5]

NB : Resistant bacterial strain by definition is a non-susceptible strain , that is categorized "resistant " or "intermediate"
The distribution of these carbapenemases with respect to the status permitted us to see that all carbapenemase -producing strains were isolated from hospitalized patients (shown on Table 2). There was an association between ESBL producing strains and carb hospitalized status and hospital units. There was a high prevalence of ESBL-producing strains and/or carbapenemase in patients hospitalized in the pediatric wards and intensive care unit.
The high prevalence of ESBL and carbapenemase-producing strains in hospitalized patient in the pediatric (36.36%) and intensive care units (36.36%) may be due to the fact that   The distribution of these carbapenemases with respect to the status permitted us to see that all producing strains were isolated from hospitalized patients (shown on Table 2). There was an association between ESBLproducing strains and carbapenemase, hospitalized status and hospital units. There was producing strains and/or carbapenemase in patients hospitalized in the pediatric wards and intensive care unit.
The high prevalence of ESBL and producing strains in hospitalized patient in the pediatric (36.36%) and intensive care units (36.36%) may be due to the fact that ESBL and carbapenemase enzymes are produced mostly by nosocomial strains responsible for some hospital outbreaks [19] and in the units with prolonged hospitalization [20]. All these suggest that frail individuals are the most exposed.
Indeed, it has been observed that the acquisition of ESBL and carbapenemase concerns patients with prolonged hospitalization and exposure to invasive devices [21]. Other risk factors may include malnutrition, hemodialysis, total parenteral nutrition, ICU admission or prior hospitalization. Some studies have reported that the restriction of the use of antibiotics has ESBL and carbapenemase enzymes are produced mostly by nosocomial strains responsible for some hospital outbreaks [19] and the units with prolonged hospitalization [20]. All these suggest that frail individuals are the most Indeed, it has been observed that the acquisition of ESBL and carbapenemase concerns patients and exposure to invasive devices [21]. Other risk factors may include malnutrition, hemodialysis, total parenteral nutrition, ICU admission or prior hospitalization. Some studies have reported that the restriction of the use of antibiotics has  Table 3 shows that ESBL phenotypes n = 27 (90%) and carbapenemase n = 9 (81.8%) were the most observed by Klebsiella pneumoniae pneumoniae.

Phenotypic classification of strains
The class D carbapenemase (OXA-48) was first described in K. pneumoniae and was often associated with other β-lactamases, especially ESBL [23]. In Turkey, several outbreaks of nosocomial infections have been associated with strains producing this type of carbapenemase [20,23]. To our knowledge, no data are available on the clinical impact of cephalosporinases whose spectrum is partially expanded to carbapenemes (Class C).
As shown on Fig. 2, the sensitivity of strains to antibiotics depends on the method used. Out of the 29 strains resistant to cefotaxime by agar diffusion method, only 23 were confirmed by determining the minimum inhibitory concentration (MIC); the other 6 were categorized as "sensitive" or intermediate by the latter. Table 4 describes the characteristics of some Klebsiella strains with respect to the specimens, the hospital unit from which it was isolated, the diameter of the zone of inhibition and MIC values of some beta lactams (ceftazidime, cefotaxime, cefepime and impenem).

CONCLUSION
The interpretation of the AST has enabled the establishment of a high prevalence of expanded spectrum β-lactamase and consequently leading to an increase in the presence of carbapenemase producing Klebsiella spp. This could lead to therapeutic failure in case of treatment with beta-lactamines antibiotics. Therefore this trend needs to be monitored.

CONSENT
All the patients recruited for the study signed the consent form.

ETHICAL APPROVAL
An ethical clearance for this study was obtained from the National Ethic Committee for Human Research in Cameroon.