Extended-spectrum beta-lactamase-and carbapenemase-producing Enterobacteriaceae clinical isolates in a Senegalese teaching hospital : A cross sectional study

Extended-spectrum beta-lactamaseand carbapenemase-producing Enterobacteriaceae clinical isolates in a Senegalese teaching hospital: A cross sectional study Makhtar Camara, Mamadou Thierry Mane, Awa Ba-Diallo, Assane Dieng, Halimatou Diop-Ndiaye, Farba Karam, Seynabou Lo-Lo, Habsa Diagne-Samb, Safietou NgomCisse, Coumba Toure-Kane, Aïssatou Gaye-Diallo, Souleymane Mboup and Cheikh Saad Bouh Boye

Although ESBLs have been described in a range of Enterobacteriaceae, these enzymes are predominantly found in the bacterial species of Klebsiella pneumoniae and Escherichia coli (Pitout and Laupland 2008).
ESBL-producing strains of E. coli and K. pneumoniae are increasingly reported all over the world, and are important pathogens in community-and hospital-onset infections (Paterson and Bonomo, 2005).ESBLproducing Enterobacteriaceae are associated with lifethreatening infections, increased morbidity and mortality and healthcare-associated costs (Pitout, 2010).
Extensive use of expanded-spectrum antibiotics including β-lactams is one of the most important risk factors associated with high prevalence of ESBLs (Chopra et al., 2015;Shukla et al., 2004;Oteo et al., 2010).
Carbapenems are generally stable against ESBLs and still mainly used as treatment of last resort in infections caused by MDR Gram-negative bacilli (Morosini et al., 2006).The emergence of carbapenemase-producing Enterobacteriaceae (CPE) is causing an unprecedented public health threat leaving few treatment options, and consequently leads to high clinical mortality rates (Tzouvelekis et al., 2012).
Carbapenemases are the most versatile family of βlactamases and are able to hydrolyze carbapenems and other β-lactams (Queenan and Bush 2007).The most important mechanism of carbapenem resistance in Enterobacteriaceae is the production of carbapenemases, although resistance can also result from the synergistic activity between AmpC-type or ESBL combined with decreased outer membrane permeability (Pitout et al., 2015;Ruppé et al., 2015).
The recognition of MDR isolates is a major laboratory challenge and their inappropriate or delayed detection may have negative impacts on patients' management and on the implementation of infection control measures (Nordmann and Poirel, 2014).To our knowledge, the prevalence of MDR Enterobacteriaceae is not well documented in Senegal.Therefore, this study was designed to determine the prevalence of ESBL-and carbapenemase-producing Enterobacteriaceae and its antibiotic susceptibility profiles at Aristide Le Dantec University Teaching Hospital in Dakar, Senegal.

Bacterial isolates
A total of 1205 non-duplicate clinical isolates were collected from January to December 2016.Clinical specimens were cultured on Eosin Methylene Blue (EMB) agar (Merck, Germany), and incubated at 37°C for 24 h.Clinical isolates were identified using standard biochemical galleria (for E. coli, K. pneumoniae, Salmonella species, Shigella species) or Api 20E (for Citrobacter freundii, Serratia marcescens, Morganella morganii, and Providencia stuartii) for Enterobacteriaceae.
ESBL production was detected by double-disk synergy test with disks of amoxicillin-clavulanic acid surrounded at a radius of 30 mm by cefotaxime, ceftriaxone, ceftazidime and aztreonam.Bacterial suspensions at a concentration of 10 7 CFU/ml were inoculated on Mueller-Hinton agar.
Carbapenemase producing strains were detected with an inhibition zone diameter of <25 mm with ERT antibiotic disk as in CASFM ( 2016).

Statistical analysis
Differences in continuous and categorical variables between groups were analyzed with non-parametric Mann-Whitney U and chisquared tests, respectively.The level of significance for all statistical tests was set at p < 0.

Prevalence of ESBL-and carbapenemase-producing Enterobacteriaceae (CPE)
During the study period, a total of 1205 Enterobacteriaceae stains were tested for ESBL and carbapenemase production.The overall prevalence of ESBL-and carbapenemase-producing Enterobacteriaceae was 26.2 and 5.1%, respectively.3.8% of these pathogens were, however, both ESBL and carbapenemase producers.
The demographic characteristics of the study population are shown in Table 1.Male patients predominated either in ESBL-producer and CPE or in non ESBL-producer and non-CPE isolates.There was no significant difference regarding age between patients infected by these two groups of pathogens.

ESBL-
and carbapenemase-producing Enterobacteriaceae (CPE) strains are increasingly reported worldwide pathogens in community-and hospital-onset infections (Paterson and Bonomo 2005;Tzouvelekis et al., 2012), suggesting the need for continuous surveillance of antimicrobial resistance (AMR) patterns.In this study, the prevalence of ESBL-and CPE and its antibiotic susceptibility patterns in Aristide Le Dantec Teaching Hospital in Dakar, the major university hospital of Senegal was investigated.The rate of ESBLand carbapenemase-producing strains was found to be 23.6 and 5.1%, respectively.
In this study, ESBL production was mainly detected towards E. coli (45.2%) and K. pneumoniae (26.3%).Similar results have been reported from USA (Ajao et al., 2013) and India (Taneja et al., 2010), showing ESBL prevalence rates from 60 to 71% for K. pneumoniae and 35.0 to 42% for E. coli.The majority of ESBL producers were recovered from urine specimens (56.6%) and pus (22.7%).This is consistent with data reported in other studies (Obeng-Nkrumah et al., 2013;Severin et al., 2010).Our findings confirm the reports of Pitout et al. (Pitout and Laupland 2008) showing higher frequencies of ESBL-producing Enterobacteriaceae among patients with severe infections including UTIs, suppurative infections, bacteremia, and intra-abdominal.Indeed, ESBL-producing Enterobacteriaceae have been associated with serious nosocomial infection outbreaks that lead to prolonged hospital stay, increased morbidity and mortality, and consequently increased healthcare associated costs with limited therapeutic options (Pitout, 2010).Increasing rate of community-acquired infections caused by ESBL-producing Enterobacteriaceae has, however, been recently reported (Lonchel et al., 2012), representing a potential reservoir for ESBL producers.
ESBL-producing strains were more dominant among patients admitted in urology, surgery, internal medicine, and intensive care units, as reported elsewhere (Obeng-Nkrumah et al., 2013;Shu et al., 2010).
In this study, all strains were resistant to cefotaxim or ceftriaxone (99.9%).These ESBLs are plasmid mediated β-lactamases resistance and are associated with coresistance to other classes of antibiotics (Paterson and Bonomo 2005).This would explain the high rates of resistance to non-β-lactam antibiotics observed in our study, including tetracycline, sulfamethoxazole/ trimethoprim complex, chloramphenicol, and ciprofloxacin, which are comparable to rates found in other studies (Lin et al., 2012;Simner et al., 2011).Low rates of amikacin and fosfomycin resistance were detected, which is in agreement with findings from Korea (Lee et al., 2012), Taiwan (Liu et al., 2015), and Japan (Wachino et al., 2010).Carbapenems are generally stable against ESBLs and still mainly used as treatment of last resort in infections caused by multi-drug resistant bacteria.As reported elsewhere (Liu et al., 2015), the result of the present study data showed that imipenem remains very active against ESBL-producing E. coli and K. pneumoniae clinical isolates.However, ESBLproducing Enterobacteriaceae carbapenem resistant are emerging worldwide.This might be due to acquisition of cabapenem-hydrolyzing β-lactamases (Nordmann et al., 2011) or a combination of plasmid-mediated AmpC βlactamase and loss of an outer membrane protein (Dahmen et al., 2012), limiting thus the drug treatment choices.The most prevalent CPEs detected in this study were E. coli ( 45.2%), K. pneumonia (27.4%), and E. cloacae (16.1%), particularly isolated from urines (58%), and pus (19.3%).All these isolates were resistant to βlactam antibiotics.In addition, high resistance rates were observed with tetracycline (90.3%), sulfamethoxazole/trimethoprim complex (85.7%), chloramphenicol (82.3%), and ciprofloxacin (72%).Interestingly, amikacin remained fully active, while fosfomycin and colistin were respectively effective only in 66.7 and 60% of these MDR strains.In fact, only few remaining antibiotics are currently in use to treat infections caused by carbapenemase-producing Gramnegative bacilli including, colistin, tigecycline, amikacin, fosfomycin, and temocillin.Appropriate combination therapy with 2 or more drugs is superior to monotherapy and associated with better survival rate (Tumbarello et al., 2015;Daikos et al., 2014;Zarkotou et al., 2011).

ESBL-
and carbapenemase-producing Enterobacteriaceae strains are important pathogens in community-and hospital-onset infections.Emergence of carbapenem-resistant clinical isolates underscores the need for continuous surveillance of antimicrobial resistance patterns.This study shows high prevalence rates of ESBL-producing isolates and emerging prevalence of CPE with limited therapeutic options, suggesting a need for continuous MDR surveillance patterns and antibiotic combination recommendation, particularly in hospital settings.

Table 1 .
Demographics and specimen type characteristics from patients infected with ESBL-and carbapenemase-producer and non-producer isolates.

Table 2 .
Distribution of Enterobacteriaceae strains ESBL-and CARB-producer and non-producer isolates.

Table 3 .
Susceptibility rate for ESBL-and carbapenemase-producing isolates to different antibiotics.