Prevalence and resistance profile of extended-spectrum β-lactamases-producing Enterobacteriaceae in Ouagadougou, Burkina Faso

1 Laboratoire des Sciences Appliquées et Nutritionnelles (LabSAN), Université Ouaga 1 Pr Joseph KI-ZERBO, 03 BP 7021, Ouagadougou 03, Burkina Faso. 2 Laboratoire National de Santé Publique, 09 BP 24, Ouagadougou 09, Burkina Faso. 3 Centre Hospitalier Universitaire Yalgado OUEDRAOGO, 03 BP 7021, Ouagadougou 03, Burkina Faso. 4 Centre Hospitalier Universitaire Pédiatrique Charles De Gaulle, 01 BP 1198 Ouagadougou 01, Ouagadougou, Burkina Faso. 5 Hôpital Saint Camille de Ouagadougou, 09 BP 444, Ouagadougou 09, Burkina Faso. 6 Institut Pasteur de Côte d’Ivoire, 01 BP 490, Abidjan 01, Côte d’Ivoire. 7 Department of Microbiology, University of Lagos, Akoka, Lagos State, Nigeria.


INTRODUCTION
Bacterial resistance to antibiotics is on the raise worldwide in healthcare setting and in community which tend to be posing a lot of challenges to the effective treatment of infections. Resistance of pathogenic bacteria to β-lactam antibiotics, a group of antibiotic mostly used for the treatment of bacterial infections because of their broad antibacterial spectrum and excellent safety profile has taken a great threatening dimension with the emergence Extended-Spectrum Β-Lactamase (ESBL) producing Enterobacteriaceae (Abdallah et al., 2015). The ESBLs first described in 1983 in Germany arose from a single nucleotide polymorphism in the bla SHV genes that altered specificity to oxyimino-cephalosporins. Overtime there has been a wide spread of ESBLs with an ever evolving ability to hydrolyze penicillins, first, second and third generation cephalosporins and monobactams but not carbapenems (Lukac et al., 2015;Tekiner and Ozpinar, 2016). In Africa, there has been various reports of ESBL producing Enterobacteriaceae (ESBL-E) implicated in causing infections across all ages. Sangare et al., (2017) noted the very high and increasing frequency of ESBL-E in their report on the prevalence of ESBL-E in teaching hospitals in Mali. In a similar study Oduro-Mensah et al., (2016) reported an overall 37.96% of 137 Enterobacteriaceae clinical isolates exhibiting ESBL phenotype in Ghana with Klebsiella spp. and Escherichia coli taking a lead. To further substantiate this, Farra et al. (2016) identified the high rate of faecal carriage of ESBL-E in healthy children in Bangui Central African Republic which portend a high risk of continuous dissemination of multi-drug resistant pathogen with grave consequences to the general health of the public. However there is paucity of information and extended study of ESBL producing Enterobacteriaceae (ESBL-E) in Ouagadougou of which study conducted has been restricted to single health centers (Zeba et al., 2007;Métuor-Dabiré et al., 2014;Ouedraogo et al., 2016). Hence this study aimed to determine the prevalence of ESBL producing Enterobacteriaceae in three of the major health centers (Yalgado Ouedraogo Teaching Hospital (CHU-YO), Charles De Gaulle Paediatric Teaching Hospital (CHUP-CDG) and Saint Camille Hospital (HOSCO)) in Ouagadougou and to describe their resistance to antibiotics commonly used in the treatment of Gram negative bacterial infections.

Study site
This cross sectional study was conducted between November 2014 Kpoda et al. 1121 to October 2016 to determine the prevalence and susceptibility of Enterobacteriaceae to β-lactams, aminoglycosides and quinolones in Ouagadougou. Three major health centers Yalgado Ouedraogo Teaching Hospital (CHU-YO), Charles De Gaulle Paediatric Teaching Hospital (CHUP-CDG) and Saint Camille Hospital (HOSCO) in Ouagadougou were chosen for the study because they received the highest number of patients and cases in the city. Ouagadougou is the capital city of Burkina Faso with a population of about 2 million people. CHU-YO is the largest medical institution located in Ouagadougou. Over 150,000 patients are annually attended to in these three health care facilities.

Detection of ESBL strains
The screening and phenotypic tests for ESBL strains were performed in line with CLSI guidelines on Muller-Hinton agar. In this test, a disc of amoxycillin+clavulanic acid (20 + 10 μg) was placed at the centre of the Petri dish already inoculated with the test strain while cefepime (30 μg), cefotaxime (5 μg) and ceftriaxone (30 μg) discs were placed at a distance of 20 -25 mm (centre to centre) from the amoxycillin+ clavulanic acid disc on the same dish. Zones  of inhibition between the third generation cephalosporin discs and amoxicillin+clavulanic acid were observed after 18-24 h incubation at 37°C. Extension of inhibition zone around one or more cephalosporin discs nearest to the amoxycillin+clavulanic acid, was considered ESBL positive (CLSI, 2005).

Statistical analysis
Data was analyzed using ANOVA one-way. Chi-square test (χ2) was used to establish statistically difference in proportions for categorical data and statistical significance was set as P values of ˂ 0.05. The statistical analysis was performed using GraphPad Prism version 5.01 (GraphPad Software, Inc.).

Distribution of strains according to origin and clinical samples
Four hundred and eighty-six isolates were obtained from all three collection centers.

ESBL-producing strains
Out of 486 isolates tested, 187 (38.5%) were ESBLproducing, 127 (67.9%) from CHU-YO, 44 (23.5%) from CHUP-CDG and 16 (8.6) from HOSCO (Table 3). Plate 1 shows double disc synergy and a key hole phenomenon that was exhibited by Klebsiella pneumonia, and Table 4 shows the ESBL species distribution according to the sample. Difference between the proportions of ESBL isolates from the 3 sites was statistically significant (p <0.0001). Furthermore, the difference between the ESBL bacteria isolated was not statistically significant (p=0.1260) with respect to age. In addition, 81 (43.3%) ESBL-E isolates were obtained from patients on antibiotic treatment, of which 16.7% (31/187) of antibiotics used were β-lactams.

Resistance profile of ESBL isolates to other antibiotics
The rate of resistance of ESBL isolates to other antibiotics is shown in

DISCUSSION
In this study, we determined the prevalence of ESBL producing Enterobacteriaceae (ESBL-E) and their resistance to antibiotics commonly used in the treatment of Gram negative bacterial infections in three major health care facilities in Ouagadougou. Enterobacteriaceae remains the major pathogens causing communityacquired and hospital-acquired infections including infections of the gastrointestinal tracts, urinary tract, sepsis, meningitis and medical device-associated infections (Mathlouthi et al., 2016). Urine, of all 6 clinical sample types analyzed gave the highest number of Enterobecteriaceae of which 50.1% were from Males. One hundred and eighty-seven (38.5%) of the 486 isolates obtained were ESBL producing with 21.0% from male. This is in line with the reports of Siraj et al. (2014) in Ethiopia and Ouedraogo et al. (2016) in Burkina Faso in which urine yielded a higher number of bacterial isolates. Hijazi et al. (2016) also reported a similar finding in Lebanon with male children having a higher colonization frequency (33.9%) of ESBL-E in contrast to their female counterparts that had a frequency of (15.9%). However this observation is a deviation from the normal trend of having more bacterial isolates from female urine samples since they were more at risk of acquiring urinary tract infection compared to their male counterparts (Ameri et al., 2014 (Ndir et al., 2016). Antibiotic resistance is a global problem which varies across countries as a result of hygiene levels in hospital and antibiotic management policies. As shown in this study, resistance of Enterobacteriaceae to regularly used antibiotics is unflinching and ever evolving. There was 86.8% resistance to amoxicillin, 35.2% resistance to amoxicillin + clavulanic acid. This rate of resistance could be attributed to selective pressure since these antibiotics has overtime been a first line drug in the treatment of bacterial infections. Isolates also displayed a remarkable resistance to cephalosporins tested. There was an average resistance of 58.5% to cefotaxime and cefepime, while resistance to ceftriaxone and cefoxitine was 61.9 and 26.1% respectively (Table 2). Mathlouthi et al. (2016) affirm this finding in their report of isolates from Tunisian and Libyan hospitals with 80% resistance to ceftazidime, cefotaxime, amoxicillin + clavulanic acid, amoxicillin and ciprofloxacin. Our report of 38.5% prevalence of ESBL-E in this study is relatively high; however a similar study by Ouedraogo et al. (2016) in Burkina Faso recorded a higher prevalence of 58%. This variation in findings could be explained by the size, duration and area where the two studies were conducted. The clinical impact of ESBLproducing pathogens on morbidity and mortality in infectious diseases in both children and adults as well as their economic burden are well documented (Lukac et al., 2015). Thus, ESBL-E is a threat that should be tackled head on. Resistance of ESBL-E to aminoglycoside was    ciprofloxacin. For other antibiotics, we observed a high rate of resistance to trimethoprim-sulfamethoxazole (87.2%). Both ESBL-E and non ESBL-E isolates were susceptible to imipenem and fosfomycin. Bourjilat et al. (2011) observed a similar trend in Morocco with all ESBLproducing isolates having total susceptibility to imipenem and fosfomycin. Also, Patwardhan and Singh (2017) in India reported 1,223 (96.5%) ESBL-producing Gram negative isolates that were susceptible to fosfomycin. This gives a glimpse of hope as this two antibiotics are still very much active against ESBL-E and can serve as a ready remedy when clinicians are confronted with multidrug resistant ESBL-E. Conversely there should be caution in the use of these molecules as resistance to imipenem is beginning to emerge (Haidar et al., 2017). In Burkina Faso, as in many other African countries, the lack of antibiotic surveillance system, unfavorable hygiene conditions in hospitals, may be attributed to the spread of ESBL, as has been reflected in this study.

Conclusion
This study demonstrates the high prevalence of ESBLproducing Enterobacteriaceae in Ouagadougou. The spread of ESBL strains reduces the successful treatment ESBL bacterial infections. Nevertheless, ESBL bacteria remained susceptible to imipenem and fosfomycin, which are often drugs of choice for severe infections. This therefore highlights the need for routine detection and systematic reporting of ESBL bacteria in Burkina Faso to avoid therapeutic failures and the spread of these bacteria for effective management of bacterial infectious diseases. Clinicians must be cautious in the prescription of antibiotics. Furthermore, antibiotic policy use is needed to limit the emergence and spread of ESBL strains.