Epidemiology and molecular characterization of Enterobacteriaceae producing extended-spectrum β-lactamase in intensive and extensive breeding animals in Burkina Faso

¹Bacteriology Laboratory Centre Muraz, Muraz Center, Mamadou Konate Avenue, 01 BP 390 Bobo Dioulasso 01, Burkina Faso, ²Bacteriology Virology Laboratory Teaching Hospital Souro Sanou, Teaching Hospital Souro Sanou, Governor William Ponty Avenue, 01 BP 676 Bobo-Dioulasso 01, Burkina Faso, ³Montpellier Teaching hospital- Hospital Arnaud de Villeneuve, Department of Bacteriology 191 Dean Gaston Giraud Avenue, 34,295 Montpellier Cedex 5, France, ⁴Infectious Diseases and Vectors Laboratory, Ecology, Genetics, Evolution and Control, Jean-François Breton, Montpellier, France, ⁵Department of Infectious Diseases Souro Sanou Teaching Hospital, Souro Sanou Teaching Hospital, Governor William Ponty Avenue, 01 BP 676 Bobo-Dioulasso 01, Burkina Faso, ⁶Teaching and Research Laboratory in Animal Health and Biotechnology (LARESBA), Institute of Rural Development, Nazi Boni University of Bobo-Dioulasso, 01 BP 1091 Bobo-Dioulasso 01, Burkina Faso, ⁷Bacteriology, Virology Laboratory Charles De Gaulles Teaching Hospital, Charles De Gaulles Pediatric Teaching Hospital, 979 Tensoba Boulevard, 01 BP 1198 Ouagadougou 01, Burkina Faso

^&Corresponding author
Soufiane Sanou, Bacteriology Laboratory Centre Muraz, Muraz Center, Mamadou Konate Avenue, 01 BP 390 Bobo Dioulasso 01, Burkina Faso

Introduction    

Antimicrobial resistance (AMR) is a global public health threat. The first global surveillance report on antibiotic resistance (ABR) published by WHO in 2014, shows that 45% of deaths in both Africa and South-East Asia were due to multi-drug resistant (MDR) bacteria [1]. Antimicrobial use, misuse, or overuse in clinical medicine is a major contributing factor in the development of AMR in human populations [2-4]. As in clinical medicine, antimicrobials are also widely used in domestic animals and livestock [5]. Sub-therapeutic doses of antimicrobials are used for growth promotion in some countries [6]. This antimicrobial exposure is thought to be an important selective pressure for AMR in animals [7]. Conversely, studies have also shown that various multidrug-resistant bacteria lineages from animals also appear in humans [8,9]. Generally, the AMR bacteria colonize the gut of animals and might play an epidemiological role in the spread of resistance between Foods Producing Animals (FPA) and humans, either through direct contact or consumption of contaminated meat [10].

According to the Food and Agriculture Organization of the United Nations (FAO), worldwide, pork (36.3%) chicken (35.2%), and beef (22.2%) are the most common meat sources [11]. Production of extended-spectrum β-lactamases (ESBLs) is the most common mechanism of resistance to third-generation cephalosporins among Enterobacteriaceae [12,13]. These resistant strains are considered a significant public health issue due to the limited therapeutic [1] options and increased morbidity and mortality associated with them [2]. The true prevalence of ESBL is not well-known in Africa and is probably underestimated because of the paucity of studies in human health, animal health, and the food chain on the continent [14]. Here, we investigate the epidemiology and molecular characterization of Enterobacteriaceae producing extended-spectrum β-lactamase in intensive and extensive breeding animals in Burkina Faso.

Methods     

A descriptive cross-sectional study was carried out for four (4) months from March to June 2017 in the Bobo Dioulasso area.

Samples collection: we investigated stool samples collected from healthy animals (cattle = 251; pigs = 250, local poultry = 153 and exotic poultry = 244) in one (1) slaughterhouse (for cattle and pigs), five (5) livestock farms, and one (1) poultry market (for poultry) (Figure 1). For cattles and pigs, samples were collected aseptically with a swab from the colon immediately after slaughtering the animal at the abattoirs. For poultry, we made a cloacal swab. The concept of antibiotic supplementation was investigated through a questionnaire addressed to farm staff.

Isolation and identification: the swab were immediately incubated in broth heart brains. After 12-hours of incubation at 37°C, broths were subcultured on Hektoen media supplemented with 4μg/ml cefotaxim at 37°C for 24h as previously described [15,16]. Each type of colony has been purified on Hektoen media supplemented with 4μg/ml cefotaxim at 37°C for 24h and the strains were stored at -80°C until their identification by MALDI-TOF at Arnaud De Villeneuve teaching hospital at Montpellier (France). The strain E. coli ATCC 25922 was used as negative control and the strain K. pneumoniae ATCC 700603 was used as a positive control.

Susceptibility test: antimicrobial susceptibility was tested with the disk diffusion method on Müller-Hinton agar. The following antibiotics were tested: amoxicillin, amoxicillin-clavulanic acid, aztreonam, cefalexin, cefepim, cefotaxim, cefpodoxim, cefoxitin, ceftazidim, piperacillin, piperacillin-tazobactam, ticarcillin, ticarcillin-clavulanic acid, imipenem, Ertapenem, nalidixic acid, ciprofloxacin, levofloxacin, amikacin, gentamicin, tobramycin, fosfomycin, chloramphenicol, and trimethoprim-sulfamethoxazol. Results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints (Version 5.0) [17]. Extended-spectrum β-lactamase production was detected by using the combined double-disk synergy method [18]. For this purpose, Cefepim disc and Ticarcillin + Clavulanic acid disc were used because of the possibility of associated cephalosporinase production. Deoxyribonucleic acid (DNA) was extracted from one single colony for each isolate in a final volume of 100 μL of distilled water by incubation at 95°C for 10 min followed by a centrifugation step. The presence of, bla_CTX-M (CTX-M group 1, 2, 8, 9, and 25), bla_TEM, bla_SHV, and bla_OXA-1-like genes was assessed by multiplex PCR according to a previously published method [19]. Deoxyribonucleic acid from reference bla_CTX-M, bla_TEM, bla_SHV-positive strains were used as positives controls. E. coli phylogenetic grouping was performed using the PCR-based method described by Clermont et al. [20]. Polymerase chain reaction products were visualized after electrophoresis on 1.5% agarose gels containing ethidium bromide at 100 V for 90 minutes. A 100 bp Deoxyribonucleic acid ladder (Promega, USA) was used as a marker size. Polymerase chain reaction products were purified using the ExoSAP-IT purification kit (GE Healthcare, Piscataway, NJ, USA) and sequenced bi-directionally on a 3100 ABI Prism Genetic Analyzer (Applied Biosystems). Nucleotide sequence alignment and analyses were performed online using the BLAST program available at the National Center for Biotechnology Information [21].

Statistical analysis: data were analyzed with Stata/IC version 13.0. The difference in the proportion of ESBL-producers between poultry groups was assessed using the chi-square test. A p-value < 0.05 was considered statistically significant.

Ethics approval and consent to participate: Burkina Faso does not have an ethics committee for animal studies, so we obtained written permission from the provincial department of animal resources of Houet (covering our study area) to conduct our study.

Results     

We identified three (3) enterobacterial species producing ESBL: E. coli (278/315), K. pneumoniae (36/315), and Citrobacter amalonaticus (1/315). Enterobacteriaceae producing ESBL was detected in 41.03% of cattle (103/251), 69.6% of pigs (174/250). Among the poultry, the Enterobacteriaceae producing ESBL was detected in 0.8% of poultry in intensive breeding (02/244) and 19.6% of poultry in extensive breeding (30/153) (p-value ˂10-3 ) (Table 1). The PCR detected three resistance genes carried: one ESBL (Bla _CTX-M15) and two beta-lactamase genes (Bla TEM, Bla Oxa - 1 like) (Table 2). Phylogenetic groups could not be determined in all E. coli strains. The E. coli strains were distributed in nine phylogroup differents. The phylogroup A was the most represented among all the animals: 56.34% (40/71) among cattle, 58.50% (86/147) among pigs, and 60.71% (17/28) among poultry (Table 2).

The notion of antibiotics supplementation: this notion was found among poultry extensive and intensive breeding.

Antibiotic susceptibility patterns of Enterobacteriaceae isolates: the overall resistance of the isolates to antibiotics, (Figure 2), shows that resistance to amoxicillin (100%), Ceftazidim (99.05%), and Cotrimoxazol (86.39%) was high while resistance to Chloramphenicol (5.67%), Netilmicin (2.1%), and Amikacin (0.3%) was low. All the isolates were susceptibles to Ertapenem and Imipenem.

Discussion     

In this study, we investigated for the first time in Burkina Faso, the prevalence of ESBL in extensive and intensive breeding animals. Our findings show a high prevalence of ESBL in livestock in Burkina Faso (41.03% among cattle, 69.6% among pigs, and 19.6% among poultry in extensive breeding). E. coli was the most common enterobacterial species producing ESBL (278/315). One ESBL gene, belonging to the CTX-M-1 group, has been found in our study; it was the blaCTX-M-15 gene. This blaCTX-M-15 gene was often associated with the blaTEM gene and the BlaOxa-1-like gene. This study reiterates the finding in other studies worldwide, that have reported antibiotic resistance among bacteria especially E. coli isolated from animals [12,22,23]. Earlier studies reported that Klebsiella species and E. coli are the most species that produce ESBLs [24]. E. coli is considered an indicator, being a commensal bacterium ubiquitous in animals and capable of providing relevant hints on the spread of antibiotic resistance [25].

The ESBLs belonging to the CTX-M family of enzymes have been reported worldwide from a variety of different food-producing animals [12,26-28], and these animals are recognized as reservoirs of ESBLs producers [27]. In the majority of the surveys, blaCTX-M-15 was the most common ESBL gene detected worldwide in animals and humans [12,29,30]. This result has been also found in Burkina Faso among human clinical samples and fecal carriage [31,32]. Then E. coli strains belonged majority to phylogroup A in our study. The majority of african studies showed also a dominance of phylogroups A and B1 among healthy animals, human clinical samples, and human fecal carriage [12,31,32].

In our study, the prevalence of ESBLS was statistically higher among poultry extensive breeding than poultry intensive breeding and the notion of antibiotics supplementation was found among both. This notion of antibiotics supplementation could not be found among pigs, cattle (at slaughterhouse), and poultry (in the poultry market). Indeed, the animals intended for slaughter come from the urban livestock farms of Bobo-Dioulasso (38%) and the villages near and far on the periphery of Bobo Dioulasso (62%) [33]. There are two main poultry production systems in Burkina Faso, the extensive and the intensive system. The former, using local poultry, is well spread throughout the country. The intensive system, restricted to some peri-urban areas, uses exotic breeds. Rural poultry production is important and widely distributed among smallholder farms [34]. Despite in our study, like in several countries, cephalosporins are not used for animals, but the high prevalence of ESBL-producing bacteria remains [35,36]. This suggests that there are additional sources for the contamination with ESBL-producing bacteria in livestock [37]. Our situation may be explained by environmental contamination. Indeed, ESBL-producing bacteria may spread to the environment by waste products from human activities and animal production [38]. In Burkina Faso, extensive livestock farming is mainly practiced. In this extensive system, animals reared in partial confinement with supplementation, feed in the environment during pasturage (cattle) or their divagation (pigs, poultry) [33,34,39,40].

In this study, the antibiotics more used in supplementation were oxytetracyclin (Cyclin) and colistin. In Burkina Faso, antimicrobials are used in livestock in four areas: therapeutics, metaphylaxis, prophylaxis or prevention, and as a growth factor. A study conducted in 2016 found that 93.65% of farms used antimicrobials as growth promoters [41]. This practice, forbidden in Europe and North America, is also widespread in Africa [42-44]. In addition to their use as supplements, antibiotics are used as self-medication by 74.60% of livestock farmers in Burkina Faso [41]. And according to the evolution of clinical signs, 69.84% of farmers then use technical agents for veterinary care of the animals [41]. Gram positive bacteria, yeast, and fungi were not considered in this study. We were unable to determine the multi locus sequencing typing (MLST), conjugation experiment, and the supports of resistance genes.

Conclusion     

This study has revealed, for the first time, the high prevalence of fecal carriage of CTX - M -15 ESBL among pigs, cattle, and poultry in breeding in Burkina Faso. This study strongly indicates the urgent need to establish integrated national programs of surveillance of antimicrobial use and occurrence of ESBL-producing bacteria and other antimicrobial-resistant bacteria (with zoonotic potential) among people, livestock, and the environment in Burkina Faso.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

SS, ASO, SG and JPH, conceived and designed the experiments. SS and AZ collected the samples and done the preliminary experiments. SS, ML and OOM realized the experiments. SS, AP, JZ, RT/O, ASO, GAO, JPH and SG Contributed to the writing of the manuscript. All authors read and approved the final manuscript.

Acknowledgments     

We express sincere gratitude to all the individuals of slaughterhouse, the veterinary services of Bobo dioulasso, and Mr Rouamba Jeremi for their support during the study. We also acknowledge all the bacteriology laboratory actors of Arnaud de Villeneuve hospital at Montpellier, France.

Tables     

Table 1: distribution of Enterobacteriaceae producing ESBL carried by animals

Table 2: analysis of antibiotic resistance genes carried and distribution of E. coli phylogroups

Figure 1: mapping of samples

Figure 2: susceptibility patterns of Enterobacteriaceae isolates of animals on the classes of antibiotics tested

References