Investigating antibiotic resistance in enterococci in Gabonese livestock

Background and Aim: The emergence of antibiotic resistance is a major problem worldwide. Antibiotics are often used to prevent or treat infections in livestock. This study aimed to investigate antibiotic resistance in enterococci in Gabonese livestock. Materials and Methods: We collected 174 animal samples (46 laying hens, 24 swine, 62 cattle, and 42 sheep) from farms in four provinces of Gabon. Bacterial strains belonging to the genus Enterococcus were obtained using selective media and polymerase chain reaction targeting the tuf gene. Antibiotic susceptibility was determined by the disk diffusion method on Mueller-Hinton agar. Results: Enterococci were present in 160 of the samples (97%), distributed as follows: laying hens (100%, 41/41), swine (100%, 22/22), small ruminants (88%, 37/42), and cattle (100%, 60/60). Resistance to cephalothin/cephalexin, streptomycin, and rifampicin (RIF) was high, and resistance to vancomycin (VAN), erythromycin, and tetracycline was moderate. A high diversity of resistance was found in Haut-Ogooué and Estuaire provinces. Laying hens and swine showed moderate levels of resistance to ciprofloxacin and penicillin, while sheep and cattle had high levels of resistance to RIF. All species showed a high level of resistance to VAN. We found various patterns of multiple resistances in the isolates, and the multiple resistance indexes ranged from 0.2 to 0.8. Conclusion: This study shows that livestock in Gabon can be considered potential reservoirs of resistance.


Introduction
The problem of antibiotic resistance plays an important role in the world due to the emergence and dissemination of resistance genes, mainly in human and animal hosts [1]. Antibiotic resistance in livestock is due to the use of antibiotics as therapeutic, prophylactic agents and growth promoters [2,3]. Antibiotic misuse could lead to the emergence of antibiotic-resistant bacteria in livestock and thus create reservoirs of resistance genes [4,5], potentially transmitted to humans through direct or indirect contact [6,7]. However, data on antibiotic resistance in low-and middle-income countries are scarce, making it challenging to establish antibiotic stewardship systems [8]. Thus, studies characterizing antibiotic resistance in animals are essential in these countries.
Antibiotic resistance is most often investigated in Enterococcus spp. because of the plasticity of their genome and the persistence of this genus in the environment, which allow it to acquire antibiotic resistance genes and colonize different ecological niches [9]. Enterococci are ubiquitous in the intestinal tract of farm animals but constitute a small proportion of bacterial ecological diversity [10]. In particular, the species Enterococcus durans, Enterococcus hirae, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus faecalis, and Enterococcus faecium are often found in the digestive tract of farm animals [11,12]. Enterococcus species have intrinsic resistance to aminoglycosides (a low-level), penicillins (a low-level), vancomycins (VANs) (E. gallinarum and E. casseliflavus), polymyxins, and streptogramins [13]. The presence of other resistances in Enterococcus species could be the result of antibiotic use, thus allowing its use as a bacterial model for the evaluation of the selection pressure created by the consumption of antibiotics in livestock.
In Gabon, studies of the presence of antibiotic resistance in hospitals [14][15][16] and mammals [17] have shown high rates of several families of antibiotics associated with resistance genes. Another study of antibiotic resistance has revealed a high prevalence to ampicillin and cephalosporins in ready-to-eat chickens [18].
However, antibiotic resistance has not been characterized in Gabonese livestock. Such studies are needed to complement the data already available for hospitals and in the environment. Thus, this study aimed to investigate antibiotic resistance in Enterococci in Gabonese livestock.

Ethical approval and Informed consent
This study was conducted in Gabon and approved by the Gabonese Ministry of Agriculture, Livestock, Fisheries, and Rural Development (General Direction of livestock, Authorization N°0052/SG/DGE). All samples from farm animals were collected after obtaining verbal consent from the farm manager.

Study period and location
The study was conducted from December 2018 to January 2020. Twenty farms were sampled in four provinces of Gabon (Estuaire, Haut Ogooué, Ngounié, and Nyanga) [ Figure-1].

Culture, isolation, and purification of colonies
Each sample was cultured on D-Coccosel (bioMérieux, France) and Slanetz-Bartley (bioMérieux) agars, which are specific culture media for enterococci, at 37°C for 18-24 h. The selection of individual suspect colonies was made according to color and morphology. Black colonies on D-Coccosel (bioMérieux) and white colonies on Slanetz-Bartley (bioMérieux) were grown on an enrichment medium at 37°C for 18-24 h.

Molecular identification of selected isolates
DNA was extracted using the Booling method described by Peng et al. [19] and quantified using a NanoDrop (Nanovue plus, UK). Genus determination was performed by amplifying a conserved sequence of the tuf gene using the primers: 5'-TACTGACAAACCATTCATGATG-3' and 5'-AACTTCGTCACCAACGCGAAC-3' described by Iweriebor et al. [20]. The polymerase chain reaction (PCR) mix consisted of 3 µL of template DNA and 17 µL of reaction mixture consisting of ×1 buffer, 0.2 mM dNTPs, 2.5 mM MgCl 2 , 0.2 mM of each primer, 50 µL of nuclease-free water, and 0.5 U/mL Taq polymerase for a final volume of 20 µL/tube. The PCR program was 3 min of initial denaturation at 95°C, followed by 35 cycles of denaturation at 95°C for 30 s, hybridization at 55°C for 30 s, elongation at 72°C for 60 s, and final elongation at 72°C for 7 min. The amplicons obtained were revealed after migration by electrophoresis on the 1% agarose gel at 100 V for 40 min with red gel and observed under ultraviolet light (ALLIANCE 4.7 transilluminator Merton, France). After confirming the presence of the required PCR products on the gel, some amplicons were sent to Macrogen (Amsterdam, Pays-Bas) for Sanger sequencing. Analysis and identification of these sequences were carried out online using the BLAST program available on the National Center for Biotechnology Information website (http://www.ncbi. nlm.nih.gov).

Statistical analysis
The multiple antibiotic resistance index (MARI) was calculated following Krumperman [23] and the multidrug resistance (MDR) profile was described as resistance to a minimum of one antibiotic in a minimum of three antimicrobial classes. Statistical analyses were performed using R software (version Ri386 3.5.1, Foundation for Statistical Computing, Vienna, Austria). The Chi-square test was used to test the relationship between province surveyed and prevalence. We considered differences significant at p<0.05.  Table-1).

Susceptibility to antibiotics by animal species
We found a high prevalence of resistance to TET in laying hens (78%) and swine (41%) but a low prevalence for cattle (8%) and small ruminants (1%). RIF resistance had a high prevalence in small ruminants (67%) and cattle (83%) but a moderate prevalence in laying hens (19%) and swine (36%). Moderate prevalence of resistance to ERY was found for laying hens (39%) and small ruminants (16%), whereas there was a high prevalence of resistance to VAN in swine (95%) and moderate in small ruminants (32%), laying hens (34%), and cattle (20%). Among antibiotics tested in laying hens and swine, CEP resistance was high in swine (100%) and moderate in laying hens (51%). In contrast, a moderate prevalence of resistance was found for penicillin (15 and 14%, respectively, for laying hens and swine). STR and CIP resistance showed moderate prevalence in laying hens (61% and 32%) and swine (27% and 68%) ( Table-2).

Multiple antibiotic resistance profile
The MDR profile of the Enterococcus revealed resistance to a minimum of three antimicrobial classes with a MARI of 0.2-1 Fifty-two (32%) isolates were resistant to multiple drugs (Tables-3 and 4). Two sheep isolates had a MARI of 0.5-0.8 and were resistant to three antibiotic classes only, whereas 18 cattle isolates had a MARI of 0.5-0.6 but no MDR detected (Table-4 (Table-4) whereas from 0.8-0.5 in Ngounié and was 0.6 in Nyanga (Table-3). Twentyfour isolates were MDR in Haut-Ogooué, while 16 isolates were MDR in Estuaire. Two MDR isolates were obtained in Ngounie and none in Nyanga (Table-3).

Distribution of enterococci
Antibiotic resistance is a major problem in the world today in the environment, animals, and humans. The emergence of resistance in livestock is most often the result of antibiotic consumption [20]. Studies of resistance in livestock are important to assess the emergence and spread of resistance in farms. This study investigated data on antibiotic resistance in the Enterococcus genus in Gabonese livestock. The high prevalence of these bacteria was obtained in all species studied. Our results are not surprising as several studies have shown a similar prevalence in farm animals [24][25][26] and Enterococcus is ubiquitous in humans, animals, and in the environment [13,27].

Resistance prevalence Enterococci
A high prevalence of resistance to CEP (68%) and moderate for STR (49%) was obtained in enterococcal isolates. These results are similar to those from South Africa [20,28], Nigeria [29], and Angola [30] and could be explained by intrinsic resistance to clinically achievable concentrations of these antibiotics in enterococci [31]. In fact, Enterococci exhibit intrinsic resistance for cephalosporins caused by low expression of penicillin-binding proteins and poor uptake of antibiotics, enzyme-mediated resistance, or sterically hindered ribosome target sites for aminoglycoside.
Among the other resistances observed, a high prevalence of resistance to RIF (57%) and moderate to VAN (35%) and ERY (16%) was found. Similar results were obtained in Tanzanian [32,33] and Nigerian [34] livestock for RIF. Several authors have suggested that this resistance could be the result of the transmission of bacteria from humans to farm animals through the consumption of human waste or contaminated water when the animals roam or during transhumance [32,35,36].
The rpoB gene encoding the b subunit of RNA polymerase is responsible for observed resistance to RIF [37]. For resistance to VAN and ERY, it should be remembered that these antibiotics are not used in veterinary medicine in Gabon. However, they have been linked to the emergence of resistance in Europe due to their use as a growth promotor in European livestock [38][39][40][41]. The mechanism of resistance to glycopeptides relies on binding to the D-Ala-D-Ala pentapeptide terminus which binds to VAN, thereby modifying the terminus to D-Ala-D-Lac or to D-Ala-D-Ser [42]. Resistance to MLS B is through three mechanisms: methylation of 23SrRNA, active efflux, and inactivating enzymes [31]. The ermB and vanC gene is the most common acquired resistance to ERY and VAN in African livestock [5,43]. This result could be due to persistence of resistance in animals originating from Europe, where a high prevalence of this resistance was observed in the previous years [40][41].
TET, which is widely used in veterinary medicine due to its broad spectrum of action on a variety of pathogens, had a moderate prevalence of resistance in our study (20%). This prevalence is high compared to studies in Nigeria [44] and Ethiopia [45] and may be related to the frequent use of these drugs in veterinary medicine, which could increase the number of resistant strains [46]. The main mechanisms of resistance include efflux pumps, modification of ribosomal RNA, and inactivation of the antibiotic. The tet(M) gene coding for ribosomal protection is frequently detected in African livestock [47][48][49].

Prevalence of resistance in enterococci by province
The identification of areas where resistance is emerging is an important step in the investigation of resistance for antibiotics used in veterinary medicine [50]. In our study, a diversity of antibiotic resistance was observed in the provinces of Haut-Ogoouée and Estuaire. These results could be related to the higher number of livestock found in these provinces due to the high demand for animal protein [51,52]. The use of antibiotics in animal husbandry is more important in places with high human density and directly correlates with resistance in livestock.

Antibiotic susceptibility of the genus Enterococcus in farm animals
In Gabon, the most exploited livestock are chickens, swine, sheep, and cattle. In this study, CIP resistance was moderate for laying hens (32%) and high for swine (68%). Similar prevalences have been observed in Tunisia [49] and South Africa [28]. These results are surprising because quinolones are not indicated for the treatment of infections in Gabonese livestock. This result could be due to the transfer of mobile genetic elements in the intestinal tract of animals leading to acquired resistance. AMP showed moderate resistance in laying hens (15%) and swine (14%). Similar results were obtained in Tanzanian livestock [35]. AMP is used to treat Enterococci infection (e.g., urinary tract infection or non-endocarditis infection) in hospitals [53]. A high rate of resistance to VAN was linked in all species of animals studied. It is necessary to determine its resistance by other tests (minimum inhibitory concentration and PCR test) to confirm resistance to the glycopeptides. However, E. gallinarum and E. casseliflavus species carry acquired resistance to VAN. A species description would be necessary to confirm the absence of these two species in our study.

Phenotypic profile of resistance
MARI and MDR were higher in laying hens and swine in our study and in Egyptian [54] and Nigerian livestock [25]. This result could be due to the health requirement of these animals compared to cattle and  small ruminants, which are grazing animals compared to swine and laying hens. Haut-Ogooué and Estuaire had higher indexes and multiple resistances than the provinces of Nyanga and Ngounié. This result reflects the high use of antibiotics in these provinces with a high risk of contamination in humans or the environment such as wastewater [54,55]. Laying hens and swine in Gabon present in the Estuaire and Haut-Ogouée provinces could be considered as reservoirs of antibiotic resistance genes.

Conclusion
This study investigated antibiotic resistance in livestock farms in Gabon. In sum, Gabonese livestock can be considered potential reservoirs of resistance genes that could be disseminated in the environment. Our study complements data characterizing resistance in humans, animals, and the Gabon environment. Molecular characterization of the resistance obtained would allow a better description of the circulation of resistance genes. Description of the species of Enterococcus spp. associated with the various resistances would also be useful.

Authors' Contributions
OED: Conceptualization of the study, investigation, data curation, formal analysis, and methodology. BL and MMR: Review and editing of the manuscript. OR: Supervision of the study. KBS: Project administration. All authors read and approved the final manuscript.