Members of the family Enterobacteriaceae are important pathogens of humans and animals causing various infections for example in humans which include septicemia, pneumonia, peritonitis etc. (Makharita et al., 2020). They have been reported to be the main causes of nosocomial infections (Centers for Disease Control and Prevention [CDC], 2013). In animals, members of the family Enterobacteriaceae can be divided into three groups depending on their pathogenicity. They include major animal pathogens like Escherichia coli, opportunistic pathogens that occasionally cause infections in animals like Proteus spp., Enterobacter spp., Citrobacter spp. and organisms of uncertain importance for animals, e.g., Erwina spp., Leclercia adecarboxylata (Veterian Key, 2016). Several virulence factors involved in disease occurrence in members of the Enterobacteriaceae family have been previously studied which include invasion, hemolysins, siderophores and toxins which are controlled by specific virulence genes (Pearce et al., 2006).

Antibiotic resistance is a serious health concern not only among human pathogens but also in isolates found in other habitats. Many resistant pathogenic bacteria and commensals are found in different hosts, or in the environment (Taylor et al., 2001). The connections between humans, animals, and their environment allow the transfer of bacteria and mobile genetic elements between different compartments (Woolhouse and Ward, 2013). Antibiotic resistance genes are emerging environmental contaminants, as they can be transferred to other bacteria present in their environment (Krishnasamy, 2018). Multidrug resistance has increased all over the world which is considered a public health threat. Several recent investigations have reported the emergence of multidrug resistant bacterial pathogen from different origins including humans, birds, cattle, and fish (Algammal et al., 2020, 2021a,b). This increases the need for routine application of antimicrobial susceptibility testing to detect the antibiotic of choice as well as screening of the emerging MDR strains. There are different mechanisms of resistance in members of the family Enterobacteriaceae. They include modification of the target, efflux phenomenon, impermeability, and enzymatic inactivation (Kotsyuba et al., 2014). Understanding resistance mechanisms in Enterobacteriaceae including how they develop and are transmitted is important for developing ways to monitor resistance spread effectively and also have effective treatment options. The various resistance determinants can be exploited to produce possible safe and potent novel antimicrobial drugs with new mechanisms of action.

Livestock animals are linked to humans through the food chain and the environment they share (Marshall and Levy, 2011). Over the years, antimicrobials have been applied for treatment of diseases in food producing animals, but also for growth promotion and prophylactic purposes. Multiple countries including developed and developing nations like Nigeria have introduced bans on the non-therapeutic use of antimicrobials. However, the coordinated surveillance and monitoring of antimicrobial use and resistance especially in developing countries like Nigeria is still limited, resulting in an increase of resistant pathogens. Most of the main classes of antibiotics used in human medicine are represented in a list of antibiotics tagged as “critically important” for livestock animals by the World Organization for Animal Health (OIE, 2007). Antibiotic residues are released into the environment as a result of inadequate absorption and metabolic activities carried out by food animals. Antimicrobials added to food for livestock animals can be disseminated as a result of leaching and release by urine and feces (Krishnasamy, 2018). The presence of antibiotic resistance in Enterobacteriaceae as well as other bacteria has been well documented in human, animal and environmental isolates in developed countries (Yates et al., 2004; Hoyle et al., 2005; Aarestrup et al., 2009; Fischer et al., 2012; Spoor et al., 2012). In low/middle income countries like Nigeria, research focus is majorly on antimicrobial resistance in human pathogens with only a few detailed reports on animal and environmental isolates (Adelowo and Fagade, 2009; Olowe et al., 2015; Ayeni et al., 2016; Ngbede et al., 2017; Adelowo et al., 2018; Shivakumaraswamy et al., 2019). The aim of this study was to investigate antibiotic resistance and characterize the resistance mechanism in Enterobacteriaceae isolated from bovine animals and the environment in Edo state Nigeria using whole genome sequencing.

Enterobacteriaceae are typically found in animals and the environment (Otokunefor et al., 2018) and have been reported as reservoirs of resistance genes that could be passed to other bacterial cells in the system. The use of antibiotics in livestock production results in an increase in antibiotic resistant strains in people (Bevan et al., 2017). Sewage, humans, companion, and domestic animals and the industries have been previously reported as sources of resistant microorganisms to the environment in African settings (Moremi et al., 2017). Determination of antimicrobial resistance in bacteria using phenotypic characterization is very important for therapeutic purposes, but further genetic characterization may sometimes seems appropriate. Whole-genome sequencing plays a key role in enhancing our understanding of how bacteria evolve, are transmitted, and monitoring of antimicrobial resistance. Regardless of the source of isolation in this study, the phenotypic antimicrobial susceptibility testing revealed high frequency of resistance to trimethoprim and cefoxitin. Recently, plasmid mediated FOX-1 AmpC ß-lactamases were detected in Escherichia coli isolates from cows in Nigeria (Ejikeugwu et al., 2018). A recent review on antimicrobial use revealed a high level of antimicrobial usage of tetracyclines, aminoglycosides and penicillins in animal production systems in Africa because they belong to the cheapest antibiotics (Kimera et al., 2020). The previous report showed high prevalence of antimicrobial resistance including multidrug resistance in the environment. Reports from developing countries especially in Africa are of major public health concern as only limited diagnostic and therapeutic options are available (Kimera et al., 2020). The presence of antimicrobial-resistant (AMR) and multidrug- resistant (MDR) bacteria that colonize the gut of animals might play an important epidemiological role in the spread of antimicrobial resistance between livestock animals and humans, either directly or through consumption of contaminated food. Several mechanisms of bacterial resistance to antimicrobial agents have been previously reported (Kumar and Varela, 2013; Sun et al., 2014). They include antibiotic efflux, antibiotic target alteration, antibiotic inactivation, antibiotic target replacement and antibiotic target protection. In this study various resistance determinants that encode antimicrobial efflux pumps were detected both in the resistant and multidrug resistant isolates. Over-expression of such determinants has been connected to a rise of clinically important multidrug-resistant pathogens (Nikaido, 1994). The regulatory system modulating antibiotic efflux (Resistance-Nodulation-Division multidrug efflux pumps) for example has been previously reported as an important determinant of multidrug resistance in Gram-negative bacteria (Fernando and Kumar, 2013). Resistance determinants that encode multidrug efflux pumps of the major facilitator superfamily were also detected in this study and have also been previously reported to contribute to the emergence of multidrug-resistant organisms (Kumar et al., 2020). Results of this study underline the wide distribution and importance of this resistance mechanism.

Some African studies identified antimicrobial resistance genes [bla_CTX–M, bla_TEM, bla_SHV, bla_OXA, aac(6′)-lb-cr, tet(A), tet(B), sul1, sul2, qnr] in bacteria from humans, animals, and environmental sources (Hamza et al., 2016; Ribeiro et al., 2016; Eguale et al., 2017; Seni et al., 2018), indicating that drug resistant pathogens have high tendency to spread widely (De Boeck et al., 2012; Ekwanzala et al., 2018; Oloso et al., 2018). Results from this study correlate with the previous African reports as bla_TEM, aph(6)-ld, tet(A), tet(D), sul1, sul2, qnr, dfrA1, dfrA5, dfrA14 were some identified resistance genes in the resistant Enterobacteriaceae isolates. Significantly, no extended spectrum beta-lactamase (ESBL) gene was detected in the bovine and environmental Enterobacteriaceae isolates. This is in contrast with a recent report on human clinical Enterobacteriaceae isolates collected from the same geographical location that had a high rate of ESBL genes detected among the isolates (Jesumirhewe et al., 2020).

The role of Enterobacteriaceae as a reservoir for ESBL genes and other resistance determinants is enhanced by the presence of IncF plasmid family type which evolves quickly by replicon diversification and acquisition of antibiotic resistance traits (Carattoli, 2013). Various plasmid replicon types detected among the resistant isolates in this study indicate their importance for dissemination of antibiotic resistance. For example, in this study, resistance genes mediating quinolone resistance and transmitted by plasmids, oqxA, qnrD, and qnrS1 were detected in the resistant animal and environmental isolates indicating that resistance to quinolones was promoted through the plasmid-mediated determinant. Plasmid-mediated quinolone resistance (PMQR) determinants play an important role in the transmission of resistance among bacterial isolates (Santhosh et al., 2017). The predominating presence of IncF plasmid replicon type in the animal and environmental isolates increases the possibility of acquiring more resistance determinants as well as the emergence of novel resistance determinants (Amos et al., 2014). Efflux pumps have been previously reported to be encoded by plasmid borne genetic elements which plays an important role in the transmission of multidrug-resistance (Nikaido, 1994; Nelson and Levy, 2011).

Most developing countries in Africa like Nigeria have been reported to lack effective antimicrobial surveillance systems or are at different stages of developing them (Alonso et al., 2017; Kimera et al., 2020). Monitoring the emergence and spread of antibiotic resistant isolates may assist in developing strategies for treatment and prevention of infections especially for animals which have limited choice of prophylactic and therapeutic antimicrobials. A systematic antimicrobial resistance surveillance using more advanced techniques like WGS is required especially in African settings to detect new antibiotic resistance mechanisms and to determine the virulence of multidrug resistance pathogens for risk assessment and prevention of infection (Hamza et al., 2016; Grundmann and Gelband, 2018). Data from WGS could assist in planning effective interventional measures.

This study provides detailed genomic characterization of antibiotic resistance in bovine animal and environmental Enterobacteriaceae isolates for Nigeria. A number of previous Nigerian studies described the detection, frequency/prevalence of resistance to antibiotics in livestock and the environment using phenotypical techniques (Ayandiran et al., 2014; Onuoha, 2017; Otokunefor et al., 2018; Ugwu et al., 2018). Only a few Nigerian studies have been able to explore the genetic mechanisms of antibiotic resistance in bovine animals and the environment (Olowe et al., 2015; Sharma et al., 2017; Adelowo et al., 2018; Ayandiran et al., 2018; Ejikeugwu et al., 2018) which is important to understand the dissemination of resistant isolates. Further studies to characterize prevalent clones and plasmids that harbor antibiotic resistance genes are required.

Whole genome based cgMLST phylogenetic analysis of the bovine Escherichia coli isolates suggest a probable clonal spread of the isolates of different sequence types and plasmid replicon types. ST10 is an important multilocus sequence type detected in three animal Escherichia coli isolates in this study. Previously, ST10 Escherichia coli have been detected in chicken and have been reported as antibiotic resistant ESBL-producers (Chen et al., 2016). Previous reports show Escherichia coli ST10 to be commonly associated with other animals and humans (Ewers et al., 2012; Chen et al., 2016). This is consistent with our results as the isolates were found in feces of bovine animals. One limitation of this study is that samples obtained were not sufficient to get a large number of isolates to analyze possible links between the bovine and environmental isolates. Although resistance determinants were detected in both the animal and environmental isolates it would be important to further analyze isolates to characterize the possible linkages between animals and the environment.

We report a wide dissemination of antibiotic resistant and multidrug resistant Nigerian bovine and environmental Enterobacteriaceae isolates. The emergence of these resistant strains is of public health concern indicated by the difficult treatment options in infections they cause. Very little data especially genotypic studies of isolates from animals and the environment exist for surveillance of antimicrobial resistance in most developing countries like Nigeria. It is necessary for developing countries like Nigeria to carry out surveillance systems involving a “One Health” approach which would be important to monitor transmission events. Support from global antimicrobial resistance networks should be sought to assist in developing and implementing antimicrobial resistance surveillance under one health approach.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

CJ conceived, designed, performed the experiments and wrote the manuscript. CJ, BS, FA, and WR analyzed the data. All the authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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