Introduction

Enterococci are commensal bacteria of the gastrointestinal tract of animals and humans (Zischka et al. 2015; Wurster et al. 2016). They are usually associated with nosocomial infections such as endocarditis, bacteremia, wound and urinary tract infections (O’Driscoll and Crank 2015). Two species, Enterococcus faecium and Enterococcus faecalis, are usually implicated in these infections (Osman et al. 2016). Enterococci are known to easily acquire and transfer virulence and antimicrobial resistance determinants by means of mobile genetic elements from and to other animal and human commensals and pathogens alike (Lozano et al. 2015a; Oliveira et al. 2016).

Genetic similarity has been reported to exist between animal strains and those causing human nosocomial infections (Yilmaz et al. 2016; Hermanovska et al. 2016). Cases of human infections by animal strains in addition to the transfer of virulence and resistance determinants from animal strains to human strains have also been reported (Hammerum 2012; Nilsson 2012; Bourafa et al. 2015). This finding raises a major public health concern in Nigeria, where the practice of using untreated poultry and cattle manure for fertilizing agricultural lands especially vegetable beds is common (Ajayeoba et al. 2016). Also of concern is that most of these vegetables are consumed raw without prior heat treatment (Gabre and Shakir 2016).

Previous studies have shown that the prevalence of antimicrobial resistance and virulence traits among Enterococcus from animals and humans vary between geographical location and antimicrobial regimes (Kwon et al. 2012; Liu et al. 2012; Hidano et al. 2015).

Zaria, Nigeria, is a large agricultural city characterized by poultry, cattle and vegetable farming, the products of which are transported to other parts of the country for consumption. Approximately 95% of the manure emanating from these animals is used for fertilization of agricultural land. Zaria, Nigeria, belongs to the tropical agro-ecological zone with a climatic condition that favours the animal husbandry. The livestock industry plays an important role in the economic and social lives of the individuals and country and is characterized by widespread rearing of cattle, poultry, pigs and small ruminants in small and large scale. In addition, wastes in the form of manure from these farms (especially cattle and poultry farms) are also directly used for fertilization of agricultural land (Yusuf et al. 2016).

A number of studies have reported the prevalence and characteristics of antimicrobial resistance among enterococci from human clinical samples, food and animals in some parts of Nigeria (Oguntoyinbo and Okueso 2013; Amaechi and Nwankwo 2015; Anyanwu and Obetta 2015; Ayeni et al. 2016); however, there is a paucity of information from Zaria in Northern Nigeria. Because of the risk of potentially harmful enterococcal strains being transmitted through the food chain and the contribution of enterococci to the spread of antimicrobial resistance and virulence, it is important to evaluate the prevalence and antibiotic resistance of strains found in poultry, cattle and their manure, which play a relevant role in the dissemination of enterococci (Liu et al. 2012; Daniel et al. 2015). Therefore, monitoring the occurrence of resistant and virulent enterococci from these sources is of utmost importance to ensure food safety and public health. Thus, the present study is expected to provide an insight into the occurrence, species diversity, resistance and virulence potential of enterococci from cattle, poultry and manure in Zaria, Nigeria.

Materials and methods

Sampling

Cloacal/rectal swab and manure samples of poultry and cattle were collected between January and July 2014 from farms located within Zaria (11.0° N, 7.7° E), Nigeria. Altogether, 390 samples originating from 130 chickens, 130 cattle and 130 manure (n = 65 cattle manure and n = 65 poultry manure) were collected. Ten cloacal/rectal swabs and five manure samples were collected from each farm and transported in ice packs to the Diagnostic Microbiology Laboratory of the Department of Veterinary Microbiology, Ahmadu Bello University, Zaria, Nigeria, where they were processed for Enterococcus spp. isolation.

Isolation and identification of enterococci

Standard bacteriologic culture methods were used to isolate and identify Enterococcus from the cloacal/rectal swabs and manure samples (Manero and Blanch 1999; Schwaiger et al. 2010). Briefly, each sample was enriched in 9 ml of bile esculin azide broth (Hardy Diagnostics, USA) and incubated at 45 °C for 48 h. After incubation, a loopful of the broth was streaked onto bile esculin azide agar (Hardy Diagnostics, USA) and the plate incubated at 37 °C for 24 h. Presumptive Enterococcus colonies (black-brown) were further subcultured to purity on tryptone soya agar (Oxoid, UK) followed by biochemical characterization using Gram’s stain; catalase production; growth in 6.5% NaCl; hydrolysis of l-pyrrolidonyl-β-naphthalamide; fermentation of d-xylose, mannitol, l-arabinose, ribose, pyruvate, sucrose, sorbitol, d-raffinose and sorbitol; pigment production; motility; and acidification of methyl-α-d-glucopyranoside. The E. faecium and E. faecalis identified were further processed for antimicrobial susceptibility, virulence trait expression and carriage of the genes encoding these traits.

Antibiotic susceptibility testing

Antimicrobial susceptibility testing and interpretation were carried out as recommended by the Clinical and Laboratory Standards Institute (CLSI 2015). Antibiotics tested included vancomycin (30 μg), gentamicin (120 μg), streptomycin (300 μg), ampicillin (10 μg), ciprofloxacin (5 μg), tetracycline (30 μg), chloramphenicol (30 μg), erythromycin (15 μg), qunupristin/dalfopristin (15 μg) and rifampicin (5 μg). Multidrug resistance (MDR) was defined as resistance of an isolate to at least one agent in each of three or more antimicrobial groups/classes.

Assay for virulence traits

β-Haemolytic activity and the ability to produce gelatinase and biofilm by the isolates were assessed using tryptone soya agar supplemented with 5% horse blood (Iseppi et al. 2015) and 13% nutrient gelatin broth and Congo red agar (Kaiser et al. 2013), respectively.

Detection of antibiotic resistance and virulence genes

The total DNA template was extracted from the isolates as described by Laird et al. (1991) with slight modifications. PCR amplification of tetracycline resistance (tetK, tetL, tetM and tetO), erythromycin resistance (ermB) and virulence (gelE, asa1, and cyl) genes was carried out using primers (Table 1) and conditions previously described by Ng et al. (2001), Sutcliffe et al. (1996) and Vankerckhoven et al. (2004), respectively. PCR products were analysed by electrophoresis on 2% agarose gel following staining with ethidium bromide.

Table 1 Primer sequences for the amplification of antibiotic resistance and virulence genes from Enterococcus
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Conjugation experiments

Conjugation experiments were carried out as described by Tremblay et al. (2012) using E. faecalis JH2-2 strain (rifampicin resistant) as the recipient strain and some of the isolates (rifampicin susceptible) as donor strains. Brain heart infusion agar plates containing rifampicin (30 μg/ml), tetracycline (10 μg/ml) and erythromycin (15 μg/ml) were used for recovery of transconjugants.

Results

Isolation rate of Enterococcus

One hundred and sixty-seven Enterococcus species distributed across 9 different species were recovered from the 390 samples collected. The isolation rate of enterococcal species from manure of chickens (64.6%) was higher than that from cloacal swabs of chickens (48.5%), manure of cattle (33.8%) and rectal swabs of cattle (30.8%). E. faecium was the most predominant species isolated from all sample types (Table 2).

Table 2 Occurrence and species distribution of Enterococcus species isolated from poultry and cattle farms
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Antibiotic susceptibility profile

Table 3 shows the susceptibility of the isolates to different antimicrobial agents tested in this study. Fourteen (12.4%) of the isolates were sensitive to all 10 tested antibiotics, and each of the remaining 99 (87.6%) strains were resistant to at least one antimicrobial agent tested in this study. Also, all the isolates were susceptible to vancomycin. The highest frequency of resistance (61.0%) was observed to tetracycline and erythromycin while the least frequencies of resistance were to quinupritin/dalfopristin (4.4%) and chloramphenicol (8.0%). High-level gentamicin (HLGR) and streptomycin (HLSTR) resistance was observed among 32.7 and 28.3% of the isolates, respectively. Among the 99 resistant strains, 60 (53.1%) expressed multiple drug resistance (resistance to three or more antimicrobials) (Table 4) presenting as 23 different antimicrobial resistance patterns. CN/ERY/STR/TET was the most predominant (n = 11) resistance pattern (Table 5).

Table 3 Antibiotic resistance profile of Enterococcus faecium and E. faecalis isolated from poultry and cattle farms
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Table 4 Multidrug resistance (MDR) among Enterococcus isolated from poultry, cattle and manure in Zaria, Nigeria
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Table 5 Multidrug resistance patterns of Enterococcus species isolated from poultry and cattle farms
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Detection of virulence and antibiotic resistance determinants

Haemolysin, gelatinase and biofilm activity was detected in 21.3, 10.6 and 83.2% of the isolates (Table 6). The molecular identification of virulence-associated genes revealed the presence of asa1, gelE and cylA as well as genes associated with resistance to tetracycline (tetK, tetL, tetM and tetO) and erythromycin (ermB).

Table 6 Virulence traits expressed by Enterococcus isolated from poultry and cattle farms in Zaria, Nigeria
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Discussion

Antibiotic-resistant bacteria have frequently been detected among animals, animal products and their related environment (Ali et al. 2014). Animals are reported to contribute to the spread and persistence of antimicrobial resistance in the community. Enterococci are an important aetiologic agent of hospital-associated infections (Khan et al. 2015). Therefore, faecal carriage of antimicrobial-resistant and virulent Enterococcus species among animals poses a major public health threat as these strains could be transferred to humans via consumption of contaminated food which then contributes to the spread and persistence of antibiotic-resistance bacteria in the general population and environment (Daniel et al. 2015; Woolhouse et al. 2015). The study of enterococci among cattle and poultry and their manure (which is widely used for fertilization of farmlands) has the potential to disclose important information on the role of animals as reservoirs and disseminators of resistant and virulent strains of the organism. In this study, the predominance of E. faecium, a species frequently associated with nosocomial infection, could be related in part to its commensal status, ruggedness and ability to survive and adapt to different selective pressures such as antimicrobial use in the animal production environment. The species detected in this study have also been reported in other studies from different parts of the world (Jackson et al. 2011; Kwon et al. 2012; Klibi et al. 2013; Ali et al. 2014; Iweriebor et al. 2015, 2016; Ben Said et al. 2016; Steoien-Pysniak et al. 2016). Host factors such as diet have been reported to affect the composition of the microbiota and, therefore, the species diversity among gut commensal bacteria (Ho et al. 2013). Therefore, the herbivore diet of the cattle in contrast to the poultry might also explain in part the difference in the species distribution observed between these sources in this study.

The detection of E. faecium in this study is significant as this species together with E. faecalis is the most predominant species among enterococci causing infection worldwide (Billington et al. 2014; Kajihara et al. 2015). These two species have also developed resistance to a wide variety of clinically important antibiotics, and are a leading cause of healthcare-associated infections (Ben Sallem et al. 2016). When the isolates (E. faecium and E. faecalis) were subjected to antimicrobial susceptibility testing, they demonstrated varying frequency of susceptibility to the different antibiotics tested. The highest frequencies of resistance were to erythromycin and tetracycline especially among isolates from poultry sources. Excessive and unjustified use of antimicrobials in livestock production provides selective pressure which is reported to accelerate the emergence of resistant bacteria strains. Tetracycline and macrolides are the most widely used drugs in animal production environments in Nigeria (Oluwasile et al. 2014; Adesokan et al. 2015; Olonitola et al. 2015; Ayeni et al. 2016). This may partly explain the high percentage of resistance observed to these classes of drug. Studies from different parts of the world have also reported the incidence of erythromycin and tetracycline resistance among enterococci from animals, humans and food. These antibiotics and their analogues are cheap, easily affordable and commonly used in animal production sometimes without prescription either for therapeutic, prophylaxis, or growth-promoting purpose (Adesokan et al. 2015; Ayeni et al. 2016). Resistance of enterococci to tetracycline and erythromycin is commonly mediated by the tet (tetK, tetL, tetM and tetO) and erm (ermB) genes, respectively, which were detected among isolates in this study and other studies (Hidano et al. 2015; Klibi et al. 2015). These genes are located on mobile genetic elements (MGE) specifically the Tn916 family and can be horizontally transferred easily (Jurado-Rabadan et al. 2014). Although tetracycline and erythromycin are not the drugs of choice for treatment of enterococcal infection, resistance to them is still of great clinical importance because they are effective for the treatment of other bacterial infections (Arias et al. 2010). Therefore, the ease with which enterococci can disseminate erythromycin and tetracycline resistance traits to other bacteria raises serious clinical and public health concern. The absence of resistance to vancomycin may be attributed to this agent or its analogue avoparcin in livestock production in Nigeria as is the case in other countries where this agent has not also been used (Hammerum 2012).

Cell wall inhibitors (ampicillin, vancomycin) and aminoglycosides (gentamicin) used in combination are the drugs of choice for the treatment of enterococcal infection (Arias et al. 2010). However, resistance to these agents was observed in this study: ampicillin (45.1%) and the aminoglycosides gentamicin (32.7%) and streptomycin (28.3%). All the high-level-streptomycin-resistant isolates were also found to be resistant to a high level of gentamicin. Gentamicin is a good predictor of resistance to other aminoglycosides with the exception of streptomycin (Abamecha et al. 2015). Resistance to any of these two agents may result in a loss of the synergistic bactericidal activity achieved by their combined use, therefore narrowing the spectrum of the therapeutic armamentarium available to clinicians for the treatment of enterococcal infections (Hollenbeck and Rice 2012). Resistance to aminoglycosides as observed in this study has also been reported by other authors (Liu et al. 2012; Martin et al. 2015).

Multidrug resistance was observed among majority of the isolates and was more predominant among isolates from poultry. MDR isolates were resistant to as few as three and as many as seven different antimicrobials and up to six different antimicrobial classes. In Nigeria, poultry is often more exposed to antimicrobial agents for the purpose of prophylaxis or chemotherapy or as a growth promoter with a resultant development of resistance among commensal bacteria. The high rate of MDR isolates detected in this study is a reflection of the extensive use of broad-spectrum antibiotics in poultry production in this locality. The MDR pattern observed was also similar to those reported among enterococci from animals, humans and food in different countries (Ristori et al. 2012; Chung et al. 2014; Iweriebor et al. 2015, 2016; Pruksakom et al. 2016). These MDR isolates pose a serious threat to public health as the same class of antibiotic is being used in the treatment of most bacterial diseases in humans (Daniel et al. 2015; Ventola 2015).

The location and spread of multiple resistance genes through mobile genetic elements (MGE) such as transposons, integrons and plasmids are a well-recognized problem worldwide (Chaffenel et al. 2015; von Wintersdorff et al. 2016). The isolates tested were able to transfer some of the resistance phenotype especially tetracycline, erythromycin, chloramphenicol, gentamicin and streptomycin to the recipient strain (E. faecalis JH2-2). The transfer of resistance determinants by the isolates in this study suggests that they are located on MGE and can easily be spread to other Enterococcus species, commensals, and pathogens alike.

Enterococcus species are currently ranked third among the leading causes of nosocomial infections worldwide (Kajihara et al. 2015). The production of virulence traits and carriage of virulence genes by some of the isolates in this study therefore suggest they have the potential to cause infection. Similar observations have also been presented by other authors (Comerlato et al. 2013; Lozano et al. 2015b; Shirin et al. 2016). Biofilm production enables the organism to survive harsh environmental conditions, causes chronic infection, evade the immune system and resist the effect of disinfectants and antimicrobial agents (Kim et al. 2016). Aggregation substance, encoded by the asa1 gene, is associated with adherence to eukaryotic cells and sex pheromones which aid in conjugation; cylA encodes cytolysin (haemolysin) which lyses red blood cells while gelatinase, encoded by gelE, hydrolyzes gelatin, collagen, haemoglobin and small peptides promoting dissemination of the organism.

In conclusion, considering that enterococci are a major cause of drug-resistant nosocomial infections and can transfer virulence and antibiotic-resistance-encoding genes to other bacteria, the detection of enterococci harbouring these determinants from poultry, cattle and their manure in this study suggests livestock environment in this area may serve as a reservoir for the emergence and dissemination of virulent and resistant strains of enterococci that may contaminate food and cause serious environmental and public health problems. Although our data are restricted to a small study area, because farming practices and the pattern of antimicrobial use are similar across the country, the result may be suggested to reflect the general situation in different parts of the country. The result of this study raises serious public health concerns and suggests the need for continuous surveillance of the evolution of antimicrobial resistance and virulence among Enterococcus from livestock.