Antibiotics Susceptibility Profile of Listeria species Isolated from Untreated Abattoir Wastewater in Akinyele, Ibadan, Nigeria and Its Implication on Public Health

Introduction: The demand for meat to meet the need of increasing human population has led to increase in the abattoir activities. Untreated abattoir wastewaters which may constitute a public health threat due to the possibility of transmission of pathogens to humans are constantly being discharged into water bodies. Listeria is an emerging pathogen that is commonly associated with food infection and has been found to survive in water. Aim: This study was carried out to determine the occurrence and antibiotic resistant pattern of Listeria species isolated from abattoir wastewater in Akinyele, Ibadan. Materials and Methods: Wastewater samples were collected from the slaughter slab and drainage channels between May and June, 2015. Listeria species were isolated using Listeria Selective Agar Base with Listeria Selective Supplement and identification of the isolates was done using conventional methods. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer disk diffusion assay using single antibiotic disks including ampicillin, streptomycin, chloramphenicol, amoxicillin/clavulanate, ceftriazone, cloxacillin, ciprofloxacin, ofloxacin, tetracycline and Original Research Article Falodun et al.; BMRJ, 16(6): 1-10, 2016; Article no.BMRJ.28066 2 trimethoprim/sulfamethoxazole. Results: A total of 82 Listeria spp. were isolated comprising 58.5% and 41.5% from the slaughter slab and drainage respectively; all the isolates were resistant to ampicillin while 86.6%, 68.3% and 65.5% were resistant to ceftriaxone, amoxicillin/clavulanate and tetracycline respectively. In addition, 12.2% of the total isolates were resistant to a combination of six antibiotics (ampicillin, tetracycline, amoxicillin, ceftriazone, streptomycin, and trimetoprim/sulfamethozaxole) while only one isolate was resistant to eight or more antibiotics. Conclusion: This study revealed that the Akinyele abattoir wastewater is a potential medium for the transmission of multi-drug resistant bacteria to humans. Hence, adequate measures should be put in place for the treatment of the abattoir wastewater before discharge into the environment.


INTRODUCTION
Listeria is an emerging pathogen which is commonly associated with food borne infection [1]. Out of the eight generally recognized species that includes the following: L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, L. grayi, L. marthii and L. rocourtiae [2,3], only L. monocytogenes and L. ivanovii are both humans and animals pathogens while L. ivanovii cause disease mostly in ruminants animals, others are regarded as nonpathogenic [2,4]. However, there have been reported cases of human infection as a result of L. ivanovii, L. seeligeri, L. innocua and L. welshimeri [5][6][7]. For instance, L innocua has been reported to be occasionally associated with encephalitis in ruminants [8]. L. monocytogenes, the etiologic agent of listeriosis continues to be a pathogen with significant impact in the public economy all over the world because of the food safety concern [9]. The pathogen has also been reported to be responsible for infections such as meningitis, encephalitis and septicemia in immune-compromised individuals, abortion in pregnant women and neonatal infections with lethality around 20%-30% of all cases [7,10].
While Listeria is reported to be responsible for several food borne outbreaks in the developed world, little is known about such outbreak in the developing countries [10,11]. Although, food is the primary route of transmission of Listeria infections, studies have shown that this pathogen is able to survive in water and that wastewater is a potential reservoir for Listeria species and possible source of its transmission [1,[12][13][14]. Transmission of this pathogen via wastewater has a serious public health implication especially in the developing world in which the greater percentage of the population depends on surface water for drinking and other domestic purposes.
Most of the surface water is negatively impacted as they are constantly receiving wastewater that is untreated or not properly treated from operations like the abattoir [15,16].
The continuous drive to increase meat production to meet the ever increasing protein demand of the increasing world population goes along with pollution problem [17,18]. Abattoir wastewater generated during the slaughtering activities are of great concern because like other discharged sewage, they eventually enter natural bodies of water like ground water, streams, rivers, lakes and oceans [19,20]. These water bodies are used by humans for different purposes especially for drinking and recreation; such usage has been implicated in the transmission of pathogens to humans [21,22]. Previous reports has shown that bacteria exists either as free-living or attached cells [23] in other to resist the action of disinfection and enhance their ability to develop resistance to antimicrobial agents [1]. More so Listeria has been reported to be resistant to antimicrobial agents which were as a result of certain resistance genes that encode proteins and act to either inhibit or reduce the effect of the antimicrobials on the pathogen [1,24].
Reports have shown that the mortality rate of Listeria infections is as high as 50% among the foodborne pathogens [10], an indication that Nigerian public is vulnerable because of the high HIV/AIDS prevalence in the country coupled with the rate of drug and alcohol abuse just like some other low income countries [1,25]. However, only limited information on the prevalence and possible outbreak of listeriosis in Nigeria is available. More importantly, Listeria is not considered as a waterborne pathogen despite reports in literature that it is well established in water supply chain [12,14]. The first prevalence study of Listeria was a serological study, but the first isolation was in 1969 [26,27] in a female adult with leptomeningitis being treated with chloramphenicol and prednisone. The first neonatal listeriosis from which L. monocytogenes was isolated was from a 2-day old baby who developed Listeria meningitis after the contact with the mother. [28]. Since then the pathogen has been isolated from neonatal septicemia patients in the country [29,30]. Listeria monocytogenes was also reported to be prevalent in cattle and other animals characterised by still birth, abortion and nervous signs before death [31].
Resistance of Listeria spp. to antimicrobial agents has been reported, the most common among clinical isolates is to chloramphenicol and ampicillin [26]. A high level of resistance to clindamycin, daptomycin, fluoroquinolone and to third-and fourth-generation cephalosporins was observed in a study on Listeria spp. isolated from humans and slaughterhouse [32]. In another study, some strains of L. monocytogenes, L. innocua, L. seeligeri, and L. welshimeri were found to be resistant to oxacillin, clindamycin and daptomycin which are widely used in hospitals for the treatment of Gram-positive infections [33]. The indiscriminate discharge of untreated abattoir wastewater into the environment may portend grave danger to the health of the public because the wastewater may harbour pathogens with high level of antibiotics resistance and even multiple drug resistant organisms capable of crossinfecting human and cattle due to Increasing evidence that the use of antimicrobial in animals selects for resistant pathogens [34]. In Nigeria, there is a need to investigate emerging waterborne pathogen that is being overlooked or not investigated though they have tendency to survive and be distributed in water supply chain [1]. This study was carried out to determine the prevalence of Listeria species in untreated abattoir wastewater as well as determine the profile of the isolated strains to selected antibiotics.

Study Site
The study site was a commercial abattoir located in Akinyele-Ibadan, Oyo State, Nigeria. Ibadan is the largest city in West Africa [35] with land size covering an area of 240 km 2 , and a human population of about 3 million. The city is located on geographic grid reference longitude 3°5 1 E and latitude 7°20 1 N [36].

Sample Collection
Wastewater samples were aseptically collected into sterile polythene bottles from slabs where animals are being slaughtered and also from the drainage channel which is about 15 meters away from the slab. The samples were transported immediately to the laboratory in ice packs for microbiological analyses. Samples were collected bi-weekly over a period of six weeks (May and June, 2015).

Isolation and Identification of Listeria species
Listeria species were isolated using the method described by Akano et al. [37]. The wastewater samples analysis was done using the Listeria Selective Agar Base (Oxoid) which was prepared according to manufacturers' instruction and supplemented with a vial of Listeria Selective Supplement. Serial dilutions of the abattoir wastewater samples were done and standard pour plate technique was used by plating out 1 ml of the appropriate dilution on the listeria selective media. The plates were then incubated at 35°C for 24-48 hrs. Typical colonies of Listeria species, gray coloured isolates with dark background, were sub-cultured on Listeria Selective Agar media to obtain pure isolates. Identification of Listeria strains were carried out in accordance with standard methods of identification of bacteria of medical importance through microscopy, Gram staining and biochemical tests.
Colonies of 18-24 hour old culture was picked and suspended in a tube containing sterile normal saline (0.85% NaCl) and the turbidity adjusted to 0.5 McFarland standards. With the aid of a sterile swab stick, the suspension was uniformly spread over already prepared Mueller Hinton agar plates and with the aid of sterile forceps, the antibiotics were placed carefully on the plates which were inverted and incubated at 37°C for 18-24 hours. After the incubation period, the zones of inhibition were measured, recorded and compared/interpreted to the CLSI standards [38].
Moreover, resistance of the isolates to ofloxacin (6.1%), cloxacillin. (11.0%) and streptomycin (36.6%) was low (Table 2). Furthermore, the result of the antibiotypes (pattern of resistance to combination of antimicrobials) showed that the highest (12.2%) was to the combination of six different drugs that included ampicillin, tetracycline, amoxicillin/ clavullanate, ceftriaxone, streptomycin and Trimethoprim/ sulfamethoxazole. Out of these, 25.0% Listeria sp. were resistant while it was 4.5% (L. monocytogenes), 6.7% (L. ivanovi) and 5.9% (L. innocua). In addition the result of this study also revealed that 9.6% of the resistant isolates exhibited resistance to a combination of four drugs including: Ampicillin, tetracycline amoxicillin and ceftriaxone with the highest (17.6%) being among the L. innocua followed by the L. monocytogen (13.6%) and other Listeria species but none of the L. ivanovvi showed resistance to the four antibiotic combination. Resistance to a combination of five drugs including ampicillin, tetracycline, amoxicillin/clavullanate, ceftriaxone and chloramphenicol was 7.3% which was among L. monocytogenes (13.6%) and other Listeria species (10.8%) while none of the L ivanovvi and L. innocua showed resistant to the combination of the drugs. More so, the result of the study also showed that one (1.2%) isolates each among the other Listeria spp. showed resistant to eight or more antibiotics (Table 3).

DISCUSSION
Isolation of Listeria species from wastewater portending the organism as emerging pathogen associated with wastewater has been reported [39]. This organism has also been isolated from ruminant animals [40,41] and in 2008; strains of L. monocytogenes were isolated from broiler abattoir in Spain [42] and from pork products in India [9]. The highest occurrence of the isolates from this study which was L. monocytogenes (26.8%) followed by L. innocua (20.7%) and L. ivanovii (18.3%) is similar to the report of the study on pork and pork products where the occurrence of L. monocytogenes was higher than the other species [9]. However, the occurrence rate of these isolates was higher in this study compared to the same study; this may be because other animals including pigs are being slaughtered in the sampling site for this present study. This observation was not in agreement with another study carried out on animal faeces from the northern part of Nigeria in which the occurrence of L. monocytogenes was reported to be the least of all the listerial isolates [40].
The sensitivity of L. monocytogenes to ampicillin (0%), chloramphenicol (59.1%), streptomycin (59.1%) and trimethoprim/ sulfamethoxazole (63.6%) in this study is not in agreement with the report of another study carried out on isolates from ready to eat food from South Africa in which all the isolates (100%) were reported to be sensitive to the combination of the four antibiotics [34]. However, the observation that all the Listeria isolates were fully sensitive (100%) to ciprofloxacin is the same with the report of Ndip et al. [34]. This observation is however higher but comparable to the 91% resistant reported in South Africa [1]. The reason for the slight difference may be as a result of the studied sample. While the present study was on abattoir wastewater, the later study was on municipal wastewater. Comparing the antibiotics resistance patterns of the L. monocytogenes form this study with the same strain in another study in the country, resistance to cloxacillin at the same concentration (5 µg) was much higher (80.7%) in the study [39] than the 4.5% in the present study. Resistance of the L. monocytogenes in this study was lower (63.6%) but comparable to the 77.4% in the other study [39]. The reason for the slight difference may be due to the different concentration of the antibiotics used in the studies which was 30 µg (present study) and 25 µg [39]. With the same drug concentration, resistance of L. monocytogenes to ampicillin (100%), chloranphenicol (36.4%) and streptomycin (40.9%) in this study was higher than the one reported by Enurah and his colleagues which was 64.5% (ampicillin), 1.2% (chloramphenicol) and 35.5% (streptomycin). Resistance to tetracycline in this study was 59.1% while it was 19.36% in the other study. However, the concentration used in this study was 30 µg while that reported in the latter study was 10 µg.

CONCLUSION
In conclusion, this study has further confirmed a high prevalence of Listeria species especially L. monocytogenes in wastewater samples. This is an indication that such wastewater could serve as a public health threat especially among the immune compromised individuals like the HIV/AIDS patients that are at greater risk as it may lead to the outbreak of listeriosis disease. It was also evident that L. monocytogenes exhibited high resistant to ampicillin, ceftriaxone, amoxicillin/clavulanate and tetracycline which is suggestive that the misuse of antimicrobial drugs for therapeutic purposes in both animals and humans may lead to antibiotic resistance development. It is therefore recommended that adequate measure should be put in place for the proper treatment of the abattoir wastewater before they are discharged into the environment and further study is needed to provide better understanding of the epidemiology of listeriosis in Nigeria.