Listerial contamination of raw beef and chevon in north-central Nigeria

Background and objective: Listeria sp. is a ubiquitous and frequently isolated foodborne pathogen. The prevalence of Listeria sp in raw beef and chevon sold in Lafia Nigeria, as well as their antibiotic susceptibility profile was evaluated. Methods: A total 104 samples comprising of 52 raw beef and 52 chevon were obtained from street vendors (hawkers), Shinge abattoir, Lafia old market and Lafia Modern Market. Isolation of Listeria sp. was performed on Listeria Selective Agar, following enrichment in supplemented Listeria Selective Broth. Identification of Listeria sp. was carried out by cultural and biochemical methods. Antimicrobial susceptibility of isolated L. monocytogenes was performed by standard disk diffusion method. Chi-square test was used to determine association between contamination levels at p=0.05. Results: Seven types of Listeria sp. were isolated. L. monocytogenes and L. ivanovii were the most frequently isolated contaminants in all meat types and from all sample sources. L. monocytogenes was isolated with a frequency of 64.4% (67/104) in the meat samples. Beef samples had the highest listerial contamination with a frequency of 58.2% (78/134) compared to chevon which had a listerial frequency of 41.8% (56/134). Resistance of L. monocytogenes to streptomycin and sparfloxacin was 58.2% and 55.2% respectively. Resistance to ampicillin (34.3%) and gentamicin (20.9%) was also observed. Resistances to multiple antimicrobials were detected in 11 L. monocytogenes isolates. Conclusion: The study demonstrated that the raw meat sold in Lafia was contaminated with several Listeria sp. L. monocytogenes showed high rate of resistance to several antimicrobial agents used for the treatment of listerial infection. Appropriate regulation and monitoring of livestock rearing and meat retailing practices are advocated to safeguard the health of consumers. IMC J Med Sci 2019; 13(2): 001. EPub date: 18 July 2019


Introduction
Listeria monocytogenes is a facultative anaerobic bacterium which can grow and reproduce inside the host's cells, making it one of the most virulent food-borne pathogens. Unlike most other foodborne pathogens it can grow and multiply at a very low temperatures [1,2]. L. monocytogenes has been typed into four serotypes of which only three (1/2a, 1/2b, 4b) are involved in 95% of all human listeriosis cases [3].
consumers [6] since it remains as one of the most virulent foodborne pathogens for immunodeficient individuals. It has been extensively studied over the past few decades due to its high case/fatality rate (20-30%), chronic infection resulting in high healthcare cost and its ability to survive for longer periods under adverse environmental conditions than many other non-spore-forming bacteria [7].
In man, outbreaks usually occur following consumption of unpasteurized milk, contaminated cheeses and other dairy products. Reports of outbreaks have also followed ingestion of undercooked meat and poultry [8]. It is frequently present in the gut of cattle, poultry and pigs and can be transmitted through ready-to-eat (RTE) foods or raw meat products [9]. Listeria species are isolated from a diversity of environmental sources, including decaying vegetation, soil, water, effluents, variety of foods, and the faeces of humans and animals [10].
L. monocytogenes is a major contaminant of RTE food and food products. Packaged raw foods can represent a potential source of contamination, and listeriosis is associated with the consumption of such undercooked raw foods [11]. Major changes in food production, processing and distribution, increased use of refrigeration as a primary preservation method, changes in eating habits particularly towards ready-to-eat foods are suggested as possible reasons for the emergence of human food-borne listeriosis [12].
While several studies have reported antibiotic resistance in bacterial isolates from human beings, it is becoming evident that food produced from farm animals is no longer exempted from antibiotic resistant bacteria [13]. Thus, the food microflora is not separated from its human counterpart in cases of antibiotic resistance. The occurrence of infection by antibiotic resistant organisms makes treatment difficult and increases the period of recovery from illness [14]. This situation has been worsened by the indiscriminate use of common broad spectrum antibiotics as prophylaxis and growth promoters in animal feed, particularly in developing nations [14,15].
There has been a dearth of information on the epidemiology of listeriosis in most African countries, including Nigeria [16] with only few reports, when compared to other developed regions like Europe and United States of America [17]. This is because the organism seems not to have been given as much attention as is required [18,19]. Listeriosis is considered a serious health problem due to its high mortality rate and severity of symptoms. Despite the foregoing and the continuous observation of the emergence of antibiotic resistant strains of Listeria, there is little or no documented reports of its prevalence and its antibiotic susceptibility profile in Lafia of Nasarawa state of Nigeria.  [20]. The study was carried out from June to August which witnessed increased slaughtering of animals in commemoration of the Eid il-Fitr celebration in the month of July, 2016.

Study area and period:
Sample collection: Preliminary investigation identified the Shinge abattoir, open markets (Lafia old market and Modern market) and hawkers as major sources of retail fresh raw meat within Lafia. A total of 104 samples comprising of 52 raw beef samples and 52 raw chevon samples were collected randomly from the four identified sources in the morning hours to prevent effects of changing temperatures on microbial population. The meat samples were bought and packaged as they are sold to other consumers, appropriately labeled and transported within 90 minutes to the laboratory for analysis. Contamination of the meat samples by other materials or sources such as collector's hand was avoided.

Isolation of Listeria sp:
Isolation of Listeria from the meat samples were based on the method described by Ndahi et al. [21] and Adikwu et al. [19] with some modifications. Aseptically, 10g of each sample was added to 90 ml Listeria Enrichment broth (Oxoid, Basingstoke, UK) containing Listeria Selective Enrichment Supplement. The mixture was homogenized for 2 minutes in a blender (MasterChef) at room temperature and incubated at 30°C for 24 hours. Listeria species were isolated on Listeria Selective Agar (Oxoid) using pour plate method, by transferring 1 ml of the overnight supplement culture into molten Listeria Selective agar and incubating for 48 hours at 37°C, after which the plates were examined for the presence of listeria-like growths.

Antimicrobial susceptibility test:
Antibiotic susceptibility of the isolated L. monocytogenes was determined by the Kirby-Bauer disk diffusion method on Mueller-Hinton Agar [19,25]. The antibiotics used include erythromycin (15µg), streptomycin (10µg), co-trimoxazole (1.25/23.75µg), rifampicin (5µg), nalidixic acid (30µg), ciprofloxacin (5µg), ampicillin (10µg), gentamicin (10µg), chloramphenicol (30µg), sparfloxacin (5µg) and ofloxacin (5µg). A broth culture of at least 18 hours old was diluted using sterile distilled water and standardized to match 0.5 McFarland standards (approximately 10 8 cfu/ml). The culture was inoculated onto dried Mueller-Hinton Agar (MHA, Oxoid) plate to create a lawn. Antibiotic discs were then placed on the seeded agar surfaces and the plates incubated for 24 hours at 37°C, after which the diameter (in mm) of the inhibition zone around each disk was measured and interpreted according to the Clinical Laboratory Standard Institute (CLSI) guidelines using the break points of Staphylococcus species [25].
Statistical analysis: IBM SPSS Statistics version 22.0 (IBM Corp., Armonk, NY, USA; 2013) was used to analyse results obtained. Pearson's chi-square test was used to determine significance of associations between variables. A p-value less than 0.05 was considered statistically significant.

Discussion
Results of this study revealed a high prevalence of L. monocytogenes in raw beef and chevon sold in Lafia. The prevalence rate of Listeria species observed in this study was lower than the 95.8% prevalence rate reported in vegetable salads in Zaria, Kaduna state [24] but higher than the 39.6% and 7.8% observed in Sokoto [26] and in Makurdi, Benue state [19]. The high L. monocytogenes contamination observed in the raw meat samples was in concordance with an earlier report where 14 out of the 15 Listeria species isolated were L. monocytogenes [27]. Similarly, the high prevalence of L. monocytogenes in beef samples confirms an earlier report [17].
The present study appears to be the first investigation regarding presence of Listeria sp in retailed meat within Nasarawa state. The high prevalence of Listeria in the two widely consumed meats raises an issue of serious public health importance. It is possible that cases of listeriosis may have been misdiagnosed across health centers in the study area since they do not include investigations for listeria infection in clinical specimens. Some of the symptoms associated with the disease onset such as gastroenteritis, headache, fatigue, muscular and joint pain are similar to those of typhoid fever [28]. Moreover, not much appears to be known about this organism in Nigeria and most African countries [16].
The least common listeria isolate was L. grayi while the most observed was L. monocytogenes. This was in contrast with an earlier report [26] where L. seeligeri and L. innocua were the least and the most observed listerial contaminants respectively. Listerial contamination of beef was highest in samples from the Shinge abattoir. Contamination was higher in beef than chevon, an observation that was also reported by earlier studies [22,29].
Although not determined in this study, the difference in contamination between the two meat types might have been influenced by factors such as pH and water activity (a w ). For instance, L. monocytogenes is known to survive at a pH of <4.3 and water activity of <0.930 [30]. The high rate of Listeria contaminants identified in beef samples from this source could be due to unhygienic practices such as slaughtering and preparing of meat on bare floor, poor drainage system, use of contaminated water, poor facility maintenance, illiteracy and lack of hygiene awareness by the handlers, as well as improper storage facilities. Vending of these meats is mostly done without any covering, thus exposing the meats to high rate of microbial contamination. Adoption of proper methods during slaughtering of animals have been suggested as a means of considerably reducing presence of listeria in meats [31,32].
Chevon samples from Lafia old market had less listerial contaminants than those from Shinge abattoir. This could be due to double-washing process practiced in Lafia old market; the meats are washed after slaughtering and before sales to butchers (retailers), unlike at Shinge abattoir where this is not practiced. The practice of repeated Findings also showed that the isolated L. monocytogenes was either sensitive or intermediate sensitive to most of the antimicrobial agents tested. Susceptibility to some antibiotics and the multiple antimicrobial resistance observed in this study is similar to earlier reports [19,21,24]. Almost all the studied strains were susceptible to a wide range of antibiotics but completely resistant to nalidixic acid. This observation is in agreement with the earlier reports [33,34]. Resistance to nalidixic acid justifies addition of nalidixic acid into selective media for the isolation of L. monocytogenes. Susceptibility to ampicillin, erythromycin, chloramphenicol, co-trimoxazole and gentamicin, observed in this study is similar to that reported by Troxiler et al. [35] and Hansen et al. [36]. Listeriosis is treated usually with β-lactam antibiotics like ampicillin or penicillin alone, or combined with an aminoglycoside (usually gentamicin) [37]. However, about 20-34% of the isolated L. monocytogenes were resistant to ampicillin, gentamicin and erythromycin in this study. This portends a serious public health issue. Around Lafia, meat is prepared by roasting, apart from boiling and frying; sometimes, this may not be enough to destroy deep listerial contaminants, leaving consumers of such products at risk of foodborne diseases.
The resistance pattern observed in the present study could be attributed to the irrational use of the antibiotics in cattle and goat by the animal rearers or veterinary quacks [26]. Misuse of antibiotics as growth promoters can confer selective pressure on bacteria [38], making those increasingly resistant to conventional antibiotics. Although not determined experimentally, horizontal gene transfer among bacteria in the environment could also been responsible for antibiotic resistance as observed in this study [38,39].

Conclusion
Listeria contamination of raw beef and chevon sold in Lafia is alarming. Unhygienic practices amongst meat handlers at the collection site could be the major source of contamination. Use of contaminated water, washing without addition of disinfectant, lack of awareness, improper storage facilities, poor equipment maintenance and dirty environment were factors believed to be the major causes and sources of listerial contamination observed in this study.