Listeria species as contaminants of lettuce and its resistant genes in Benin city , Nigeria

This study was aimed at determining the level of Listeria contaminants and subsequently detects some resistance genes among the isolated Listeria species from lettuce sold in some vegetable markets in Benin City, Nigeria. Twenty-four lettuce samples were purchased from three vegetable markets in Benin City, Nigeria and examined using standard microbiological methods. Microbial characterization revealed Listeria monocytogenes and L. grayi as the predominant species isolated. Plate count analysis on Listeria selective agar revealed that lettuce sold in Oba market and Forestry market had 2 2 the highest and lowest mean count of Listeria species 224.00 x 10 CFU/g and 83.00 x 10 CFU/g respectively. Most (63.75%) of the Listeria species isolates were found to be susceptible to Ofloxacin (5 μg), Ciprofloxacin (10 μg), Streptomycin (10 μg), Gentamycin (10 μg), Pefloxacin (5 μg) whereas species harbouring tetracycline (65%) and erythromycin (60%) resistant genes. The study provides an evidence of the colonization of Listeria species in lettuce sold in Benin City which may pose serious public health threat


Introduction
Nigeria.The first documentation of Listeria in Listeria is the causative agent for Nigeria by Eyo et al., (1969) emerged after "Listeriosis" an infectious disease of public health Njoku-Obi and Njoku-Obi (1965) report using significance with vulnerable groups as serological procedures among healthy blood immunocompromised individuals (e.g.donors.Subsequently, there emerged positive HIV/AIDS, patients on chemotherapy), pregnant reports of Listeriosis in Nigeria.The first women, new-born and the elderly (Farber, confirmation of Neonatal Listeriosis in Nigeria 1991).The genus Listeria comprises of 6 species: associated with L. monocytogenes was reported by Onyemelukwe and Lawande (1982) with L. monocytogenes, L. ivanovii, L. innocua, L.
evidence of transmission from mother to newwelshimeri, L. seeligeri, and L. grayi.Though, only born.Onyemelukwe et al., (1983) and Emele L. monocytogenes has been associated with (2000) reported the association of L. outbreaks of human foodborne infection (Gilbert monocytogenes in patients with meningitis and et al., 1999) and other life-threatening illnesses septicaemia from Nigeria.In 2002, Shigella r a n g i n g f r o m s e v e r e s e p s i s t o sonnei and Listeria monocytogenes infection meningitis/encephalitis.In December, 2017, a recent outbreak was reported in South Africa were linked to lettuce consumption in diet (WHO, 2018).Consequently, there have been (salad) (Pelczar et al., 2006).Even though the threat alert to other African countries like incidence of Listeriosis is low in Nigeria with disparity in the prevalence (Mawak et al., 2008

Materials and Methods and Ajayeoba et al., 2016), very high mortality
Sample collection rate ranging from 20% to 30% have been Lettuce samples were purchased from reported from other parts of the world (Mead et three vegetable markets namely: Airport road al., 1999).Although the main route of market, Forestry market and Oba Market in transmission is via consumption of contaminated Benin City, Edo State, Nigeria.Eight samples food, other route such as mother to foetus were collected from each market site for 4 weeks transmission via placenta has been previously within the month of November 2015.Collected reported (Emele, 2000).
samples were collected in labelled sterile sample The intake of lettuce is increasing in bags and transported in ice pack to the several urban cities in Nigeria.Fresh lettuce is Microbiology Laboratory, Benson Idahosa widely applied in dietary preparations like salads University for analysis.A total of 24 lettuce or sandwiches.Studies have shown that their samples were collected throughout the sampling plant tissue are internalised by some bacterial period.pathogens (Farber, 1991).Pondei and Ogbonna (2004) reported the isolation of Listeria from Identification and Serotyping of Listeria species vegetables and irrigation water in Jos, Nigeria.
Isolation and Identification of isolates Studies on food pathogens transmitted via fresh was done by cultural, morphological and vegetables have reported the involvement of biochemical test.Distinct colonies were picked Gram-negative bacterial pathogens such as from incubated plates and pure cultures were Escherichia coli and Salmonella.Consequently, made following sub-culturing prior to there are few studies exploring the involvement biochemical test. of Listeria in contaminating fresh vegetables Serotyping was done using commercially such as lettuce in Benin City, Nigeria.With prepared Listeria antiseria with Oxoid Listeria evidence of the surfaces of raw vegetables Test Kit (Oxoid, United Kingdom).A drop of contaminated with a range of microbes due to saline was placed on a well of the reaction card, microbial population of the environment, mode after which a distinct colony from the Listeria of handling, time and condition of storage selective agar plate was collected using a sterile (Pelczar et al., 2006), it is therefore paramount to wire loop and emulsified in the drop.A drop of examine the microbial load of bacterial the test latex was added to the suspension and pathogens.
rocked for up to 2mins and examined for Antimicrobial resistance is currently the agglutination.greatest challenge worldwide.It decreases the Principles: Polyvalent antisera are prepared effectiveness of drugs that decrease morbidity against purified flagellin proteins from Listeria and mortality associated with serious and life monocytogenes (antigens A, B and C) and threatening infections and thus, compromising Listeria grayi (antigen E) are used to coat latex human health (Collignon et al., 2009).Food particles.When mixed with a suspension contamination with antimicrobial resistant containing Listeria species, the latex particles bacteria can be a major threat to public health, rapidly agglutinate to form visible clumps.The as the antibiotic resistance determinants can be Oxoid Listeria Test Kit detects all motile strains of transferred to other bacteria of human clinical Listeria species.significance.Detection of resistance genes molecularly remains a mainstay however, there Enumeration of Listeria species exist paucity in information on resistance genes Ten-fold serial dilution of each among Listeria-contaminated lettuce in Benin homogenized lettuce samples was made using City, Nigeria.
1% of sterile peptone water.This was serially Therefore, this study is aimed at diluted and were plated on Listeria selective agar determining the microbial contaminants of (Oxoid, United Kingdom) using spread plate Listeria species isolated from lettuce in vegetable method and then incubated for 24-48 hours at markets in Benin City, Nigeria and their plate were counted and sub cultured onto freshly prepared agar plates after which pure cultures of the isolates were streaked onto tryptone soy agar.
The disc were transferred aseptically and placed unto Muller Hinton plates with a sterile O forceps and then incubated at 37 C for 24hours.Resistance was recorded when there were no clear zones of inhibition around the respective disc and sensitivity was recorded when there Polymerase chain reaction products were was presence of inhibition.separated in 1.5% agarose gel which was stained with ethidium bromide.Polymerase chain reaction (PCR) products on gel were genes Genomic DNA of Listeria isolates were extracted after the bacteria was grown in tryptone soy agar for 24hours.The Listeria cells were harvested using 200µl nuclease free water after which it was subjected to denaturation by heating at 100°C for 10minutes then the supernatant containing the DNA was collected by centrifugation at 13,000x g for 1minute.

Results
contaminated lettuce of Listeria species followed Listeria by Airport road market and Forestry market.The results of species in some Listeria species isolated in this study include: lettuce vegetables sold in Benin City is shown in Listeria monocytogenes (90%) and Listeria gravi Table 2.Among the three market locations (10%).Biochemical characterization revealed sampled, Oba market had the highest microbial ).The isolates demonstrated a high speciation of Listeria: L. monocytogenes (18) percentage resistance (45%-100%) to and L. grayi (2).All the Listeria species isolated Amoxycillin, Chloramphenicol, Ceftriaxone, were Gram positive rods, catalase positive, Erythromycin and Tetracycline [Figure 1].positive with Listeria antisera test kit, produced Molecular study using polymerase chain acid with fructose, glucose and xylose.All reaction (PCR) showed the occurrence of Listeria species were coagulase negative, antimicrobial resistant genes in Listeria species oxidase negative while some produce acid with isolates from lettuce [Table 4].Out of 20 Listeria mannose and few were positive to OBIS mono species, 13 species representing 65%, were test kit.Table 3 shows Antibiotic sensitivity positive for Tetracycline genes: TetA and TetM pattern of Listeria species isolated from lettuce.
respectively while 12 species representing 60% Majority of the isolates were resistant to were positive for Erythromycin resistant gene Amoxycillin, Chloramphenicol, Ceftriaxone, (ErmB) and 2 species representing 10% were Erythromycin and Tetracycline but were sensitive positive for Daptomycin resistant gene.

Table 2 :
Listeria counts from lettuce purchased from the different Vegetable Markets 2 Key: OM=Oba market; ARM=Airport road market; FM=Forestry market

Table 3 :
Antibiotic susceptibility pattern of Listeria species isolated from lettuce