Prevalence and characteristics of Listeria monocytogenes isolates in raw milk, heated milk and Nunu, a spontaneously fermented milk beverage in Ghana

: Listeria monocytogenes is a gram-positive food-borne pathogen that causes listeriosis in humans. Currently, there is little information on the prevalence of Listeria monocytogenes in raw milk and traditional yoghurt-like milk beverage, nunu, in Ghana. The purpose of this study was to investigate the prevalence of L. monocytogenes isolates in raw cow milk, boiled milk and nunu in Ghana, and to characterize these L. monocytogenes isolates according to their serogroups, virulence potentials and antibiotic susceptibility proﬁles. A total of 254 samples comprising 114 raw cow milk, 56 boiled milk and 84 nunu were collected from dairy farms and market vendors for detection of L. monocytogenes . The overall prevalence of L. monocytogenes in raw milk, boiled milk and nunu was 5.5% (14/254). Listeria monocytogenes was prevalent in raw cow milk (8.8%; 10/114) and nunu (13.1%; 11/84), while no Listeria spp. was not detected in boiled milk. A total of 62 L. monocytogenes isolates were analysed to belong to molecular serogroups 1/2a-3a (32/62, 51.6%), 1/2b-3b-7 (14/62, 22.6%), 4b-4d-4e (9/62, 14.5%) and 1/2c-3c (7/62, 11.3%). All 62 L. monocytogenes isolates harbored the virulence-associated genes inlA , inlB , inlC , inlJ , plcA , actA , hlyA , iap and prfA . All Listeria monocytogenes in the present study were generally susceptible to the tested antibiotics, except neomycin and tetracycline, for which phenotypic resistance was observed among isolates.


Introduction
Listeria monocytogenes is a gram positive, facultative intracellular food-borne pathogen that can cause listeriosis in humans, especially in people of compromised immunity, including the elderly, pregnant women and newborns [1,2].The bacterium is widespread in nature and can survive and grow under low temperatures and pH, high concentrations of salt or bile, oxidative stress, carbon starvation, and other adverse conditions making it a potential hazard in foods [3].L. monocytogenes has been isolated from different raw and ready-to-eat (RTE) foods and in raw milk and dairy products in different countries [4][5][6].Several cases of listeriosis in humans are reported, sometimes with a high case-fatality rates [7].
Generally, L. monocytogenes isolates are susceptible to many antibiotics, especially ampicillin/penicillin which are the primary antibiotics for treating listeriosis [14,15].However, resistance to single or multiple antibiotics have recently been reported for L. monocytogenes strains isolated from various food and environmental sources [6,[16][17][18].It is, therefore, necessary to intensify research aimed at increasing the available data on the prevalence and antibiotic susceptibility of L. monocytogenes isolates from various food and environmental sources around the world.
In Ghana, cow milk may be consumed in its raw form, heated or processed by spontaneous fermentation into a yoghurt-like beverage known as nunu [19][20][21].Poor control measures in the Ghanaian dairy chain potentially exposes raw cow milk and its products to possible contamination by pathogenic microorganisms such as L. monocytogenes, which has implications for the safety of consumers of raw milk and traditional dairy products in Ghana.Appiah et al. [20] reported on the quantitative probabilistic assessment of L. monocytogenes exposure among consumers of milk in Ghana.In general, however, there is limited data that can be used for the qualitative and/or quantitative assessment of the risk of L. monocytogenes infection associated with the consumption of raw milk and traditional dairy products in Ghana.The problem of limited data is compounded by the lack of a proper surveillance systems for food-borne diseases to provide reliable information on the burden of food-borne illnesses, particularly those involving L. monocytogenes associated with milk and milk products in Ghana.Consequently, a number of illnesses or sporadic outbreaks associated with the consumption of Listeria contaminated raw milk and milk products may go unreported.Therefore, the objectives of this study were to determine the prevalence of L. monocytogenes in raw milk, heated milk and nunu in Ghana and to characterize the isolated L. monocytogenes according to their molecular serogroups, antibiotic susceptibility profiles and virulence potential.

Samples
A total of 326 samples comprising 114 raw cow milk, 56 boiled milk and 84 nunu (spontaneously fermented yoghurt-like milk beverage) were purchased from dairy farms and open markets in the Northern Region of Ghana between September 2015 and July 2016.Raw milk samples were collected within 10 min. of milking at the dairy farm while boiled milk and nunu samples were collected at point of sale in the market.Prior to sampling, all samples were stored at ambient temperatures (28 ± 2 • C).All samples were collected aseptically and immediately transported in a cool box stored at 4.0 ± 0.5 • C to the laboratory (Savana Agricultural Research Institute, Tamale-Ghana) for analysis.Samples were kept at 4 • C and analyzed within 24 h of collection.The pH of samples was determined upon delivery to the laboratory using a pH meter (Crison basic 20, Barcelona, Spain) calibrated with standard buffers.

Isolation and Characterization of L. monocytogenes
Isolation and identification of L. monocytogenes in this study was carried out as described by Becker et al. [22].Briefly, a 25 g or 25 mL of each sample was aseptically homogenized in 225 mL of Listeria enrichment broth base (CM 0862, Oxoid Ltd., Basingstoke, UK) with selective enrichment supplement (SR 0141, Oxoid Ltd., Basingstoke, UK) in Stomacher bags (Seward Ltd., Worthing, UK) for 30 s using a Stomacher (BagMixer, Buch & Holm A/S, Interscience, 78860 St Nom, France), followed by incubation at 30 • C for 24 h.Second enrichment was done by adding 0.1 mL of the overnight culture to 10 mL of Listeria enrichment broth base (Oxoid, Basingstoke, UK) with selective enrichment supplement (Oxoid, Basingstoke, UK) and incubated at 37 • C for 48 h.Subsequently, 0.1 mL of the enriched broth was surface plated on Listeria selective oxford agar (CM0856, Oxoid, Basingstoke, UK) plates supplemented with Listeria selective supplement (SR0140, Oxoid, Basingstoke, UK).Inoculated plates were incubated at 37 • C for 24-48 h.Presumptive Listeria spp.were identified on oxford agar plates after 24 h incubation as colonies with approximately 1 mm diameter, grey to black colonies surrounded by a black halo.Following 48-h incubation period, typical Listeria species colonies were approximately 2-3 mm diameter, black with a black halo and sunken center.
For biochemical identification of L. monocytogenes, up to five (5) presumptive colonies from each positive plate were streaked on tryptic soy agar (Oxoid, Basingstoke, UK) supplemented with 0.6% yeast extract (Oxoid, Basingstoke, UK) (TSA-YE) and incubated at 35 • C for 24 h.The colonies from TSA-YE plates were confirmed by biochemical tests including Gram staining, catalase, oxidase, triple sugar iron (TSI), sulphide-idole-motility (SIM), methyl-red Voges-Proskauer (MR-VP) reaction, nitrate reduction, and production of acid from rhamnose, xylose, mannitol and α-methyl-D-mannopyranoside.Listeria monocytogenes ATCC 19115 was used as a reference strain for biochemical tests and PCR analysis.

PCR-Based Identification of L. monocytogenes
Following biochemical characterization of Listeria isolates, identities of L. monocytogenes were further confirmed polymerase chain reaction (PCR).Crude DNA was extracted from Listeria isolated in this study and L. monocytogenes ATCC 19115, used as positive control, by direct boiling of a suspension of the cell lysates [23].PCR was performed to detect the presence of Listeria spp.(LI1/U1) using the specific primer sequences (5 -CTCCATAAAGGTGACCCT-3 ) and (5 -CAGCMGCCGCGGTAATC-3 ) and L. monocytogenes (LM1/LM2) primer sequences (5 -CCTAAGACGCCAATCGAA-3 ) and (5 -AAGCGCTTGCAACTGCTC-3 ) [24].The primers LI1/U1 and LM1/LM2 were used to amplify the highly conserved 16S rRNA gene (938 bp) of all Listeria spp.and listeriolysin O gene (702 bp) to detect L. monocytogenes, respectively.The PCR reaction was performed in an automatic thermal cycler (Biotron, Göttingen, Germany) under the following optimized cycling conditions: initial denaturation step of 4 min at 95 • C; 30 cycles of denaturation at 95 • C for 1 min, annealing at 52 • C for 45 s, extension at 72 • C for 2 min; and a final elongation at 72 • C for 8 min.The PCR products were separated by a submerged 1.5% agarose gel electrophoresis, stained with ethidium bromide and visualized under UV illumination.A PCR reaction mixture without DNA template was used as negative control for extraneous nucleic acid contamination.

Detection of Virulence Associated Genes in L. monocytogenes
Detection of the virulence-associated genes in L. monocytogenes was carried out in two separate multiplex PCR assays.Internalin genes (inlA, inlB, inlC, and inlJ), were detected in a multiplex-PCR using primers and cycling conditions described by Liu et al. [26].For the detection of plcA, actA, hlyA, iap and prfA, PCR assay was carried out as described by Kalorey et al. [27].

Determination Antimicrobial Resistance among L. monocytogenes Isolates
Sixty-two identified L. monocytogenes isolates were tested for their susceptibility to antibiotics using the broth micro-dilution method recommended by the standard criteria of the Clinical and Laboratory Standards Institute (CLSI) guidelines M45-A2 for L. monocytogenes [28].The antibiotics used include Amoxicillin, Ampicillin, Cefepime, Chloramphenicol, Ciprofloxacin, Clindamycin, Doxycycline, Erythromycin, Gentamycin, Kanamycin, Neomycin, Penicillin, Rifampin, Tetracycline, and Vancomycin.MIC was determined by two-fold dilution of antibiotics (in the range of 64 to 0.06 µg/mL) in cation-adjusted Mueller-Hinton broth (CAMHB, Difco Laboratories) supplemented with 5% lysed horse blood.The MIC breakpoints used for the interpretation of susceptibility were based on those proposed by Bertsch et al. [16] and Acar et al. [29].

Prevalence of L. monocytogenes in Raw Milk, Boiled Milk and Nunu
In this study, a total of 254 samples of raw milk, boiled milk and nunu were examined for Listeria spp.and Listeria monocytogenes (Table 1).Overall, 44/254 (12.2%) samples were contaminated with Listeria spp.whereas 14/254 (5.5%) samples were positive for Listeria monocytogenes.The prevalence of L. monocytogenes among the various samples were 8.8% (10/114) for raw cow milk and 4.7% (4/84) for nunu.No Listeria spp. or L. monocytogenes was detected in boiled milk.All Listeria spp.and L. monocytogenes isolates from raw milk and nunu samples were confirmed by both biochemical tests and duplex PCR (Figure 1).

Determination Antimicrobial Resistance among L. monocytogenes Isolates
Sixty-two identified L. monocytogenes isolates were tested for their susceptibility to antibiotics using the broth micro-dilution method recommended by the standard criteria of the Clinical and Laboratory Standards Institute (CLSI) guidelines M45-A2 for L. monocytogenes [28].The antibiotics used include Amoxicillin, Ampicillin, Cefepime, Chloramphenicol, Ciprofloxacin, Clindamycin, Doxycycline, Erythromycin, Gentamycin, Kanamycin, Neomycin, Penicillin, Rifampin, Tetracycline, and Vancomycin.MIC was determined by two-fold dilution of antibiotics (in the range of 64 to 0.06 µ g/mL) in cation-adjusted Mueller-Hinton broth (CAMHB, Difco Laboratories) supplemented with 5% lysed horse blood.The MIC breakpoints used for the interpretation of susceptibility were based on those proposed by Bertsch et al. [16] and Acar et al. [29].

Prevalence of L. monocytogenes in Raw Milk, Boiled Milk and Nunu
In this study, a total of 254 samples of raw milk, boiled milk and nunu were examined for Listeria spp.and Listeria monocytogenes (Table 1).Overall, 44/254 (12.2%) samples were contaminated with Listeria spp.whereas 14/254 (5.5%) samples were positive for Listeria monocytogenes.The prevalence of L. monocytogenes among the various samples were 8.8% (10/114) for raw cow milk and 4.7% (4/84) for nunu.No Listeria spp. or L. monocytogenes was detected in boiled milk.All Listeria spp.and L. monocytogenes isolates from raw milk and nunu samples were confirmed by both biochemical tests and duplex PCR (Figure 1).Among other human pathogenic microorganisms, L. monocytogenes is considered a major microbiological hazard associated with the consumption of raw cow milk [30,31].The prevalence of Listeria spp.and Listeria monocytogenes in milk and dairy products, has been widely reported [17,[31][32][33].The prevalence of L. monocytogenes in milk varies considerably among different survey reports and has been attributed to factors such as geographic (environmental) conditions, farm size, farm Among other human pathogenic microorganisms, L. monocytogenes is considered a major microbiological hazard associated with the consumption of raw cow milk [30,31].The prevalence of Listeria spp.and Listeria monocytogenes in milk and dairy products, has been widely reported [17,[31][32][33].The prevalence of L. monocytogenes in milk varies considerably among different survey reports and has been attributed to factors such as geographic (environmental) conditions, farm size, farm management practices, sampling and detection methods used among others [30].In this report, the prevalence of L. monocytogenes in raw cow milk collected from small dairy farms in northern Ghana was 8.8% while Appiah et al. [20] reported prevalence of over 42% for raw cow milk sampled from farms in Accra (southern Ghana).According to other previous survey reports, the frequency of detection of L. monocytogenes in raw cow milk can vary from 0% [34] to more than 45% [35][36][37][38][39][40].Despite the widely reported variation in the prevalence of L. monocytogenes in milk, all the surveys demonstrate that raw cow milk and dairy products can be a source of Listeria infections in humans [30,32].
Listeria monocytogenes occurs widely in agricultural and food processing environments [41] and can contaminate raw milk during milking processes [31].Additionally, L. monocytogenes is widely distributed in food processing environments with the capacity to tolerate and grow in the temperature range of 0-45 • C in media with pH range of 4.4-9.4[42][43][44].
Due to the observed high prevalence of L. monocytogenes in raw cow milk from dairy farms in this study, it is possible that the pathogen could enter the processing environments and persist during processing, resulting in the contamination of the dairy products.Nunu is produced by spontaneously fermenting raw cow milk at ambient temperature (28-35 • C) for about 18-24 h under uncontrolled conditions, bringing the pH to 4.1 ± 0.9 (Table 1).The fermented yoghurt-like milk beverage, nunu, can then be consumed within 4 days after fermentation without refrigeration or refrigerated at 4 • C for several weeks [21].The observed prevalence of L. monocytogenes in nunu may be attributed to factors such as post-processing contamination, improper handling of raw milk, or inadequate fermentation to bring the pH below tolerance levels for L. monocytogenes.Thus, the production of organic acids and bacteriocins which are important natural antimicrobials in nunu, resulting from lactic acid fermentation of milk, might not be sufficient to eliminate L. monocytogenes in the product and therefore should not replace good hygiene practices (practices) during processing.On the other hand, Listeria was not detected in boiled milk, which undergoes thermal treatment without further processing.Boiling is one method that is known to reduce or even eliminate the microbiological risk associated with the consumption of raw milk [30].

Prevalence of Virulence-Associated Markers
Irrespective of serotype, all of the sixty-two (62) L. monocytogenes tested harbored the virulence-associated genes inlA, inlB, inlC, and inlJ inlA, inlB, inlC, inlJ, plcA, actA, hlyA, iap and prfA.Listeriolysin O is reported to be the main bacterial determinant for the escape of L. monocytogenes from both primary and secondary vacuoles and is one of its main virulent factors [50,51].The detection of internalin genes (inlA, inlB, inlC, and inlJ) in almost all examined L. monocytogenes isolates from food samples have been reported [52][53][54][55].The virulence markers InlA, InlC and InlJ, all of which play various roles in L. monocytogenes infections and pathogenesis, are important virulence factors of L. monocytogenes [11].In L. monocytogenes, InlA and InlB have evidently been shown to be important for host cell invasion and virulence [56,57].Furthermore, null mutations in the internalin genes (inlA, inlB, inlC, and inlJ) result in reduced invasion or virulence in tissue culture or animal model studies [58-60], underscoring the important role of these genes in virulence and pathogenesis.In general, the presence of the internalin genes and the other virulence-associated genes in L. monocytogenes isolated from milk and dairy products in Ghana indicates that these L. monocytogenes strain could be potentially virulent.

Antibiotic Resistance among L. monocytogenes Isolates
Phenotypic resistance to 15 antimicrobial agents by 62 L. monocytogenes isolates from raw milk and nunu in Ghana are shown in Table 2. Based on the MIC breakpoints used in this study, L. monocytogenes isolates were generally susceptible to amoxicillin (100%), ampicillin (100%), erythromycin (100%), gentamycin (100%), penicillin (100%), rifampicin (100%) and vancomycin (100%).However, phenotypic resistances were observed against neomycin (61.3%) and tetracycline (24.2%), while intermediate susceptibilities were obtained for chloramphenicol, ciprofloxacin, clindamycin, doxycycline, kanamycin, neomycin, streptomycin and tetracycline.Listeria monocytogenes is naturally reported to be sensitive to a wide range of antibiotics.However, recent reports show a continuous pattern of emergence of resistant strains [6,14,48].Listeria monocytogenes from food sources have generally been reported to be highly susceptible to ampicillin, gentamycin, penicillin, rifampicin and vancomycin.On the other hand, varying prevalence of resistance to tetracycline by L. monocytogenes isolates from different food sources have also been reported [16,53,61,62].The high number of L. monocytogenes isolates showing intermediate susceptibility to ciprofloxacin (11.3%) and clindamycin (17.7%) has similarly been reported by Lyon et al. [63] and Bertsch et al. [16].Natural populations of several Listeria species have been described as intermediately susceptible to ciprofloxacin and clindamycin [64].

Conclusions
The results presented in this report demonstrates Listeria monocytogenes is prevalent in raw milk and nunu, a spontaneously fermented yoghurt-like milk beverage, in Ghana.L. monocytogenes isolates from raw milk and nunu in Ghana harbored virulence-associated genes.However, the L. monocytogenes isolates were generally susceptible to the different antibiotics tested except tetracycline and neomycin for which phenotypic resistance was observed.However, genotypic assessment of the prevalence of genetic markers associated with antimicrobial resistance for the genetic basis of resistance phenotypes observed in this study would provide a better baseline for further antimicrobial resistance molecular surveillance of L. monocytogenes milk and dairy products in Ghana.Implementation of adequate sanitation program and good hygiene/manufacturing practices (GHP/GMP) for milk production and processing of fermented dairy beverages in Ghana could reduce microbial contamination of these products.

Figure 1 .
Figure 1.Representative PCR product detecting the highly conserved 16Sr RNA gene sequence for Listeria spp.and listeriolysin O gene sequence for L. monocytogenes.Lane M, 100 bp molecular size DNA marker; lane 1, L. monocytogenes ATCC 19115 (positive control); lanes 2-7, L. monocytogenes isolated from raw cow milk and nunu; lanes 8 and 9, non-L.monocytogenes isolated from raw cow milk and nunu; lane 10, negative control (PCR mixture without DNA template).

Figure 1 .
Figure 1.Representative PCR product detecting the highly conserved 16Sr RNA gene sequence for Listeria spp.and listeriolysin O gene sequence for L. monocytogenes.Lane M, 100 bp molecular size DNA marker; lane 1, L. monocytogenes ATCC 19115 (positive control); lanes 2-7, L. monocytogenes isolated from raw cow milk and nunu; lanes 8 and 9, non-L.monocytogenes isolated from raw cow milk and nunu; lane 10, negative control (PCR mixture without DNA template).

Table 1 .
Prevalence of Listeria spp.and Listeria monocytogenes in raw milk, boiled milk and nunu.
ND: Not detected; NA: Not applicable; a Spontaneously fermented yoghurt-like milk beverage.

Table 1 .
Prevalence of Listeria spp.and Listeria monocytogenes in raw milk, boiled milk and nunu.
Product pH of Samples Number of Samples Listeria spp.(%)Listeria monocytogenes (%)ND: Not detected; NA: Not applicable; a Spontaneously fermented yoghurt-like milk beverage.

Table 2 .
Antimicrobial susceptibility profile of L. monocytogenes isolates.
a MIC breakpoint values for all antibiotics against L. monocytogenes were according to the description of Bertsch et al., 2014, except doxycycline and neomycin for which the breakpoints proposed by Acar et al., 1998 were used.b S: susceptible; I: intermediate susceptibility; R: resistant.