Detection of Pathogenic Serogroups and Virulence Genes in Listeria monocytogenes Strains Isolated from Beef and Beef Products Retailed in Gauteng Province, South Africa, Using Phenotypic and Polymerase Chain Reaction (PCR)-Based Methods

South Africa recently (2017-18) experienced the largest outbreak of human listeriosis in the world caused by L. monocytogenes following the consumption of “polony,” a ready-to-eat meat product. Most (59%) cases originated from Gauteng province, South Africa. As a follow-up study to the outbreak, we used standard bacteriological and molecular methods to determine the prevalence of pathogenic and virulent serogroups of L. monocytogenes in various beef and beef products retailed in Gauteng province, South Africa. The overall prevalence of Listeria spp. was 28% (112/400), comprising Listeria monocytogenes (9.3%), Listeria innocua (16.3%), and Listeria welshimeri (2.5%) (p < 0.001). It is crucial to have detected that the region (p=0.036), type of product (p=0.032), and temperature at storage (p=0.011) significantly affected the occurrence of L. monocytogenes in beef products. It is alarming that pathogenic serogroups 4b-4d-4e (51.4%) and 1/2a-3a (43.2%) were detected among the isolates of L. monocytogenes. Importantly, they were all carriers of seven virulence-associated genes (hlyA, inlB, plcA, iap, inlA, inlC, and inlJ). Our study also demonstrated that 16.7% of “polony” samples investigated were contaminated with L. monocytogenes. Considering that pathogenic and virulent L. monocytogenes contaminated beef and beef products retailed in South Africa, the food safety risk posed to consumers remains and cannot be ignored. Therefore, it is imperative to reduce the contamination of these products with L. monocytogenes during beef production, processing, and retailing to avoid future outbreaks of human listeriosis in the country.


Introduction
Listeriosis is a well-known food-borne infection mainly afecting humans, particularly immunocompromised, pregnant, young, and old individuals [1].Listeria monocytogenes is the leading cause of human and animal listeriosis, posing the highest risk to food safety and public health and responsible for various clinical manifestations [2].L. innocua has been generally considered nonpathogenic, and human listeriosis infections due to L. innocua constitute rare occasions.However, few reports of the implication of L. innocua in human listeriosis have been documented [3].L. innocua is also known to share the same food environment with pathogenic L. monocytogenes, with the potential for the transfer of virulence and resistance genes between both species of Listeria [4].Several global human listeriosis outbreaks have been reviewed [5].L. monocytogenes has been isolated from several types of foods that serve as vehicles for transmitting the pathogen to humans and causing listeriosis globally [6].Te types of food implicated include milk and milk products [7], vegetables [8], and meat and meat products [9].
Several virulence genes have been documented in L. monocytogenes strains recovered from beef and beef products and other food sources [13,14].Te frequency of virulence genes is higher in L. monocytogenes than in other Listeria spp [15,16] and varies with the geographical location, source, and types of samples [10].Te virulence genes possessed by strains of L. monocytogenes have been demonstrated to be responsible for the organism's pathogenicity [17,18], especially those in the Listeria Pathogenicity Islands (LIPIs) [17][18][19].Te genes perform diferent roles in the pathogenicity of L. monocytogenes, including facilitating the infectious life cycle and survival in the food processing environment by the virulence genes in the LIPI-1 and LIPI-3 clusters [20].For example, inlA, inlB, and inlC play a role in the adherence to, and internalization by the host cell, virulence genes hly, plcA, and plcB are responsible for escape from the vacuoles, htp enables intracellular replication, and actA gene facilitates cellular movement by L. monocytogenes [17,18,21].
Between 2017 and 2018, South Africa experienced the world's largest outbreak of human listeriosis [22].Whole genome sequencing (WGS) identifed L. monocytogenes sequence type 6, which originated from "polony," an RTE beef product, as the outbreak's origin [23].Matle et al. [10] reported the prevalence of L. monocytogenes to be 10.1%, 13.5%, and 19.5% for raw intact meat, RTE meat products, and processed meats in South Africa.Tere is, however, a shortage of current information on epidemiological data on the samples assessed for contamination with L. monocytogenes, the risk posed by RTE foods, and the species of Listeria other than L. monocytogenes in beef and beef products.
Of relevance to the current study conducted in Gauteng province, one of the nine provinces in South Africa, is that 57.93% of the country-wide confrmed 1060 cases in the recent human listeriosis outbreak the country originated from the province [22,24].Te current study, therefore, investigated the occurrence of pathogenic serogroups and virulence genes in L. monocytogenes and other species of Listeria in beef and beef products using phenotypic and polymerase chain reaction (PCR)-based methods.Te study characterized the isolates of L. monocytogenes regarding their serogroups and carriage of virulence genes.Te study also investigated the efects of selected variables on detecting Listeria spp.

Physicochemical Properties and Microbiological Properties of Biltong and Polony.
A total of fve categories of beef and beef products (beef burger, ""biltong", minced beef, brisket, cold beef ("polony" and Vienna) were sampled in the current study, but only two ("Biltong" and "Polony") are unique, and popularly consumed in South Africa.

"Biltong".
Biltong is an RTE beef product popularly consumed across South Africa.It is produced in dry and moist forms for consumers at sale outlets.Biltong is made from thin slices of raw muscle meat that are marinated or cured (spices and organic acids), refrigerated, and dried (air or oven) without cooking or thermal lethality step [25,26].Te production of "Biltong" is a relatively straightforward process, increasing traditional home production and smallscale production on farms and butcheries and modern manufacturing methods in the country with no established content and microbial quality control.It is a product like the American dried meat product known as Jerky [27], which, unlike the South African "Biltong," is subjected to thermal lethality step treatment before drying.Dry "biltong" in South Africa is characterized by a low water activity (a w ) ranging from 0.65 to 0.68 [28].It has been documented that the critical a w in which dry "biltong" is microbiologically stable is below 0.68 [29].On the other hand, moist "biltong" has higher moisture content, ranging from 0.85 to 0.89 [30].Te pH ranges of "biltong" in South Africa have been variable.Petit et al. [28] reported that the pH values of "biltong" varied from 5.00 to 6.26, with an average of 5.35 and 5.58 for dry and moist "biltong," respectively.Reports by others documented some "biltong" pH ranges between 4.81 and 5.83 [29,31,32].Gavai et al. [33], using the standard process for processing "biltong" assessed the efect of the drying process on the population of inoculated L. monocytogenes and reported that an internal water activity (a w ) reached <0.85 at 5-log reduction levels and ensured that conditions were lower than that which would support bacterial growth, including L. monocytogenes.To date, "biltong" (moist or dry) has not been associated with human listeriosis.
Only dry biltong was sampled and processed in the current study.

"Polony"
. "Polony," a bologna sausage, is a low-cost, readily available food popular across all socioeconomic groups in urban and rural communities in South Africa.It is produced mainly from mechanically recovered meat (beef/ pork/chicken) and processed by food manufacturers in South Africa [22].Te extensive use of meat trimmings (beef, pork, or chicken) makes "polony," an inexpensive and afordable meat product [34].Te pH ranges of "biltong" in South Africa have been variable.Te product is typically sliced and served cold."Polony" has been documented to have a shelf life of fve months and is produced in large quantities by several manufacturers for local consumption and export [23]."Polony" elicited considerable anxiety in the South African population with the implication that the RTE 2 International Journal of Microbiology product, which originated from a single facility in the country, was demonstrated to be responsible for the large outbreak of human listeriosis.An average pH of 5.35 and 5.58 for dry and moist polony, respectively, has been reported for "polony" [35].Kivikari [36] reported that the pH and bufering capacity of the raw materials used in cooked sausage or "polony" afected the fnal pH.

Study Design.
Te cross-sectional study was conducted at 48 retail outlets in Gauteng province, South Africa.Tese were selected based on information from the Consumer Goods Council of South Africa (CGCSA) (https://www.cgcsa.co.za/).Te number of samples collected from each type of outlet was proportional to its size and availability during sampling visits using a convenience sampling approach.Te sample size for the study was determined using the formula by Trusfeld [37], where P exp is the expected prevalence and d is the desired precision A P exp value of 14.7% [10]  ( For the study, a total of 400 samples were collected from retail outlets across Gauteng province, South Africa, and distributed across 48 retail outlets, as shown in Supplementary Table S1.Overall, from the 48 outlets, 8 samples were collected from 32 outlets (n � 256), while 9 samples were obtained from 16 outlets (n � 144).
Te samples were collected between October 2019 and April 2021 during a single visit to each outlet, including raw, chilled, and frozen beef and dried beef-based RTE products.

Sample Collection, Isolation, and Identifcation of Listeria spp.
For the 48 outlets from where samples were collected, at 32 (66.7%) outlets, 8 samples were collected per outlet, while at 16 (33.3%),9 samples were obtained per outlet.Te types of samples collected per outlet were based on the beef and beef products available during the visit.Te strategy for collection and the number of samples collected from each outlet are shown in Supplementary data, Table S2.Te maximum number of samples collected from the four categories of the outlets was 12 (n � 160), 8-10 (n � 128), 4-6 (n � 80), and 1-2 (n � 32) for chain, large, medium, and small retail outlets, respectively.A total of 13 types of raw and beef products (beef steak, liver, tripe, chunk, sausage, raw beef, beef "polony," Russian "polony," Vienna, dry "biltong," moist "biltong," beef patties, and beef burger) were sampled from the retail outlets (Supplementary Table S2).
Matle et al. [10] described isolating and confrming suspect Listeria spp.using phenotypic assays.For the study, the validated Listeria Precis method, according to a protocol by Termofsher Scientifc and reported by Matle et al. [10], was used with some modifcations.Ten grams of each sample was aseptically transferred into a stomacher bag containing 225 mL of ONE broth-Listeria (Oxoid, Basingstoke, UK) for selective enrichment.Te samples were homogenized in a Stomacher (Stomacher Lab Blender 400, Seward Ltd., West Sussex, UK) for 5 min at 15,493 × g speed, followed by aerobic incubation at 35 °C for 24 h for enrichment.Tereafter, for isolation of Listeria, 10 μL of selective enriched broth sample was inoculated onto Brilliance-Listeria agar (BLA) plates (Oxoid, Basingstoke, UK) and incubated at 35 °C for 24 h.Based on the phenotypic appearance of the colonies on BLA, which presumptively classifed green-blue colonies without a halo as Listeria spp.Blue colonies with white/cream halo were tentatively identifed phenotypically as L. monocytogenes.Single colonies of suspected Listeria spp.(colonies that appeared blue without a halo) and L. monocytogenes (blue colonies with a white/cream halo) were subcultured on BLA for further purifcation [38].

Extraction of DNA from Selective Enriched Broth
Cultures and Colonies.DNA was extracted by heating method, and the crude extract was used in subsequent PCR protocols [39].Briefy, 2 ml aliquots of samples in selective enrichment broth were spun at 15,493 x g for 5 minutes in a microcentrifuge (Eppendorf, South Africa).Te pellets were suspended in 200 μL of sterile distilled water, heated to 95 °C in a dry block for 10 minutes, cooled at room temperature for 5 minutes, and centrifuged at 15,493 × g for 5 minutes.Te supernatant was pipetted into sterile tubes, and the pellet was discarded.Te DNA in the supernatant was used as template DNA in PCR assays.

Determination of the Species of Listeria Using mPCR.
Te multiplex PCR was used to determine the fve species of the Listeria isolates (L.grayi, L, innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri) as described by Ryu et al. [40].Te multiplex PCR mix was prepared as follows: 12.5 μL of 2x red Taq master mix, 5 μL (Lasec, SA, Pty, Cape Town, South Africa) nuclease-free water, 5 μL DNA template, and 4 μL of 20 μM primer mix for PCR assay.Multiplex PCR was performed with an initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, and fnal extension at 72 °C for 5 min.PCR amplicons were electrophoresed on a 3.0% agarose gel using 1 × Tris-acetate-EDTA (TAE) bufer and stained with ethidium bromide.L. monocytogenes ATCC 19111, Listeria innocua ATCC 33090, L. welshimeri ATCC 35897, L. grayi ATCC 25401, L. ivanovii ATCC 19119, and L. seeligeri ATCC 35967 were used as positive controls, Campylobacter fetus ATCC 27373 as a negative control, and water as a blank.Te primer sequences used to determine the species of Listeria in the study are listed in Supplementary data, Table S3.

Determination of the Serogroups of L. monocytogenes
Isolates.Multiplex PCR was used to classify L. monocytogenes strains into serogroups by targeting genes (ORF2110, ORF2819, Imo1118, Imo0737, and prs) as described by Doumith et al. [41].Te prs gene was used as a target marker for the Listeria species.PCR mix was prepared as follows: 12.5 μL of 2x of red Taq master mix (Lasec, South Africa), 5 μL nuclease-free water, 5 μL DNA template, and 4 μL of 20 μM primer mix.Te PCR cycling conditions were as follows: initial denaturation step at 94 °C for 3 min; 35 cycles of 94 °C for 0.40 min at 53 °C for 1.15 min and 72 °C for 1.15 min, and one fnal cycle at 72 °C for 7 min and amplifcation was done in a Termocycler.Te PCR products were subjected to electrophoresis on 3% ethidium bromide-stained agarose gel for 3 h at 100 v. L. monocytogenes ATCC 19111 was used as a positive control, Campylobacter fetus ATCC 27373 was used as a negative control, and water was used as a blank.Te primer sequences used to determine the serogroups of L. monocytogenes in the study are listed in Supplementary data, Table S4.

Detection of Virulence Genes in L. monocytogenes
Isolates.Multiplex PCR, earlier described by Rawool et al. [42], was used to identify the hlyA, plcA, actA, and iap virulence genes specifc to L. monocytogenes.Te second multiplex PCR was used to determine the extra virulence genes inlA, inlB, inlC, and inlJ [43].PCR mix was prepared as follows: 12.5 μL 2× DreamTaq master mix, 5 μL nuclease-free water, 5 μL template DNA, 3 μL primer mix.Te PCR cycling conditions were as follows: Te initial denaturation step at 2 min at 94 °C, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a fnal extension at 72 °C for 10 min.Te PCR products were electrophoresed on a 3% agarose gel, and results were captured using a gel documentation system (Vacutec, SA).Te primer sequences used to detect virulence genes are listed in Supplementary data, Table S5.

Data Analysis.
Laboratory data generated for the prevalence of the six species of Listeria, serogroups, and virulence genes were analyzed using Stata software (Stata-Corp LLC, College Station, Texas, USA), and the association of variables with the detection of Listeria or selected characteristics was determined using Fisher's Exact and Chisquare tests.Te signifcant diference was evaluated using (P < 0.05), and percentages were calculated at a 95% confdence interval.A descriptive analysis was also performed using StataCorp to assess the prevalence of the six species by the locations of the outlets, type of retail outlets, type of beef and beef products, storage temperature, and product status.
Te prevalence of L. monocytogenes, L. innocua, and L. welshimeri in beef and beef products and the univariate analyses of associated factors is shown in Table 1.
Te prevalence of L. innocua was 16.3% (65/400).Te temperatures at which the beef and beef products were kept pre-sale had a statistically signifcant (P < 0.001) efect on the prevalence of L. innocua.

Discussion
Our fndings that L. monocytogenes contaminated 9.3% of the beef and beef products tested have food safety implications for consumers because L. monocytogenes, the most 4 International Journal of Microbiology important species of Listeria, is frequently associated with cases and outbreaks of human listeriosis [44].A similar prevalence of 8.3% for L. monocytogenes was reported for beef and beef products (raw beef, RTE, milled beef, ofal, and organs) sampled at retail outlets in Mpumalanga province, South Africa [45].However, Matle et al. [10] reported a higher pathogen prevalence (14.7%) in the country's meat products.Te diferences in the fndings between both studies that used the same detection methods may be due to several factors.Tese factors include that the current study was conducted on beef and beef products sampled from retail outlets in Gauteng province.In contrast, the study reported by Matle et al. [10] was done on meat and meat products (poultry, cattle, sheep, pork, and game); meat samples collected from the three major ports of the country and abattoirs, meat processing plants, butcheries, and retail outlets.Tis is because the sources of the meat products can potentially infuence the prevalence of L. monocytogenes.It has been documented that all these variables potentially afect the prevalence of L. monocytogenes in meat [46].Te study also documented, for the frst time in Gauteng province, South Africa, the prevalence of three species of Listeria (L.monocytogenes, L. innocua, and L. welshimeri) in beef and beef products according to retail outlet location, types of outlets, and beef products, and the virulence/ pathogenic characteristics of the L. monocytogenes isolates.
Te prevalence of L. innocua (16.3%) in the present study is lower than the 21.3% in RTE food samples in Johannesburg, South Africa [47].Te organism has been recovered at varying frequencies in meat products elsewhere, such as in Spain, 13.9% [48], and China, 28.9% [49].It is well established that, unlike L. monocytogenes, the most important Listeria species implicated in human listeriosis, L. innocua is considered nonpathogenic [50].Rare reports associating L. innocua with human listeriosis have been documented in immunocompromised individuals [3].Te detection of L. innocua in beef and beef products in the current study may, therefore, not have clinical signifcance for human listeriosis, but they are known to share the same food niche or environment with L. monocytogenes [48], with the possibility of transfer of genes (e.g., virulence and resistance genes) between L. monocytogenes and L. innocua.Furthermore, WGS confrmed the presence of the L. monocytogenes pathogenicity island (LIPI) in strains of L. innocua.[51].Tese fndings suggest that the pathogenic potential of L. innocua in humans cannot be ignored.

International Journal of Microbiology
Regarding the variables for the detection of L. monocytogenes in the current study, the prevalence of L. monocytogenes varied signifcantly across the district locations of the retail outlets, a fnding in agreement with published studies in South Africa [10] and Bangladesh [55].However, Ristori et al. [56] did not detect any association between the presence of L. monocytogenes in meat products and the geographical regions in Brazil.Te disparity in the types of outlets, hygienic practices at the outlets, and the degree of contamination by Listeria spp.may account for the diferences across regions.
Unsurprisingly, L. monocytogenes was detected at the highest frequency (13.2%) in chilled beef and beef products compared with those kept at room temperature and frozen temperatures.Te pathogen can survive and multiply at chilling or refrigeration temperatures, which occur at the retail level [57] and during transport.Tis may afect the number and detection frequency for Listeria.
Interestingly, the prevalence of L. monocytogenes varied signifcantly among beef and beef products.Tis may be attributed to preparation, treatments, or methods of handling, such as brisket to raw spiced beef ("biltong"), raw beef chopped with a knife or grinder (minced meat) to precooked (cold beef/delis).Tese variable preparation methods and hygienic practices can potentially increase or decrease contamination by L. monocytogenes [58].
In our study, L. monocytogenes contaminated 14.5% of minced beef samples, which is higher than the prevalence of 1% reported in minced meat in Switzerland [59] and 12.2% in Japan [60].However, considerably higher frequencies of contamination of minced meat and products by L. monocytogenes have been documented in Ireland, 29% [61], Belgium, 42.1% [62], and Brazil, 59.4% [56].Minced beef and beef products are known to be contaminated by pathogens, primarily due to the preparation methods [56].Furthermore, minced meat-borne listeriosis outbreaks have been documented [63].
Te detection of L. monocytogenes in 6.9% of the RTE products is also a food safety concern since RTE beef products have been associated with human listeriosis globally [6].However, although L. monocytogenes was detected in various brands of "biltong" sampled at several sources (home industries, butcheries, supermarkets), the combination of the curing (pH range: 4.81-5.83)and drying (a w 0.65-0.68) of the product inhibited the survival or growth of the pathogen after marination and within 96 h post-drying [28,30,31].Tese fndings were supported by the report of Gavai et al. [33] that the low internal a w (<0.85) achieved in assessing "biltong" produced by the standard process in the USA failed to support the survival of L. monocytogenes.Tis is imperative considering that poor sanitary practices exist in the preparation of "biltong," particularly during production at homes, farms, and butcheries in South Africa that facilitate product contamination.Tese fndings suggest that there is a need to standardize the output of "biltong" in South Africa to assess the potential for moist and dry "biltong" to support the contamination and subsequent proliferation of L. monocytogenes at critical control points, specifcally during curing (types and concentration of agents) and the temperature and length of drying the product.Tese factors can potentially infuence the contamination, survival, and multiplication of L. monocytogenes in "biltong."For example, all "biltong" samples tested in Botswana were negative for L. monocytogenes, as reported by Matsheka et al. [64].Te authors attributed the absence of the pathogen compared to the reported contamination of "biltong" produced in South Africa to the fact that unlike in South Africa, 6 International Journal of Microbiology the processing of "biltong" does not involve soaking of meat in cider; the spices are applied directly onto the fresh meat before solar drying in the open air.Furthermore, the mean a w of the product is 0.51 compared to the mean a w of 0.65-0.68 for the product in South Africa [28].
It cannot be over-emphasized that "Polony," another popularly consumed RTE in the country, was responsible for the largest outbreak of human listeriosis [23].Studies elsewhere have documented a lower prevalence of L. monocytogenes in RTE beef products, such as the reported 0.3% [65] and 3.7% to 5.1% in Ethiopia [66] than in our study.However, a higher prevalence of L. monocytogenes (44.3%) in RTE beef products was reported in Trinidad and Tobago [67].Te possibility of "polony" still serving as a vehicle and promoter of growth for L. monocytogenes in consumers cannot be ignored.Te presence of L. monocytogenes could be attributed to the contamination of "polony" before and during production and distribution and within the retail environments of this commodity due to the ubiquitous presence of this foodborne pathogen [68].Listeria monocytogenes contaminated 16.7% of our study's "polony" samples.
Of potential virulence and pathogenicity importance is the detection that all (100%) of the isolates of L. monocytogenes assessed in the current study were positive for seven of the virulence-associated genes, which included the genes encoding specifc virulence factors, specifcally, internalins (inlA, inlB, inlC, inlJ), hemolysin (hlyA), phospholipase (plcA), and actin polymerization (actA) in 97.3% of the isolates.Similar fndings were reported in a nationwide study on meat and meat products [10] and isolates recovered from cattle farms and cattle abattoirs [10,74].However, diversity has been reported in the detection of the virulence genes in L. monocytogenes recovered from meat and meat products in the literature, such as hlyA, prfA, and inlA in Chile [75], inlC, and inlJ in China [76], and hlyA, actA, inlA, inlB, inlC, inlJ, prfA, plcA, and iap in Turkey and Norway [77,78].Te roles played by these virulence genes in the pathogenesis of clinical listeriosis following the consumption of Listeria-contaminated meat products, particularly RTE foods, are well documented in the literature [76,79,80].Te ability of L. monocytogenes to cause listeriosis is known to be multifaceted.It has been mainly attributed to six virulence genes, prfA, plcA, hly, mpl, actA, and plcB, which are located in the PrfA-dependent virulent gene cluster known as LIPI-1 [81,82], its dependence on genomic islands and, Listeria pathogenicity islands, namely, LIPI-1, LIPI-2, LIPI-3, and LIPI-4, and internalin (inl) genes, as reported by Gilmour et al. [83] and Wagner et al. [84].
Although the present study determined the frequency of pathogenic serogroups and virulence genes among the isolates of L. monocytogenes, a limitation is that the study design did not quantify the number of L. monocytogenes per g of beef and beef products.Tis is because the number of L. monocytogenes per g of beef products is essential to assess the risk of listeriosis posed to consumers of contaminated products.Nonetheless, the potential health risk of listeriosis posed to consumers of beef and beef products cannot be ignored, considering that 9.3% were contaminated by pathogenic serogroups, particularly 4b-4d-4e and 1/2a-3a, and were all carriers of virulence genes.Also of health concern is our fnding that this pathogen also contaminated 16.7% of the "polony" samples processed.Tis is because the product was responsible for the country's recent outbreak of human listeriosis, to which Gauteng province contributed 57.93% of the cases nationwide.It is pertinent to mention that there are currently no standards (quantitative or qualitative) for L. monocytogenes in foods in South Africa.

Conclusions
In conclusion, detecting pathogenic serogroups of L. monocytogenes in 9.3% of beef and beef products, particularly in RTE foods (6.9%), should be a concern for consumers of these products.Tis is important because the country recently experienced a large outbreak of human listeriosis, with 59% of cases linked to the consumption of "polony" and RTE beef products.Te fact that L. monocytogenes was detected in some of these RTE foods in the province is indicative that food safety concerns regarding exposure to the pathogen still exist.However, to better assess the risk of listeriosis posed to consumers of contaminated food products, it is essential to determine the load of L. monocytogenes per g of the product before making appropriate recommendations.Tereafter, the WHO/FAO and FDA listeriosis policy described as a "zero tolerance" where a limit of <100 L. monocytogenes cells/g at the point of consumption is acceptable can be adopted.Te detection of L. innocua, a nonpathogen, at a higher frequency than L. monocytogenes in all the types of beef and beef products tested may have food safety relevance because it shares similar food niches with L. monocytogenes and thus may be predictive of the presence of the pathogen in foods.Overall, it is imperative to reduce or eliminate the contamination of beef and beef products by pathogens such as L. monocytogenes through good sanitary practices at slaughterhouses or abattoirs, during transportation, and at retail outlets.Finally, there is a need to conduct a whole genome sequencing and bioinformatics analysis of the isolates of L. monocytogenes using their sequence types (ST) to construct the phylogeny to elucidate the clustering or genetic relatedness of the isolates from various sources and sample types.

Table 1 :
Prevalence of L. monocytogenes, L. innocua, and L. welshimeri in beef and beef products, and univariate analysis of associated factors.

Table 2 :
Frequency of detection of L. monocytogenes serogroups by the region, size of retail outlets, and type of beef and beef products.Of a total of 37 isolates of L. monocytogenes. 2 Supermarket chain: Over 1 outlet, large: 6 or more cashiers, medium: 3-5 cashiers, and small: 1-2 cashiers.