The consumption of convenience foods, such as sliced and prepackaged ready to eat (RTE) products, has increased worldwide. The RTE meat and dairy products require no post-processing heating or other additional antimicrobial treatments before consumption, hence the contamination of pathogenic microorganisms, such as Listeria monocytogenes, could compromise the safety of the products becoming a great risk for the final consumers (Calvo-Arrieta et al., 2021).

The presence of L. monocytogenes in RTE products has been worldwide described and confirmed, including European Union (European Food Safety Authority, 2022) and South Africa (SA) (Matle et al., 2019). The contamination of RTE products can occur along the entire food production chain as L. monocytogenes is widely distributed in primary production areas and in food production environments (FPE) of food producing plants (FPP), processing and packaging plants, storage facilities and retail shops (Gupta and Achyut, 2022). L. monocytogenes is ubiquitous and able to survive at low refrigeration temperatures, low pH and high salt concentrations, but also to develop biofilms, increasing the risks of food contamination (Lee et al., 2019; Osek and Wieczorek, 2022). L. monocytogenes contamination is associated with poor food processing practices and cross-contaminations in FPP, involving mainly RTE, such as vegetables, fish, dairy and meat products (Dufailu et al., 2021).

Listeria monocytogenes is a foodborne pathogen that can cause listeriosis, a potentially severe foodborne disease in human and animals (Maury et al., 2019). In healthy people, more frequently L. monocytogenes is a cause a febrile gastroenteritis, whereas in susceptible people, such as children, elderly, immunocompromised and pregnant women, it may lead to septicemia, meningitis, abortion and eventually death (Osek and Wieczorek, 2022). Even if rare, an occurrence of respiratory infection caused by L. monocytogens has been reported in Italy (Guidi et al., 2021a).

The European Food Safety Authority (European Food Safety Authority, 2022) reported 2,183 confirmed invasive human cases of L. monocytogenes in 2021. Moreover, the European case fatality rate is high (13.7%), similar to 2020 (European Food Safety Authority, 2022), confirming listeriosis as one of the most severe foodborne diseases. L. monocytogenes outbreaks reported were linked to different food matrices, such as ice cream (Chen et al., 2017b), cheese (Chen et al., 2017a), RTE fish products (Schjørring et al., 2017) and RTE meat products (Althaus et al., 2017; Duranti et al., 2018; Gelbíčová et al., 2018; Orsini et al., 2018).

Few data are available regarding the prevalence of L. monocytogenes in RTE food products from African countries (Garedew et al., 2015; Oyinloye, 2016; Matle et al., 2019), however, one of the biggest listeriosis outbreak, with 1,060 cases, was reported in SA between 2017 and 2018, due to polony, a RTE processed meat product (Smith et al., 2019; Thomas et al., 2020).

The aims of this study were to report the presence of L. monocytogenes in Zambian RTE products, provide data on the genomic characterization of isolated strains and evaluate their similarity with strains circulating in other African countries using whole genome sequencing (WGS).

To date, very few data are available on L. monocytogenes in RTE foods from Zambia and in Zambian products. Our findings highlighted the presence of L. monocytogenes mainly in ham and polony; the latter was the same type of product involved in the SA outbreak (Smith et al., 2019). Previous studies conducted in Zambia reported the prevalence of L. monocytogenes in 74% of poultry dressed carcasses (Mpundu et al., 2022a) and 26.3% in cattle carcasses (Mpundu et al., 2022b).

Data are available from African countries, reporting the presence of L. monocytogenes in different matrices, such as foods, animal and environmental samples, in Nigeria, SA, Egypt, Ethiopia, and Botswana (Dufailu et al., 2021). In particular data related to L. monocytogenes in East Africa, Ethiopia, reported a prevalence of 6.25% in RTE food of animal origin (Garedew et al., 2015), lower than our findings. In Nigeria, a prevalence of 7% was detected in raw meat samples from Rivers State (Odu and Okonko, 2017), while 91.8% in chicken flocks and meat in Oyo (Ishola et al., 2016). In Lafia (Nigeria), the 64.4% of isolates from beef and chevon were confirmed to be L. monocytogenes (Chuku et al., 2020). Additionally, in locally made soft cheeses the 12.4% of isolates were L. monocytogenes (Oyinloye, 2016). In a study, Matle et al. (2019), conducted in meat samples from SA over several years, reported L. monocytogenes in 13.5% of RTE foods collected, higher than the one obtained in our study. The presence of L. monocytogenes in the RTE meat placed on markets could be mainly due to inadequate hygiene management systems (Matle et al., 2019), which is a problem in many African countries.

It emerged in this study that several FPP considered presented more than one RTE meat category contaminated by L. monocytogenes. The subsequent placing on the market of these contaminated products and their availability for the consumers, poses a high risk especially for vulnerable people. The risk associated with RTE products depends mainly on the effectiveness of control measures applied by food business operators (FBOs), including good hygiene and good manufacturing practices, both at processing and retail (European Food Safety Authority, 2022). In smaller retail store and local manufacturers, inadequate equipment for the preparation of RTE foods may also increase the contamination risk of the final products. In addition, inadequate procedures applied to clean and sanitize FPE (i.e., cutting boards) may increase the cross-contamination of other food products and the persistence of L. monocytogenes. Moreover, the presence of L. monocytogenes is reported in animals (Heredia and García, 2018), then the contamination of processed foods could be also related to inadequate management of animals, agriculture and farming practices, not only to the post-processing phases.

In literature is well known that listeriosis cases are predominantly associated with RTE foods, including RTE meat products (European Food Safety Authority, 2022). As reported, in the USA, the main listeriosis cases were caused by meat products sliced at retail (Forauer et al., 2021). Food products sliced in retail shops presented a higher bacterial contamination than products prepared in FPP (Forauer et al., 2021), hence RTE food preparation at retail shops could increase the risk of cross-contamination of products from different FBOs (Kurpas et al., 2018).

The WGS approach provided crucial information regarding the CCs distribution and the genomic characteristics of L. monocytogenes isolated from Zambian RTE meat foods, especially related to CC1 and CC2 clones, the main CCs detected in the RTE products tested. CC1 and CC2 are both well known to be hyper-virulent clones, often associated with clinical infections (Maury et al., 2016), their presence in RTE foods is a crucial concern for consumer safety. In literature, CC1 clone was often isolated in the pork-meat production sector and it is frequently associated with dairy sector (Félix et al., 2018). The presence of hyper-virulent clones in foods has been reported worldwide, including African countries, suggesting the L. monocytogenes adaptation to food products (Matle et al., 2020; Mafuna et al., 2021). The clustering conducted in this study with other public available African L. monocytogenes genomes, showed that one CC1 isolate from biltong sample, seems to be closely related to CC1 strain previously detected in 2016 from a SA processed meat.

According to our results, both CC1 and CC2, carried a full-length internalins, inlA and inlB, which are considered one of the most influent factors for L. monocytogenes invasiveness (Su et al., 2019). Increased hyper-virulence and bacterial colonization has been reported in isolates with a full-length inlA and a complete LIPI-1 and LIPI-3 (Maury et al., 2016; Yin et al., 2019), suggesting that some isolates have increased virulence potential (Gray et al., 2021). In addition to a complete LIPI-1, CC1 isolates also harbored a complete LIPI-3, encoding the production of Listeriolysin S, a hemolytic and cytotoxic factor also observed to be released in acid stress (Tavares et al., 2020).

Previous studies reported that CC9 and CC121 were often associated with meat products, including RTE, and FPP worldwide, also in African countries (Matle et al., 2020; Mafuna et al., 2021). As known, L. monocytogenes is able to resist under stress conditions, supporting its spread in many types of foods (Kurpas et al., 2018). In particular, CC9 and CC121 are well known to be better adapted to meat processing environments (Maury et al., 2019; Centorotola et al., 2021; Guidi et al., 2021b). Specific factors are reported to be crucial for L. monocytogenes resistance to the environmental stress. In particular, the presence of SSI-1 ensures resistances to low pH, high osmolarity, bile and nisin, while SSI-2 confers resistances to alkaline and oxidative stresses (Maury et al., 2019). According to our results, SSI-1 was observed in L. monocytogenes strains belonging to different clones, whereas the SSI-2 genes were mainly found in CC121 isolates (Harter et al., 2017). The presence of these genes increases the survival, resistance and persistence capacity of L. monocytogenes in the FPP, increasing the risk for consumers. In addition, the presence of Tn6188_qac transposon confers tolerance to BC disinfectant, mainly used for sanitation in food processing plants. In this study, in fact, CC9 was one of the main clones observed in the RTE meat products tested, and this finding indicates a need for improvement in the FPP sanitation. However, a PMSC mutation was detected in the inlA gene, confirming CC9 and CC121 clones as hypo-virulent (Maury et al., 2019; Guidi et al., 2021b). Moreover, L. monocytogenes strains showing PMSC in inlA were reported in literature, isolated both from clinical case (Magagna et al., 2023) and animal (Sévellec et al., 2020). Our results showed the CC5 as one of the main clones in RTE meat products, as reported also in China (Wang et al., 2015). In the study of Maury et al. (2016), CC5 was not classified as a hyper-virulent clone, but further studies are needed. This clone caused several outbreak cases, such as the cantaloupe case in the USA in 2011 (Lomonaco et al., 2013) and in a previous study by Zhang et al. (2019), the CC5 was reported as the second common clone in clinical L. monocytogenes strains in Beijing (China).

Regarding the minor CCs detected in this study, CC155 strains were mostly found in farm and environmental samples, but also this clone was detected worldwide both in food samples and in clinical cases (Matle et al., 2020). L. monocytogenes CC3 was reported as prevalent in a study conducted on cooked food tested in China and also in clinical samples (Wang et al., 2018). Similarly to previous data (Chen et al., 2019), and like CC1, the CC3 isolate reported in this study harbored a complete LIPI-3 that could be responsible for the increasing of virulence (Vilchis-Rangel et al., 2019). CC3 strains were also reported in poultry RTE foods during a previous outbreak in SA (Matle et al., 2020).

The comparison between L. monocytogenes detected in Zambian RTE and those selected from NCBI, available from SA, showed the presence of several relatedness’s, in particular for hyper-virulent CCs such as CC1 and CC2. This findings was crucial, highlighting how is important to continue to detect and characterize African L. monocytogenes strains in order to improve knowledge on strains circulation, surveillance and the monitoring of any possible clusters or new outbreak. About antimicrobial resistance L. monocytogenes strains of this study showed to be widely susceptible to clinically relevant classes of antibiotics (penicillin, carbapenems and quinolones) similar to previous reports (Wiśniewski et al., 2022). Our results also confirmed the intrinsic resistance to broad-spectrum cephalosporin antibiotics, commonly used in therapy of bacterial infections (Krawczyk-Balska and Markiewicz, 2016). WGS identification of antimicrobial resistance genes revealed that in all the L. monocytogenes tested, intrinsic core genes were observed, as expected, according to what is found in literature (Kurpas et al., 2020). Two strains showed to be phenotypically resistant to tetracycline, despite the absence of tet genes. This could be driven by adaptive mechanism such as the antimicrobial resistance determinants exchange with other bacteria through horizontal gene transfer or plasmid mobilization (Wiśniewski et al., 2022). Just one strains showed a resistant phenotype against trimethoprim-sulfamethoxazole concordant with the presence of sul gene. However, all the strains resulted susceptible to the antimicrobials used as first choice for the therapy of human listeriosis (Baquero et al., 2020). Listeria spp. isolated from different parts of Africa are generally susceptible to ciprofloxacin, but resistant to penicillin (Dufailu et al., 2021). Previous studies reported the increase in resistance of L. monocytogenes strains to antibiotics of different classes (Godreuil et al., 2003; Swetha et al., 2021). Keet and Rip (2021) described the presence of isolates susceptible to all antibiotics tested in RTE food, at the same time some isolates were resistant to erythromycin and tetracycline and some strains from polony were multi-drug resistant.

According to literature, the presence of genetic mobile elements, such as plasmids and prophages, increases the L. monocytogenes resistance. Interestingly, several studies indicated that some plasmids might confer increasing tolerance to stresses, heavy metals, quaternary ammonium compounds, salt, oxidative, heat, and cold stress (Mafuna et al., 2021; Schmitz-Esser et al., 2021). According to previous studies, among the hyper-virulent clones, few strains carried plasmids; on the contrary, the hypo-virulent clones normally show a higher number of plasmids (Schmitz-Esser et al., 2021). With the additional presence of disinfectant resistances, such as the bcrABC cassette and the Tn6188_qac for BC tolerance, various prophages and plasmids involved in stress response, the hyper-virulent clones could resist several stresses and probably persist in the environment for years. Data from literature highlighted the distribution of the hyper-virulent clones and their ability to contaminate, during the years and for long time, food products, food processing plants in Africa, increasing risk for human (Mafuna et al., 2021).

Although there has been listeriosis cases reported worldwide, L. monocytogenes data from Africa remains limited. Despite being a first preliminary work with a limitation in the sampling plan and a limited number of samples and isolates, this study reported our findings providing justification for a more complete surveys in future work, and helping to increase existing knowledge on contaminated RTE products circulating in Africa countries. Furthermore, this study highlighted interesting results about L. monocytogenes in Zambian RTE meat foods, confirming the presence of hyper-virulent CCs, which could play an important role in human diseases, posing a public health concern for consumers. Moreover, the presence of stress resistance factors could help these hyper-virulent clones to adapt, survive and persist over the years, as described by relatedness found in this study with other L. monocytogenes isolated in SA, during the years, from different matrices.

This preliminary study was conducted in order to test local Zambian foods available in local supermarkets, but also to allow generalizable findings. Further studies should be conducted on RTE products from Zambia and other African country, in order to deeply investigate the worldwide diffusion and the genomic characteristics of L. monocytogenes strains with particular attention on hyper-virulent clones aiming to improve worldwide surveillance and food safety.

In the future it will become crucial to improve the sampling plan, test more RTE foods and food environmental samples, but also to collect and characterize by WGS more L. monocytogenes isolates, especially from each positive sample, improving data and knowledge on L. monocytogenes in food products from Africa.

Due to the global increasing request, trade and consumption of foreign foods, it would be necessary to improve hygiene conditions and standardize procedures for hygiene and sanitation in food processing plants in Africa countries. Moreover, it could be useful to apply a better practices related to animal, agriculture and farming management in order to reduce L. monocytogenes in preharvest animals and consequentially in RTE meat products. According to One Health approach, it will be important compare available L. monocytogenes WGS data with clinical strains, in order to identify any clusters, including any potential connections to listeriosis cases and outbreaks.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

MZ, GC, GM, and FP conceptualized the study. MZ, BB, SM, and PM carried out sample collection, identification and isolation experiments. GC, ACo, ACh, and CC carried out the WGS experiment. ACh and GC carried out the bioinformatics analysis. GC, MZ, ACo, and ACh analyzed the data, organized the draft, and wrote the manuscript. FP, MT, and GM supervised the entire work. ND’A and MS acquired the funding. GC, MZ, ACo, ACh, MT, FG, CC, BB, SM, PM, ND’A, MS, FP, and GM contributed to editing the manuscript and reviewed the final version. All authors have read and approved the manuscript.

This work was supported financially by “Enhancing Research For Africa Network” (ERFAN) project, Italy, in collaboration with the Ministry of Fisheries and Livestock, Zambia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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