Ready-to-Eat Sandwich Microbiota: Diversity, Antibiotic Resistance, and Strategies to Enhance Food Safety

Abstract

Ready-to-eat (RTE) sandwiches are consumed globally due to their convenience, availability, and affordability. Sandwich processing practices and their ingredients expose the sandwiches to various sources of contamination, which can enhance their microbial diversity and introduce certain pathogenic and spoilage bacteria, thereby affecting their safety and quality. Sandwiches may not receive safe cooking temperatures sufficient to destroy food poisoning bacteria, as they are often cooked and served quickly to meet high consumer demand. Improper storage temperatures can enhance microbial growth, and frequent improper handling makes this food a good vehicle for various pathogens such as Escherichia coli, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, and norovirus. Many pathogenic sandwich-associated bacteria, such as L. monocytogenes, showed resistance to clinically important antibiotics. Sandwich microbiota have been investigated; however, their diversity, antimicrobial resistance, and importance to sandwich safety and quality have been rarely reviewed. Therefore, this review elucidates the diversity of sandwich microbiota as an impact of ingredients, handling practices, and storage, with emphasis on the importance of this diversity on sandwich safety and quality. It also discusses strategies, control measures, and recommendations to reduce the risk of contamination of sandwiches with pathogenic bacteria or their antibiotic resistance genes, thereby safeguarding public health.

1. Introduction

According to the Codex Alimentarius Commission [1], ready-to-eat (RTE) food is defined as any food (including beverages) which is normally consumed in its raw state or any food handled, processed, mixed, cooked, or otherwise prepared into a form in which it is normally consumed without further processing. Ready-to-eat foods are widely consumed worldwide due to their convenience, broad availability, and relatively low prices compared to other conventionally prepared foods. Sandwiches are cooked and served either immediately or stored for subsequent use without any treatment before consumption. Indeed, this processing practice poses some risks that could compromise the safety of the RTE. First, RTE foods are quickly cooked and served to meet consumers’ demands, meaning that RTEs may not receive a recommended safe-cooking temperature that is expected to destroy food poisoning pathogenic bacteria, such as Salmonella spp., Escherichia coli, Staphylococcus aureus, and others, if they are present. Second, RTEs are often not consumed immediately and are probably kept at a temperature above the recommended temperature (below 5 °C), reaching 25–30 °C in some places. Thus, sandwiches may stay at temperatures within the danger zone of 5–63 °C for a time long enough to allow the growth of microbes that survived the cooking temperature either because the heat treatment was not sufficient in killing them or because the contaminant can resist cooking temperature [2].

Various sandwich-associated pathogens can grow significantly under temperature abuse, posing a risk to food safety in sandwich matrices and other RTEs. For instance, the growth of L. monocytogenes is significantly influenced by temperature, with higher growth rates observed at elevated temperatures (e.g., 30 °C) compared to lower temperatures (e.g., 4 °C) [3]. Likewise, S. aureus can grow at a wide range of temperatures, with growth rates increasing with temperature. For instance, at 30 °C, S. aureus can produce staphylococcal enterotoxins in sandwiches, with maximum amounts of 0.15 ng/g after 52 h [4]. The minimum growth temperature for S. aureus in dairy products was found to be around 5.7 °C, while storing cheese at temperatures below 0 °C was found to limit maximum population density to approximately 10^2–4 CFU/g, below the international toxicity threshold [5]. E. coli strains, including O157:H7, can survive and grow at various temperatures. However, growth is more pronounced at higher temperatures. E. coli O157:H7 showed good growth in ground beef stored at 10 °C [6]. In mixed-ingredient salads stored at 15 °C, E. coli O157:H7 populations increased significantly [7]. E. coli growth is more pronounced at higher abuse temperatures. In ready-to-eat lettuce stored at temperatures above 16 °C, E. coli populations increased up to 1.1 log CFU/g [8].

Storage of RTE sandwiches at high temperatures also leads to the growth of various harmful bacteria, such as Citrobacter and Enterobacter species, making the sandwiches unfit for consumption [9]. Third, unlike conventionally prepared foods, RTE foods are heavily handled by humans from the first step of ingredient preparation until the serving step. Some researchers studied the effect of food practices of food handlers on the growth of E. coli present in tuna sandwiches stored at 4 °C and 30 °C for 6 days. Using good hand hygiene procedures, E. coli was only detected when the samples were stored at 30 °C at day 6, while its count remained unchanged from day 0 of storage at 4 °C. In contrast, poor hand hygiene practices lead to the detection of E. coli in both temperatures during storage [10].

Ready-to-eat foods include a wide range of foods that differ in their names, main contents, and other ingredients. Sandwiches are considered the typical RTE foods [11] and are processed across the globe with different names, such as shawarma in most Middle Eastern countries or kabab in some Western countries, such as Australia. Sandwiches contain main ingredients such as chicken, meat, tuna, cheese, and eggs, and other secondary ingredients such as bread, salad ingredients, spices, salt, and seasonings. The main ingredients are either barbecued, boiled, or fried and mixed with other secondary ingredients. In cheese sandwiches, the main ingredients are added to the other ingredients without any further treatment. In most developing countries, sandwiches are considered street foods that are prepared outside restaurants and cafes at temperatures that may exceed 40 °C in the summer months under unhygienic conditions and with a lack of food safety knowledge among food handlers. In addition, due to possible unsafe cooking temperature and unhygienic preparation conditions, which provide good chances of cross-contamination from food handlers, utensils, and surfaces, sandwich ingredients are sources and vehicles of pathogens [12,13].

Sandwich main ingredients, such as meat and chicken, and secondary ingredients, such as salad, can significantly contribute to diversifying its microbiota, mainly bacteria, which pose a risk for its safety. For instance, meat and poultry products are the main sources of Salmonella spp. [14], whereas salad ingredients such as fresh produce are the main vehicles of sanitary pathogens such as E. coli [15]. Moreover, a sandwich is prepared manually by a human who is the main source of S. aureus, and these can harbor virulence genes. Other sandwich-associated bacteria, such as E. coli, were found to harbor the virulence gene stx [16]. As a result, sandwiches are involved in causing many food poisoning outbreaks [17]. Sandwich microbiota has been widely studied in terms of bacterial counts and type; however, the diversity, antibiotic resistance, and food safety aspects were rarely evaluated, highlighted, and reviewed. Therefore, this review aims to address the microbiota of sandwiches, focusing on their diversity, antibiotic resistance, and importance for food safety. It also addresses the health implications for contamination of sandwiches with pathogenic and antibiotic-resistant bacteria, as well as the strategies and control measures to mitigate these risks.

In the culinary context, a sandwich typically refers to a food item consisting of two slices of bread with various fillings in between [18]. In this review, the term sandwich is used broadly to reflect the diversity of ready-to-eat products described in the literature. Across different regions and studies, “sandwiches” encompass foods consisting of bread or a bread-like carrier (e.g., buns, rolls, flatbreads, pita) combined with fillings such as cooked, cured, or processed meats (e.g., deli slices, hot dogs, shawarma, cheeseburgers), poultry, seafood, eggs, vegetables, cheeses, and sauces. They share common characteristics: a multi-component, ready-to-eat assembly with diverse ingredients, minimal further processing before consumption, and potential for microbial complexity arising from multiple contamination sources. The sandwich types included in this review were selected to represent this broad diversity, covering different bread matrices, protein sources, preparation methods, and retail environments reported across global studies. Therefore, the chosen categories capture the major forms of RTE sandwiches encountered in both commercial and household settings.

2. Commensal Microorganisms Associated with Sandwiches

Commensal microbes are non-pathogenic microorganisms that naturally inhabit various environments. The background commensal microbiota in sandwiches can vary significantly depending on the type of sandwich, ingredients, and preparation conditions. High aerobic plate counts reaching 6.4 CFU/g were found in various sandwiches, indicating significant microbial presence [19]. The Enterobacteriaceae family includes various commensal bacteria such as Enterobacter, Klebsiella, and Citrobacter, which can be found in food products, including sandwiches. High levels of aerobic plate counts and Enterobacteriaceae indicate poor hygienic conditions during sandwich preparation and storage. For example, aerobic plate count values in sandwiches were found to be satisfactory or acceptable but increased over time during storage [20]. Common genera found in salad include Enterobacter, Acinetobacter, Klebsiella, Pantoea, Achromobacter, Microbacterium, and Acidovorax [21]. Lactic acid bacteria (LAB) are frequently found in cooked meat products and are considered beneficial due to their role in fermentation and preservation. They are the dominant group in the microbiota of modified atmosphere packaged sliced cooked meat products, including ham, turkey, and chicken [22].

Cheese harbors a diverse range of commensal bacteria. When cheese is used as an ingredient in sandwiches, these commensal bacteria can be transferred to the sandwich. LAB are the most prevalent microorganisms in dairy products. They include species such as Lactocaseibacillus casei, Lactocaseibacillus paracasei, and Lactocaseibacillus rhamnosus, which are dominant in many ripened cheeses and contribute significantly to flavor development [23]. Corynebacterium spp. are commonly found on the surface of smear-ripened cheeses and contribute to the organoleptic properties of the cheese [24]. Staphylococcus vitulinus is a non-pathogenic strain that is part of the superficial flora of some cheeses [25]. Micrococcus spp. are also found on the surface of various cheeses and contribute to the cheese’s microbial diversity [24]. Bread may harbor various microbes, such as LAB, yeasts, Acetobacteraceae, and Enterococcus, that can potentially be transferred to sandwiches [26]. Sauces can also introduce a variety of commensal microorganisms. LAB, such as Lactobacillus and Pediococcus, can be present in soy sauce [27] as well as yeasts [28].

3. Spoilage Microorganisms Associated with Sandwiches

Spoilage microorganisms are responsible for the deterioration of food quality, leading to off-odors, off-flavors, and textural changes. Sandwiches contain various ingredients that can be a source of many spoilage microbes. The counts of yeasts and molds reached 4.2 CFU/g in “falafel” sandwiches [19]. Key spoilage microorganisms include Pseudomonas, LAB, Brochothrix thermosphacta, yeasts, and molds. Pseudomonas spp. are aerobic, Gram-negative bacteria known for their spoilage potential in various food products, including meat, fish, and dairy, which are used as ingredients in sandwiches [29,30]. They produce extracellular enzymes such as proteases and lipases, leading to protein and fat degradation [29]. Pseudomonas fragi, for instance, is noted for its strong degradation activity in fish, resulting in higher levels of total volatile basic nitrogen and protein oxidation [30]. B. thermosphacta is a significant spoilage organism in meat products, particularly under aerobic storage conditions. It produces metabolites like acetoin, contributing to off-odors and spoilage [31]. In co-culture with Pseudomonas spp., B. thermosphacta can enhance spoilage, leading to higher microbial counts and more pronounced spoilage indicators [32].

While LAB can inhibit other spoilage organisms, they themselves can be spoilage agents in certain conditions, such as in dairy products [33]. LAB thrive in nutrient-rich environments, such as those provided by the ingredients in sandwiches (meat, cheese, vegetables), leading to their proliferation and spoilage activity [34]. Certain LAB strains, such as Lactobacillus spp. and Leuconostoc spp., can produce slime, leading to a ropy texture in meat products, which can also affect sandwiches containing meat. LAB can grow at refrigeration temperatures, which are typical for sandwich storage. For instance, Leuconostoc mesenteroides, which was isolated from cooked meat, could grow well at 4 °C, though at a slow rate [35]. Yeasts and molds are significant contributors to food spoilage, including sandwiches. They produce enzymes that break down lipids and proteins, resulting in off-flavors and odors. Yeasts and molds can tolerate a range of environmental conditions, such as low pH, low water activity, and the presence of preservatives. This extremotolerance allows them to survive and propagate in sandwiches, which often have varied ingredients and storage conditions [36]. For instance, Hyphopichia burtonii, Wickerhamomyces anomalus, and Saccharomycopsis fibuligera were isolated from industrial gluten-free bread, in which they produce dust-type spots of white powdery and filamentous colonies typical of the spoilage produced by chalk yeasts [37].

4. Pathogenic Microorganisms Associated with Sandwiches and the Influence of Ingredients, Preparation, Handling, and Packaging

Sandwiches are often prepared and handled at room temperatures ranging from 25 °C to 35 °C. This temperature range enhances the growth of most pathogenic mesophilic bacteria, such as S. aureus, Salmonella sp., and E. coli [18], with humans being a potential source for these bacteria [14,18]. Therefore, many previous studies reported the presence of pathogens and high microbial loads in RTE foods. For instance, poultry products such as chicken and eggs are the main sources of Salmonella sp. Meat and meat products are sources of pathogenic E. coli and L. monocytogenes [18]. Moreover, bread products are a good potential vehicle for Bacillus cereus originating from soil [19].

Fresh produce vegetables, which are commonly used in sandwiches, were found to harbor S. aureus and various opportunistic pathogens belonging to Enterobacteriaceae, such as E. coli, Klebsiella pneumoniae, and Enterobacter cloacae [15], indicating possible contamination from human and animal wastes. Sandwich-associated microbes, which are used to evaluate their safety and quality, can be affected by their ingredients, cooking temperature, hygiene status of handling, and holding temperature before serving. Although aerobic colony count was found to be satisfactory in different sandwiches in Greece [14], Asiegbu et al. [15] reported a serious concern about the safety of sandwiches served in South Africa. Likewise, in Malaysia, Latchumaya et al. [16] found that sandwiches had low microbial quality. Moreover, due to their load of pathogens, Abd-El-Malek et al. [17] raised concerns about the safety of certain types of sandwiches in Egypt. Table 1 shows possible sources and vehicles of microbial contaminants in sandwiches.

Table 1.

Possible sources and vehicles of microbial contaminants in sandwiches.

Stage/Source	Food/Surface	Role	Most Possible Contaminant	References
Ingredients	Meat	Source	Listeria monocytogenes, E. coli, Shiga toxin-producing E. coli (STEC)	[18,19]
	Poultry products	Source	Salmonella sp.	[38]
	Tuna	Source	Clostridium botulinum	[39]
	Salad (tomato, cucumber, cabbage, lettuce)	Vehicle	Noroviruses, hepatitis A, E. coli, Shiga toxin-producing E. coli (STEC), Salmonella sp.	[40,41]
	Bread	Source	Bacillus cereus	[42]
	Mayonnaise	Vehicle	Salmonella sp.	[43]
	Spices	Vehicle	B. cereus	[44]
Preparation	Cutting board and knife	Vehicle	Serves as a vehicle for all the above microbes	[12,13]
Handling	Food handler	Source	Staphylococcus aureus, Noroviruses, hepatitis A, E. coli, Shiga toxin-producing E. coli (STEC), Salmonella sp.	[45,46]

Sandwiches such as shawarma are traditionally prepared by slicing meat or chicken, stacking it on a vertical rotisserie with layers of animal fat on the top of the rotisserie for enhancing flavor, slowly roasting until the color of the meat or chicken turns brown, mixing with other ingredients, stuffing in bread, wrapping in paper, and serving. Initially, fat melts and penetrates through cuts, providing a good shelter for microbes from thermal process killing. In fact, bacteria attached to meat surfaces, such as Salmonella Typhimurium and Campylobacter jejuni, have shown increased heat resistance during cooking, surviving longer than predicted by standard thermal death values. Thus, melting oil could protect bacteria present in meat shawarma by enhancing their thermal resistance and aiding in their recovery post-thermal treatment [47].

The temperature of safe cooking is not regularly checked, and the readiness of roasted meat or chicken for serving is indicated by changing the color of the meat or chicken from red to brown. At temperatures lower than 60 °C, myoglobin remains relatively stable, and the meat retains its red or pink color due to the presence of deoxymyoglobin and oxymyoglobin. Cooking meat at higher temperatures leads to the denaturation of myoglobin, resulting in the formation of metmyoglobin. Thus, the color of roasted muscle foods such as meat, chicken, and seafood results from heat-induced changes in myoglobin, such as the oxidation of oxymyoglobin and deoxymyoglobin to metmyoglobin (brown), which does not indicate whether the food reached a safe internal temperature sufficient to kill pathogens. Metmyoglobin formation increases with higher temperatures and prolonged exposure to oxygen [48]. The transition from red/pink to brown is often used as an indicator of doneness and safety. However, this can be misleading. For example, meat cooked in high-oxygen environments can appear brown even at lower temperatures (before reaching the safe internal temperature of 71.1 °C), potentially leading to premature browning and a false sense of safety. Therefore, color change alone is a poor indicator of safety, and a thermometer should be used to ensure proper cooking [49]. Thus, it could be expected that some vegetative and spore pathogens could survive the roasting stage. Moreover, wrapping papers of sandwiches were found to be contaminated with bacteria belonging to the Bacillaceae, Staphylococcus, and Pseudomonas genera [50]. Mayonnaise is a popular seasoning and flavor ingredient in most sandwiches, yet in most sandwich preparation sites, it is made with raw eggs despite regulatory prohibitions. Consequently, mayonnaise was found to cause some food poisoning outbreaks [43]. The possible sources, vehicles, and factors affecting the microbial contamination of sandwiches are indicated in Figure 1.

Figure 1.

Possible sources, vehicles, and factors affecting microbial contamination of sandwiches [51,52,53,54,55,56].

5. Pathogenic Microorganisms in Cook–Serve and Cook–Chill Sandwiches

Based on their ingredients, handling status, and handling temperature, bacteria were the main microbial group targeted in previous microbial research of sandwiches. The main bacterial genera that were investigated qualitatively and quantitatively included B. cereus, S. aureus, L. monocytogenes, Salmonella sp., E. coli, and Shiga toxin-producing E. coli. Therefore, this review focuses on these genera. Moreover, it highlights the importance of sandwich microbiota in food safety and food poisoning outbreaks. Additionally, it elucidates the limitations in the previous studies in the safety and quality evaluation parameters and the need for the determination of more virulence factors in sandwich-associated bacteria. It is also worth noting that although the included studies originate from different countries, several contextual factors can explain variations in microbial contamination. Food service environments differ in hygiene standards, staff training, ingredient sourcing, and the extent of manual preparation. A study from Iran reported good microbial quality in grilled meat and chicken sandwiches possibly due to good preparation practices and effective control and monitoring by food health experts which likely contributes to the satisfactory microbial quality of food products [57]. However, studies from Egypt [58], Jordan [19], and South Korea [59] showed high microbial counts and detection of pathogens in various types of sandwiches, indicating that the current practices may not be sufficient. In addition, a warm and humid climate can support microbial growth in these countries.

5.1. Sandwich Prepared Under the Cook–Serve System

Most studies have focused on the microbiota of cooked and immediately served sandwiches, as it is the most common sandwich consumption mode. For instance, to evaluate the effect of Hazard Analysis and Critical Control Point (HACCP) on ensuring the safety of sandwiches, a HACCP-implemented premise sandwich study in Greece did not find the typical pathogens, including L. monocytogenes, Salmonella sp., and S. aureus. The total bacterial and Enterobacteriaceae counts were found at an acceptable level [20]. Assessing the microbial quality of different street-vended sandwiches, the cheeseburger was found to contain the highest incidence of L. monocytogenes, whereas Salmonella sp. was found in hotdog sausages [60]. This indicates the effect of the main ingredients (cheese and sausages) on the predominant pathogen type.

Moreover, to emphasize the effect of the sandwich’s main ingredient, namely poultry and meat products, on the prevalence of its pathogenic microbes, L. monocytogenes dominated the Listeria sp. in the shawarma prepared from meat and poultry products by 63% and 60%, respectively [58]. Among other pathogens, Salmonella Enteritidis, S. Typhimurium, Shigella flexneri, and Shigella dysenteriae were found in liver and minced meat sandwiches [61]. Both S. aureus and B. cereus were found in 88% of a supermarket’s sandwiches in Taiwan [62]. Moreover, in evaluating the microbial quality of street food in Brazil [63], the prevalences of E. coli and B. cereus were 18% and 15%, respectively, in hot sandwiches.

In the quantitative aspect, cheese sandwiches were found to contain a higher aerobic mesophilic bacterial count than the fermented sausage sandwiches, which could elucidate the impact of the processing on the sandwiches’ total bacterial load [64]. Moreover, evaluating the microbial quality of kibda (liver) sandwiches, the total bacterial and Enterobacteriaceae counts were found in averages of 6 log CFU/g and 4 log CFU/g, respectively [53]. In an RTE microbial surveillance system in the UK conducted for about 2 years, which collected data of 3391 RTE foods, 15.4% of kebab sandwiches were unacceptable based on total bacterial count [65].

5.2. Cook–Chill Sandwich

A cook–chill sandwich is cooked and stored at a low temperature until served. The microbiota of this type of sandwich could be affected by storage temperature, besides the previously mentioned factors. In fact, the microbiota of this type of sandwich has not received enough attention compared with cook–serve sandwiches, although some pathogens, such as L. monocytogenes, grow normally at chilled temperatures. Moreover, the recommended chill-stored temperature, e.g., 0–3 °C [66], is not followed in many retail stores, especially in developing countries. Thus, storing a cook–chill sandwich in the danger zone, 5–63 °C, which prevails in many developing countries, permits mesophilic pathogens such as S. aureus and Salmonella sp. to grow. In a small study, total bacterial and Enterobacteriaceae counts were found to be high in cook–chill egg, chicken, tuna, and meat sandwiches, and most of the sandwiches were found at the marginal level of acceptability [67]. In another small study, 24 cook–chill sandwiches of different foods, pathogenic Listeria ivanovii, L. monocytogenes, S. aureus, Yersinia sp., and Citrobacter sp. were identified by Analytical Profile Index (API) strips [11,60]. Both studies have limitations of pathogen coverage and confirmation of the identity of the pathogen by reliable methods, such as genotypic methods. Table 2 summarizes the main pathogens isolated from different types of sandwiches.

Table 2.

Pathogens identified in different types of sandwiches.

Sandwich Type	Pathogen	References
Cheeseburger	Listeria monocytogenes	[60]
Hotdog sausages	Salmonella sp.	[60]
Chicken and meat shawarma	L. monocytogenes	[58]
Liver and minced meat	Salmonella Enteritidis, S. Typhimurium, S. Dublin, Shigella flexneri and S. dysenteriae	[61]
Seafood	Staphylococcus aureus, Bacillus cereus	[62]
Hotdog	E. coli and B. cereus	[63]
Chicken	Methicillin-resistant S. aureus	[68]
Deli slices	L. monocytogenes	[69]
Shawarma	S. aureus	[70]
Meat shawarma	S. aureus	[71]
Meat	Escherichia coli, Klebsiella pneumoniae	[72]
Eggs	E. coli, K. pneumoniae	[72]

Some examples of microbial hazards, growth potential, and risk factors in sandwiches prepared under the cook–serve system as compared to the cook–chill system are given in Table 3.

Table 3.

Microbial hazards, growth potential, and risk factors in sandwiches prepared under cook–serve and cook–chill systems.

System	Examples of Microbial Hazards	Growth Potential	Risk Factors	References
Cook–serve	Escherichia coli	Generally low if proper cooking and handling are maintained	Cross-contamination from food contact surfaces and chefs’ hands.	[73]
	Campylobacter	High risk if hygiene is not maintained	Non-compliance with hygiene standards on food contact surfaces and chefs’ hands.	[67]
	Total coliforms	Can grow if temperature control is inadequate	Poor hand hygiene and improper sanitation practices.	[67]
Cook–chill	Clostridium perfringens	High if cooling is not rapid and effective	Inadequate cooling processes.	[74]
	Listeria monocytogenes	Can grow at refrigeration temperatures	Extended storage duration and temperature abuse.	[75,76,77]
	Bacillus cereus	Can grow if temperature control is inadequate	Improper holding temperature and cross-contamination.	[69]
	Staphylococcus aureus	Can grow if post-cooking handling is poor	Addition of ingredients after cooking and poor sanitation.	[78,79]
	Salmonella	Low if proper cooking and handling are maintained	Cross-contamination and improper sanitation.	[78]
	E. coli	Can grow if temperature control is inadequate	Extended storage duration and inadequate chilling.	[20]

6. Sandwich-Associated Food Poisoning Outbreaks

Most of the sandwich-associated pathogens are part of the common foodborne pathogens that have caused different food poisoning outbreaks globally at high rates. For instance, L. monocytogenes caused a historical food poisoning outbreak in South Africa from June 2017 to April 2018, where processed meat products contaminated by L. monocytogenes poisoned 937 individuals, resulting in 193 deaths [20]. In 2008, a hospital food poisoning outbreak caused by B. cereus in Oman poisoned 58 people. The symptoms included diarrhea and vomiting [80]. S. aureus poisoned 24 persons in Italy in 2015, in which three sea-positive toxin-producing strains of S. aureus were found in the dessert, environment, and cooks, suggesting that the outbreak originated from food handlers [81]. Salmonella sp. has dominated food poisoning outbreaks as a causative agent in recent years, and according to the World Health Organization (WHO), a Salmonella chocolate outbreak affected 11 countries with 151 cases in 2022. The implicated species was S. Typhimurium sequence type 34 [82].

Shiga toxin-producing E. coli (STEC) has also been associated with sandwiches. In the United Kingdom, an outbreak of STEC O145:28 was traced back to pre-packed sandwiches and wraps containing lettuce. This outbreak resulted in 288 cases, with 49% of the affected individuals hospitalized and 10% requiring emergency care [83]. Another outbreak in Scotland involved STEC O26:11, where 32 cases were associated with consuming pre-packed sandwiches from a national food chain franchise. The common ingredient in these sandwiches was a mixed salad of Apollo and Iceberg lettuce and spinach leaves [84]. In Japan, about 3000 students were poisoned by E. coli serotype O7:H4 carrying the astA gene from the consumption of seaweed [85].

Sandwiches with different main ingredients, such as chicken, eggs, and meat, are implicated in a large number of outbreaks. In Taiwan, for instance, 27 people were poisoned by eating online-sold foods. Salmonella sp. from egg origin was suspected as the cause of this outbreak [17]. Egg-made mayonnaise is a common dressing item in sandwiches, which could be suspected as a source of food poisoning-causative pathogens. In fact, a sandwich food poisoning outbreak in the UK with 68 cases was caused by S. typhimurium DT4 from consumption of sandwiches containing mayonnaise made from eggs [86]. Moreover, at the media level, reports of sandwich-associated food poisoning are widespread. For instance, in 2019, Reuters reported three deaths from hospital sandwiches in the UK. In Hong Kong, Food Safety News reported an outbreak linked to sandwiches that infected 200 people [87]. The Limited Times reported that chicken sandwiches or smoked salmon sandwiches were suspected of poisoning eight individuals in Hong Kong in 2022. Moreover, sandwiches caused a listeriosis outbreak in the USA with a total of 10 people infected and one death [88]. In Spain, a meat-based sandwich was associated with an outbreak of 60 infected people; E. coli or Clostridium perfringens were possible causes [89].

Outbreaks from sandwiches are commonly caused by a combination of factors such as pathogen contamination, poor food handling and hygiene practices, breaches in cold chain management, environmental contamination, and specific high-risk ingredients. L. monocytogenes is frequently found in sandwiches, including hospital settings, leading to severe infections in vulnerable populations [90]. Infected food handlers are a significant factor, with outbreaks often traced back to asymptomatic or symptomatic food handlers who contaminate food during preparation. For example, a restaurant S. Enteritidis outbreak was associated with an asymptomatic infected food worker in the United States of America. Moreover, poor hygiene practices, such as not using gloves or improper handwashing, contribute to the spread of pathogens through cross-contamination [91]. Breaches in the cold chain have been linked to the growth of L. monocytogenes in sandwiches [90]. Outbreaks have been linked to contamination in the food preparation environment, including equipment and surfaces. An example of that is the outbreak that was caused by B. cereus and C. perfringens among hospital workers in Alaska in 2021 [92]. Ingredients like egg and poultry are frequently associated with Salmonella outbreaks [93], while sandwiches containing salad ingredients, soft cheese, and mayonnaise are more prone to contamination with Listeria [90].

7. Sandwich Microbial Impact on Its Safety and Quality

The safety and quality of sandwiches have been evaluated by comparison of total bacterial counts, the counts and presence of foodborne pathogens, with the recommended levels in food regulations. Nevertheless, evaluation parameters varied from one study to another. It is also important to note that the cited studies employ different enumeration and detection methods, food matrices, inoculum levels, and storage conditions. To account for this, we interpret each dataset within the context of its respective methodology and focus on identifying consistent patterns rather than making direct numerical comparisons. For instance, Kokkinakis et al. [17] based pre-packed sandwich acceptability on an E. coli count of <2 log CFU/g, and an Enterobacteriaceae count of 4 log CFU/g was considered borderline. Other pathogens, which were considered safety indicators, included L. monocytogenes, Salmonella sp., and S. aureus; none of them were detected. Khater [20] assessed the quality and safety of liver and kofta sandwiches by counting total aerobic plate, coliform, staphylococci, fungal, proteolytic, and lipolytic microbes. Based on the microbiological guidelines for RTE food and the counts in the previous microbial groups, the contamination level was acceptable in 80% of the liver sandwiches.

In evaluating the bacteriological quality of different sandwiches, Al Harbi et al. [16] considered a count of <5 log CFU/g set by the New South Wales Standards as a criterion of acceptability, and accordingly, none of the sandwiches met this count. Moreover, a combination of qualitative and quantitative microbial parameters such as the counts of aerobic mesophilic bacteria, yeast and molds, B. cereus, coagulase-positive staphylococci coliforms and detection of E. coli, Salmonella sp., and L. monocytogenes were used to evaluate the microbiological quality of sandwiches served in hospitals and schools, and according to these parameters, the hygienic conditions of sandwiches were evaluated to be very poor [94]. Contrary to the former study, Bae and Park [53] used S. aureus count as a sole criterion to evaluate sandwich acceptability. Besides S. aureus, Hanashiro et al. [63] added B. cereus as a second bacterium criterion to evaluate the microbial quality of street sandwiches, and accordingly, improving safety was recommended.

On the other hand, certain regional and national standards have been issued to evaluate RTE food safety and quality based on the microbial count and type. For instance, the Gulf Standardization Organization (GSO) Standards request sandwiches with salads to be free from Salmonella sp. and E. coli O15:H7, but allow E. coli at a count of 2 log CFU/g [95]. Food Standards Australia and New Zealand, for example, limit Enterobacteriaceae, B. cereus, and S. aureus counts at <2 log CFU/g [59]. Canadian Standards, however, request E. coli count to be <1 log CFU/g, but agree with Food Standards Australia and New Zealand in B. cereus and S. aureus counts [96].

8. Sandwich-Associated Pathogen Virulence Genes

Microbial importance is determined by specific species or strain and their capability to perform specific tasks, such as metabolizing food components to produce products that spoil food or introduce virulence genes to cause foodborne diseases. Certain strains, subtypes or serogroups of the pathogens that have been found in sandwiches in previous studies have been found to harbor specific virulence genes. Among Bacillus spp., for instance, B. cereus strains harboring virulence genes, such as nhe, hbl, and cytK, were found to be associated with gastrointestinal disorders [97].

Based on the virulence factors and the associated food poisoning outbreaks, E. coli was classified into five main groups, namely enterohemorrhagic (EHEC) with the typical Shiga toxin-producing genes; enterotoxigenic (ETEC) with heat-stable genes, sth, stp and lt; enteropathogenic (EPEC) with intestinal microvilli-attaching genes, eaeA and bfpA; enteroaggregative (EAEC) with an intestinal surface adherence gene, aggR; enteroinvasive (EIEC) with a fever and watery diarrheal gene, ipaH; and diffuse-adherent (DAEC) with a HEp-2 cell adherence gene, daaE [98,99].

E. coli Shiga toxin-producing (EHEC) strains are dominating the E. coli food poisoning strains. Many standards limited the E. coli Shiga toxin-producing strains to E. coli O157:H7; nevertheless, with new emerging pathogens, many E. coli Shiga toxin-producing strains, which are abbreviated as STEC, such as O26, O45, O103, O111, O121, and O145, are currently causing sporadic food poisoning outbreaks around the globe. Shiga toxin-producing E. coli was found to harbor different genes such as eae, aaiC, aggR, stx2a, stx1, and stx2; however, eae, stx1 and stx2 and stx2a genes were found to be involved in a wide spectrum of gastrointestinal disorders ranging from non-bloody diarrhea to hemolytic uremic syndrome. All STEC strains were found to contain stx gene and several subtypes of stx, such as stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g, were reported [16,98,100,101,102].

Among the 21 Listeria species, L. monocytogenes is considered the main pathogenic species, which can cause infection in humans and animals. Moreover, 13 serotypes of L. monocytogenes were reported, in which serotypes 4b and 1/2a are widely associated with human listeriosis [103]. The serotype 1/2a was found to produce InlA, the gene which is suggested to have a critical role in human listeriosis. InlA was found in 96% and 65% of the clinical and food strains, respectively [104]. Along with InlA, which facilitates L. monocytogenes attachment to the host cell, prfA and hly genes were found to facilitate the intracellular pathogenesis potential of L. monocytogenes [103]. Staphylococcal food poisoning was widely found to be caused by a wide variety of se genes ranging from sea to see and seh worldwide; however, the sea gene was found to be involved in the most staphylococcal food poisoning outbreaks [81,105,106,107]. Within these genes, sea and see were found mostly in food and clinical samples [105]. Almost all Salmonella species are pathogenic and have been associated with the most frequent salmonellosis from different sources around the globe. Table 4 summarizes the pathogens’ main virulence genes.

Table 4.

Common virulence genes in sandwich-isolated pathogens.

Pathogen	Virulence Gene	References
Bacillus cereus	Nhe, hbl, CytK	[97]
Staphylococcus aureus	sea to see and seh	[96,105,106,82]
Listeria monocytogenes	InlA, prfA, hly	[103,104]
E. coli (ETEC)	sth, stp, lt	[98,99]
E. coli (EPEC)	eaeA, bfpA	[98,99]
E. coli (EAEC)	aggR	[98,99]
E. coli (EIEC)	ipaH	[98,99]
E. coli (DAEC)	daaE	[98,99]
E. coli (STEC)	stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, stx2g	[93,17,98,100,108]

9. Antibiotic Resistance in Sandwich-Associated Bacteria

Antibiotics are chemical substances produced by various microbes. They are utilized to treat infections in humans and animals or as a prophylaxis measure to prevent them [109,110]. Unwise use of antibiotics, such as utilizing sub-therapeutic concentrations to enhance animals’ growth, may contribute to the development of antibiotic resistance in animal-associated microbes and to the build-up of antibiotic residues in the edible animal tissues [100]. “Resistance” refers to the microbial inherited capability to grow at increased concentrations of antibiotics, irrespective of the period of the treatment. It is usually determined by evaluating the minimum inhibitory concentration (MIC), which is the lowest concentration required to inhibit the visible growth of a microbe for a particular antibiotic. The term “resistome” is used to refer to the collection of genes involved in various mechanisms of antibiotic resistance [111]. Infections caused by antibiotic-resistant bacteria (ARB) can increase morbidity and mortality rates because of the possibility of treatment failure. These infections are also costly [74]. The increased bacterial antibiotic resistance burden is attributed to the tremendous use of antibiotics in agriculture, animals, and humans [112].

Microbial antibiotic resistance is well documented in different pathogenic microbes isolated from different foods. This resistance has been attributed to many factors, such as climate change, which has received special attention. In fact, a clear association between antibiotic resistance increase and climate change was found and emphasized in many studies [113,114,115,116]. For instance, an increase of 1 °C in ambient temperature increased certain pathogens’ antibiotic resistance, such as Pseudomonas aeruginosa, by 1.06-fold [113]. This association was attributed to the impact of temperature increase due to climate change on increasing microbial growth and adaptation, as well as pathogenic gene exchange and antibiotic resistance among microbial populations [113,117]. Antibiotic resistance in sandwich bacteria was widely investigated worldwide [11,118,119,120,121]. The pathogens that showed the most antibiotic resistance included S. aureus, L. monocytogenes, and E. coli [11,118,119]. Certain antibiotics which showed resistance in previous studies, such as amoxicillin, metronidazole, and tetracycline, are clinically important in Oman and other countries [122].

9.1. Prevalence of Antibiotic Resistance in Ready-to-Eat Foods and Sandwich-Associated Bacteria

The prevalence of ARB associated with RTE sandwiches is a significant concern, as these foods can serve as vectors for resistant pathogens. Nevertheless, there is limited data in the literature concerning antibiotic resistance in sandwiches as compared to other RTE foods and sandwich ingredients. This highlights the need for stringent food safety measures and routine surveillance, including larger geographical locations and large-scale studies, to mitigate the risks associated with ARB in sandwiches. A study evaluating bacterial load in RTE sandwiches from vending machines and supermarkets in Modena, Italy, found that 50% of the 54 bacterial isolates were pathogenic. These included L. ivanovii, L. monocytogenes, S. aureus, Yersinia spp., Citrobacter spp., and Enterococcus spp. Two Enterococcus faecium isolates exhibited resistance to vancomycin, and one isolate showed resistance to both ampicillin and erythromycin. One S. aureus isolate had resistance to erythromycin (2 µg/mL) and oxacillin. Additionally, five Listeria isolates demonstrated resistance to erythromycin. Aeromonas hydrophila was resistant to imipenem, and Citrobacter showed resistance to amikacin [52]. These findings highlight the potential health risks associated with consuming contaminated sandwiches.

Studies from Iran [123] highlight the role of both ready-to-eat (RTE) foods and raw sandwich ingredients as important reservoirs of antibiotic-resistant foodborne pathogens. S. aureus has been detected in a variety of RTE products in Tehran, with notable levels of contamination and widespread resistance to commonly used antibiotics, including penicillin, tetracycline, gentamicin, and several others. These findings indicate that consumers of RTE foods may be exposed to multidrug-resistant S. aureus through improperly handled or prepared items. Complementary investigations [124] of raw kebab and hamburger meat that are frequently used in sandwich preparation also revealed high contamination rates with E. coli, Salmonella spp., L. monocytogenes, and S. aureus. Many of these isolates carried clinically important resistance determinants such as bla_SHV, bla_TEM, and mecA, highlighting the circulation of β-lactamase-producing and methicillin-resistant strains within the meat supply. Together, these studies demonstrate that both raw meat components and RTE products commonly used in sandwiches can serve as significant vehicles for the transmission of antibiotic-resistant bacteria, underscoring the need for improved hygiene, handling, and monitoring practices across the sandwich production chain.

Evidence from Egypt [72] further demonstrates the substantial public health risk posed by antibiotic-resistant S. aureus in RTE sandwiches. In an assessment of beef burger and hot dog sandwiches, coagulase-positive S. aureus was detected in the majority of samples, with a large proportion classified as multidrug-resistant. Notably, the presence of methicillin-resistant S. aureus (MRSA) and even vancomycin-resistant S. aureus (VRSA) highlights the circulation of highly resistant strains in foods that are consumed without further cooking. The isolates exhibited extensive resistance to multiple antibiotic classes, suggesting significant antimicrobial selection pressure within the food chain. The detection of MDR, MRSA, and VRSA in popular sandwich items underscores the potential for RTE foods to act as vehicles for clinically important resistant pathogens and highlights the urgent need for stricter hygiene controls, improved handling practices, and surveillance measures to protect consumers

Several studies report the presence of Extended-Spectrum β-Lactamases (ESBLs)- and AmpC β-Lactamases (AmpC)-producing Enterobacteriaceae in ready-to-eat (RTE) sandwiches, indicating that these foods can act as vehicles for antibiotic-resistant pathogens. For example, in Algeria [44], E. coli, K. pneumoniae, and K. oxytoca isolates recovered from street-vended sandwiches frequently carried CTX-M, SHV, and AmpC β-lactamase genes, suggesting circulation of multidrug-resistant clones within the food chain. The detection of identical ESBL/AmpC-producing strains in sandwiches from different city locations also highlights widespread contamination likely originating from shared ingredients, inadequate hygiene during preparation, and cross-contamination during handling or packaging. Overall, these findings underscore the need for improved control measures and monitoring of RTE sandwiches to reduce consumer exposure to resistant pathogens. A summary of antibiotic-resistant foodborne pathogens recovered from sandwiches is provided in Table 5.

Table 5.

Antibiotic resistance in various foodborne pathogens recovered from sandwiches.

Sandwich Type	Location	Bacteria	Antibiotic Resistance	References
Shawarma (chicken with tahini sauce and vegetables)	Klang Valley, Malaysia	Staphylococcus aureus	Ampicillin, penicillin, ciprofloxacin, tetracycline, kanamycin, trimethoprim, trimethoprim–sulfamethoxazole, gentamicin, cephalothin	[70]
Kofta, luncheon, burger, shawarma, hawawshi, liver, sausage	Zagazig, Egypt	S. aureus	Kanamycin, penicillin, neomycin, oxacillin, erythromycin, ampicillin, nalidixic acid	[72]
Meat, chicken, and fish	Sharkia Governorate, Egypt	Escherichia coli	Erythromycin, amoxicillin–clavulanic acid	[121]
Meat (kebab, hamburger)	Iran	E. coli, Salmonella spp., S. aureus, Listeria monocytogenes	Amoxicillin, penicillin, cefalexin	[124]
Shawarma (chicken and beef)	Jordan	E. coli, Salmonella spp., Citrobacter freundii, S. aureus	Tetracycline, streptomycin	[125]
Various (tuna and tomato, ham and cheese, tomato and mozzarella cheese, tuna and eggs, turkey and vegetables, shrimp and pink sauce, raw ham, smoked cheese and tomatoes, cooked ham and mushrooms, tuna and onions, cooked ham and artichokes, raw ham and eggplants)	Modena, Italy	S. aureus, Listeria spp. Yersinia spp. Citrobacter spp.	Amikacin, ciprofloxacin, ampicillin, oxacillin, imipenem, tetracycline, erythromycin, vancomycin	[11]

9.2. Mechanism of Antibiotic Resistance in Foodborne Bacteria

The correlation between the increased use of antibiotics and the emergence of ARB is widely acknowledged [112]; however, antibiotics, ARGs, and the mechanisms responsible for the transmission of ARGs between bacteria have been present for millions of years before humans started to use antibiotics [126]. In their natural niches, bacterial populations are usually heterogeneous in their susceptibility to antimicrobial agents [127]; however, when they are challenged with antibiotics, resistant cells outcompete the susceptible counterparts in a ‘Darwinian’ fashion [110]. The function and the origin of antibiotics and ARGs cannot be certified. Their existence in the natural environments, including those that have not been visited previously by humans, can be explained by two main reasons: (1) Some bacteria may produce antibiotics to compete with other microorganisms for the limited nutrients. These antibiotic producers, at the same time, develop mechanisms to counteract the effect of the antibiotics they synthesize, rendering them resistant to antibiotics [128]. (2) Antibiotics may be involved in or perform significant functions for the bacterial cells that produce them, such as signaling [129] and biofilm formation [126]. Nevertheless, other bacteria produce other compounds that have functions involved in cell signal transduction, homeostasis, and metabolism, but these molecules can also resist high concentrations of antibiotics. The existence of ARGs in extreme environments that are not known to be polluted by humans, such as deep Greenland ice core and clean Antarctic water, is an example [129]. Bacterial resistance to antibiotics can be an intrinsic criterion [130], or it can be developed through gene acquisition, accumulation of mutations, or both, utilizing various mechanisms [98,112,126].

9.2.1. Intrinsic Bacterial Resistance to Antibiotics

Intrinsic bacterial resistance occurs when bacteria resist a particular antibiotic due to structural or functional characteristics of these bacteria [93]. Intrinsic resistance is not related to the use of antibiotics; instead, it is a natural, complicated process exhibited in all bacteria, especially environmental bacteria, including food bacteria. The outer membrane, which acts as a permeability barrier in Gram-negative bacteria, and efflux pumps are the most common intrinsic antibiotic resistance mechanisms [131]. Efflux pumps have the potential to pump antibiotics or other antimicrobial substances, such as disinfectants, outside the cell. They utilize adenosine triphosphate (ATP) or proton-motive force (PMF) to obtain energy to complete this process [20]. Efflux pumps are grouped into two main families: (1) the major facilitator superfamily (MFS) and (2) the small multidrug resistance (SMR) family [132]. Many genes are involved in the intrinsic resistance mechanism [130,131], which can be targeted by manufacturing antibiotics to inactivate specific pathogens [93]. Various serotypes of E. coli were found in RTE sandwiches, with intrinsic resistance to antibiotics like erythromycin. Being a Gram-negative bacterium, E. coli has an outer membrane that prevents macrolides like erythromycin from entering the cell. Moreover, efflux pumps also contribute to reduced susceptibility [121]. Staphylococcus spp. and Enterococcus spp., which are widely isolated from sandwiches, use efflux pumps to expel antibiotics, contributing to intrinsic resistance. Acinetobacter spp., known for their intrinsic resistance to multiple antibiotics, were also isolated from RTE sandwiches. These bacteria are particularly problematic in healthcare settings due to their resistance profiles. Interestingly, intrinsic genes coding for antibiotic resistance could be the origin of acquired resistance, particularly in the genus Acinetobacter [133]. This can happen through horizontal gene transfer (HGT) when intrinsic resistance genes are mobilized and transferred to other bacteria via plasmids, transposons, and integrons or through incorporation of the intrinsic resistance genes into mobile genetic elements, which facilitate their spread among different bacterial populations [134].

9.2.2. Acquired Bacterial Antibiotic Resistance

Acquired bacterial antibiotic resistance can occur via three main mechanisms: (1) modification in the antibiotic target site by post-translational modification or mutations, (2) decreasing the intracellular level of antibiotic by utilizing efflux pumps or reducing permeability, or (3) antibiotic inactivation by hydrolysis or modification [130]. Natural selection and evolution largely depend on mutations that occur in bacterial DNA. Bacteria are haploid for their genes with short generation times, which leads to the accumulation of mutations, thus increasing the potential of the emergence of antibiotic-resistant phenotypes [135]. The rate of gene mutation is affected by factors including environment and population dynamics, microbial genetic makeup, and cell physiology. For a full resistance to occur, a mutation should occur in multiple genes, because a single antibiotic has genetic redundancy in its target sites. For instance, the antibiotic fluoroquinolone targets the enzymes topoisomerase II and IV that are important for supercoiling of bacterial DNA and are encoded by the genes parA, parC, gyrA, and gyrB. Mutations should occur in at least two of these genes or all of them for the development of a bacterial phenotype that is fully resistant to fluoroquinolone [136].

A bacterial ‘hypermutable’ phenotype may arise when bacteria have defects in their DNA mismatch repair system, leading to potential accumulation of significant mutations [135]. Mutational resistance can develop to some antibiotics, such as streptomycin, fusidic acid, and rifampicin, when they are used during a treatment course against certain bacteria, and thus the combination of these antibiotics with these bacteria should be avoided [137]. Studies showed that exposure to osmotic pressure and freezing stress significantly affects the antibiotic resistance of S. enteritidis and S. typhimurium. Prolonged freezing (96 h) increased antibiotic resistance, while shorter freezing periods (24 h) decreased it [138]. This suggests mutation-based antibiotic resistance can arise in food-associated bacteria such as Salmonella and Campylobacter, especially under environmental stresses (e.g., salt, low pH, heat, and preservatives). However, detecting natural mutation events in sandwich-associated bacteria and identifying the specific genes involved remain active research areas that require further investigation.

9.2.3. Antibiotic Resistance Development by Horizontal Gene Transfer

Lateral or HGT is the process of transferring antibiotic resistance between different bacterial cells. Transformation, transduction, and conjugation are the three principal mechanisms for HGT. Transformation occurs when naked DNA that contains resistance determinants is released to the environment and taken up by a recipient bacterial cell [126], which should enter a ‘competence’ state. Then, particular recognition sequences are recognized in the DNA for successful transformation [136]. DNA released from dead cells to the environment was observed to persist and to be protected by soil particles from DNase [95]. Transduction happens when a bacteriophage is involved in transferring antibiotic resistance genes from one bacterial cell to another [126,136]. Lysogeny is the process of integrating the genes into the chromosome of the recipient bacterial cell [126]. Bacteriophages can mediate the transfer of antibiotic resistance genes among Enterobacteriaceae, including non-typhoidal Salmonella and Shiga toxin-producing E. coli. This mechanism significantly contributes to the spread of resistance genes in foodborne pathogens [139]. Conjugation involves the transfer of antibiotic resistance genes that are located on a plasmid or conjugative transposon that occurs when these elements are moved from a donor cell to a recipient one via a mating bridge [126,136]. In fact, the food environment may facilitate HGT involving various species. For example, a transposase gene was transferred from Enterococcus to Streptococcus thermophilus, and a plasmid was transferred from S. thermophilus to E. faecium [140]. A fourth process of HGT has been mentioned, which occurs through DNA-containing membrane vesicles that are released from the surface of bacterial cells and acquired by other bacteria [141]. Outer membrane vesicles (OMVs) from Campylobacter coli were demonstrated to transfer both plasmid-encoded and chromosomally encoded antibiotic resistance genes to Campylobacter jejuni. This transfer is independent of natural transformation and involves direct fusion between OMVs and recipient bacterial membranes. Campylobacter is an important foodborne pathogen whose resistance to antibiotics poses a serious threat to public health [142].

Transposons are important genetic elements in the context of HGT. They are special genetic elements that have the capability of excising themselves from their genetic loci into new loci either in the same bacterial cell or in other bacteria, even those of different taxa. They can be transferred by the three described basic mechanisms of HGT and are known to play a significant role in the development of antibiotic resistance. This is because they have special gene sequences called “integrons”, which are particularly important in the dissemination of antibiotic resistance genes between bacteria [136]. Resident plasmids can be mobilized by conjugative transposons [143]. Additionally, circular gene cassettes can be moved among bacteria. Multidrug resistance can occur because of the integration of multiple gene cassettes [136] that are transferred together [11,129]. A superintegron may have more than 100 antibiotic resistance genes [11].

The three main steps that determine the fate of an antibiotic-resistant gene in an environment are the acquisition, maintenance, and spread of the gene [118]. Initially, the biological cost of having an antibiotic resistance gene is high [144], but some mechanisms of resistance have a low fitness cost and thus the wild antibiotic-sensitive bacterial types will not outcompete the antibiotic-resistant bacteria [129]. This is because, to lessen the negative effects of the resistance mutations, compensatory mutations occur in antibiotic-resistant mutants [135]. The presence of plasmids and transposons as mobile transfer elements carrying antibiotic resistance genes was demonstrated in many foodborne bacteria [110]. Figure 2 summarizes the mechanisms and transmission pathways of antibiotic resistance in foodborne bacteria.

Figure 2.

Summary of the mechanisms and transmission pathways of antibiotic resistance in foodborne bacteria.

ARB have been isolated from a wide range of habitats, including humans, animals, plants, natural ecosystems, and food environments [145]. This broad distribution underscores the role of commensal, spoilage, and pathogenic bacteria in disseminating ARGs and highlights the food chain as an important conduit linking environmental and human reservoirs of resistance [146]. The multilayered matrices of ready-to-eat foods such as sandwiches, which contain diverse ingredients supplying nutrients, moisture, and structurally complex surfaces, can create favorable microenvironments for HGT among resident microbial communities. Evidence from fresh-produce studies illustrates this risk. Chopped lettuce has been shown to support HGT of ESBL genes, such as bla_SHV-18, between K. pneumoniae strains, with transfer frequencies increasing under mild temperature abuse (15–24 °C). Notably, gene transfer on lettuce occurred at even higher frequencies than in liquid media, indicating that fresh produce can constitute an effective environmental surface for the propagation of antibiotic resistance [147].

Chicken meat has been shown to carry phages capable of transferring ARGs. Approximately 24.7% of phages isolated from chicken meat were able to transduce resistance to antibiotics such as kanamycin, chloramphenicol, tetracycline, and ampicillin into E. coli [148]. This suggests that phage-mediated HGT is a significant pathway for spreading ARGs in chicken meat. In dairy products like cheese, HGT can occur through conjugative plasmids. For instance, in Minas Frescal cheese, integrons carrying ARGs for β-lactams, tetracyclines, quinolones, and sulfonamides were detected, indicating potential HGT events [149]. These integrons facilitate the transfer of resistance genes among bacteria in the cheese matrix. In pickled vegetables, metagenomic sequencing has identified the presence of ARGs and mobile genetic elements that facilitate HGT. For example, Levilactobacillus brevis in pickled vegetables carried MDR genes and transposable elements, indicating active HGT processes [150]. Within sandwiches, commensal or spoilage bacteria may thus serve as intermediate hosts facilitating ARG exchange with pathogenic species, which can then cause infections or exchange ARG with other pathogenic or non-pathogenic microbes. A well-known example is the global dissemination of the blaCTX-M-15 gene, encoding a dominant ESBL enzyme. Originally located on the chromosome of environmental Kluyvera spp., this gene mobilized onto plasmids and subsequently spread to diverse clinical pathogens worldwide, emerging as the most significant acquired resistance mechanism to third-generation cephalosporins since its first identification in the mid-1990s [151].

Many studies have demonstrated the presence of phenotypic and genotypic antibiotic resistance among bacteria isolated from RTE sandwiches [145]. Beyond their presence in the final product, these resistance determinants may persist and exert effects after ingestion. Certain strains can act as commensals, opportunistic pathogens, or primary pathogens that colonize the human gastrointestinal tract, where infections may occur even long after exposure to contaminated food [152]. Importantly, ingested ARB and their associated genes can contribute to the intestinal resistome, particularly when resistance determinants persist and integrate into the gut microbial community [153]. These bacteria or free DNA may also serve as vehicles for the horizontal transfer of ARGs within the gut [154] or among microbial populations present on sandwich ingredients such as fresh produce [147]. Because RTE sandwiches typically undergo minimal or no heat treatment, they are more likely to introduce viable ARB and intact ARGs into the gastrointestinal tract compared to foods exposed to higher thermal processing, where DNA degradation is more extensive [152]. Consequently, RTE foods can facilitate the spread of ARB or ARGs beyond the point of consumption, with potential implications for public health, including reduced treatment efficacy, higher healthcare costs, and increased morbidity and mortality associated with resistant infections [155].

9.3. Public Health Implications of Antibiotic-Resistant Bacteria in Sandwiches

Sandwiches can be a silent vehicle for spreading MDR bacteria. Antibiotic resistance in RTE foods, including sandwiches, poses significant public health risks. The presence of ARB in these foods can lead to severe health consequences for consumers, particularly those who are immunocompromised or elderly. Consumption of RTE foods contaminated with ARB can lead to foodborne illnesses that are difficult to treat due to resistance. This can result in longer hospital stays, higher medical costs, and increased mortality rates [156]. Moreover, antibiotic resistance can enhance the pathogenicity of bacteria, increasing the severity of infections. For example, resistant strains of E. coli and S. aureus can produce toxins that exacerbate food poisoning symptoms [71,125]. A study examined 140 RTE sandwiches (burger, hawawshi, kofta, liver, luncheon, shawarma, and sausage) in Egypt for enterotoxin producers and MDRSA (multidrug-resistant S. aureus). One or more staphylococcal enterotoxin genes were detected in 72.7% of S. aureus, and the mecA gene was detected in 81.8% of coagulase-positive S. aureus. These bacteria also showed resistance to various antibiotics such as kanamycin (100%), penicillin and neomycin (92%), oxacillin and erythromycin (84%), and ampicillin and nalidixic acid (68%) [71].

Likewise, in a study conducted to investigate the presence of MDRSA in 60 RTE shawarma sandwiches in Malaysia, 60% of the samples harbored S. aureus, with 80.6% of them being resistant to at least one antibiotic. Resistance was demonstrated for ampicillin (69.4%), penicillin (69.4%), ciprofloxacin (47.2%), tetracycline (33.3%), kanamycin (22.2%), trimethoprim (5.6%), trimethoprim–sulfamethoxazole (2.8%), gentamicin (2.8%), and cephalothin (2.8%). Moreover, 33.3% of the isolates were MDR and were biofilm producers [52]. These results show the potential of ARB present in RTE foods to harbor various virulence factors and their genes, complicating infections and treatment outcomes, as well as making their eradication more difficult. Therefore, vulnerable populations, such as the elderly and immunocompromised individuals, are at higher risk of severe outcomes from infections caused by resistant bacteria [11]. Moreover, the consumption of contaminated RTE foods can facilitate the spread of antibiotic resistance genes among human populations, exacerbating the public health crisis [157]. RTE foods contaminated with resistant pathogens have been linked to outbreaks of foodborne illnesses, highlighting the need for stringent food safety measures [83]. Various foodborne pathogens, such as L. monocytogenes [158], E. coli O157:H7, and S. aureus [159] capable of causing outbreaks, have been demonstrated to be MDR. Thirty-nine V. parahaemolyticus isolates were recovered from 511 RTE Chinese foods from 24 cities, and the isolates exhibited resistance to ampicillin (51.3%), cefazolin (51.3%), and streptomycin (89.7%).

9.4. Strategies, Control Measures, and Recommendations to Reduce Risk of Antibiotic Resistance in Sandwiches and Other RTE Foods

To mitigate the risk of contamination of sandwiches and other RTE foods with pathogenic bacteria, particularly those exhibiting antibiotic resistance traits or their genes, various strategies and frameworks can be targeted. First, it is crucial to strengthen surveillance systems and regulatory frameworks to monitor and control antibiotic resistance in the food supply chain in every country [5,157,160]. Antibiotic resistance is a global challenge, and the development of resistance in one country can spread to other countries. In alignment with the efforts of the international organizations (the World Health Organization, the World Organization for Animal Health, the Food and Agriculture Organization of the United Nations, and the Codex Alimentarius Commission) in establishing standards for resistance surveillance programs, it will be important to establish integrated surveillance programs that harmonize laboratory testing methodologies and antimicrobial-use reporting across different sectors, including human health, animal health, and food production [161,162].

To ensure consistency and comparability of data across nations, it is advisable to utilize existing frameworks like the Global Antimicrobial Resistance and Use Surveillance System (GLASS) [163,164]. It is necessary to implement a comprehensive monitoring systems that cover the entire farm-to-fork continuum, including sampling from humans, animals, and food products [162] as well as to develop real-time surveillance and clinical decision-support systems, such as HAITooL, to monitor antibiotic usage and resistance rates, facilitating early identification of outbreaks and supporting proper antibiotic prescription [165]. Moreover, the surveillance program can consider wastewater surveillance as a complementary tool for tracking antibiotic-resistant genes and bacteria, providing early warnings of emerging threats [166].

Second, implementing responsible and safe food production practices, including the prudent use of antibiotics in agriculture, can help mitigate the spread of resistance [157,167]. In this case, developing regulatory frameworks can be powerful to enforce stringent policy interventions governing antibiotic usage in food production, including registration management policies, usage monitoring systems, and integrated surveillance programs [160]. Policy makers can also learn from successful stewardship strategies in European nations, such as banning antibiotics for disease prevention, benchmarking antibiotic utilization, and setting national reduction targets [168]. Tackling the antibiotic resistance problem also needs international collaboration and coordinated action to address antibiotic resistance, leveraging the One Health approach to integrate efforts across human, animal, and environmental health sectors [169]. To ensure that regulatory measures are contextually appropriate and effective, it is important to address economic dependencies and cultural understandings of risk that drive global antibiotic consumption and resistance [170].

Third, raising awareness about the risks associated with antibiotic-resistant bacteria in RTE foods and promoting safe food handling practices among consumers is essential [157]. This can be achieved by enhancing consumer awareness and responsibility through verified labeling and digital innovations that support informed choices regarding antibiotic use in food products [168] and conducting public educational campaigns to improve knowledge and behaviors related to antibiotic use and resistance, leveraging interactive and engaging methods [171]. Moreover, targeted education programs can be implemented for various stakeholders, including students, healthcare professionals, and the general public, to improve understanding and practices related to antibiotic use. Diverse educational tools, such as board games, e-learning, and environmental experiments, to engage different audiences and promote responsible antibiotic behavior, can be used [169]. By integrating these strategies, surveillance systems and regulatory frameworks can be significantly strengthened to monitor and control antibiotic resistance in the food supply chain, ultimately contributing to global public health and food safety. The strategies to reduce the risk of antibiotic resistance in sandwiches and other RTE foods are summarized in Figure 3.

Figure 3.

Summary of the strategies to reduce the risk of antibiotic resistance in sandwiches and other RTE foods.

It is also important to note that effective control of pathogenic and ARB in RTE foods requires targeted interventions. Several validated strategies have demonstrated success in reducing ARB and ARGs in RTE food systems. Hurdle technologies that combine pH reduction, lowering water activity, and applying organic acids have been shown to suppress L. monocytogenes in RTE meats and sandwiches [172]. High-pressure processing (HPP) is widely recognized for its ability to inactivate pathogens while preserving sensory quality, with documented reductions of up to 5-log CFU for various foodborne pathogens [173]. Antimicrobial packaging incorporating nisin or plant-derived compounds has effectively reduced Listeria growth on RTE cheeses and fresh produce during storage [174]. In ingredient preparation, interventions such as peracetic acid washes, chlorine dioxide, and UV-C treatment significantly reduce microbial loads on fresh produce commonly used in sandwiches [175,176]. Improved sanitation protocols, cross-contamination controls, and temperature management along the preparation chain further minimize risks [177].

Bacteriophage therapy can be effective against multiple serotypes of ARB in RTE foods like sandwiches. Several commercial phage preparations (e.g., those targeting L. monocytogenes and Salmonella) have been approved for application on RTE meats, fish, fresh produce, and dairy products. These phages can be applied as surface sprays or dips and have demonstrated significant log-reductions in pathogen levels without affecting sensory quality. Importantly, phage therapy offers high specificity, minimizing disruption to beneficial microbiota and reducing selective pressure associated with chemical sanitizers [178]. Incorporating these techniques into sandwich preparation systems along the food chain can reduce the risk of consuming sandwiches containing ARB or their genes, thereby protecting consumer health. In addition, global surveillance systems such as GLASS can be operationalized by integrating AMR monitoring into raw-material testing, facility-level environmental sampling, and post-processing verification.

10. Previous Studies’ Limitations

Despite its widespread consumption as a typical RTE food in many countries around the world, there is still a lack of comprehensive studies regarding the microbiota of sandwiches that include commensal, opportunistic, and pathogenic microbes in many countries, such as Oman. Most investigations focused on pathogenic bacteria that are mostly involved in causing outbreaks. In general, previous studies investigating the microbiota of sandwiches have faced several limitations, which can be categorized into methodological, environmental, and analytical challenges. Regarding methodological limitations, many studies have limited sample sizes and often focus on specific types of sandwiches or ingredients, which may not represent the full diversity of sandwich types and their microbiota. For instance, studies often examine a narrow range of sandwich types, such as falafel or pre-packed sandwiches, without considering the wide variety of sandwiches consumed globally [19,20]. The conditions under which samples are collected and stored can significantly impact the microbial load. For example, the storage location and temperature can affect the bread’s structure and moisture content, which in turn influences microbial growth [179]. Additionally, the time between sample collection and analysis can vary between studies, leading to changes in the microbial community [19]. The techniques used to detect and enumerate microorganisms can vary, leading to inconsistencies in results. Different studies use various media and methods for microbial enumeration, which can affect the detection of specific microorganisms [19,20]. For example, the use of different agar media types for counting mesophilic aerobes, coliforms, and S. aureus can yield different results and thus interpretations [19].

Environmental limitations, such as the hygienic conditions of the environment where sandwiches are prepared and sold, can greatly influence the microbial load. Studies have shown that sandwiches from street vendors or certain restaurants often have higher microbial counts due to poor hygienic practices [58]. This variability makes it challenging to generalize findings across different settings. Moreover, there is the issue of ingredient variability. The type and quality of ingredients used in sandwiches can introduce variability in microbial communities. Ingredients like hummus and tahini salad have been shown to contribute significantly to the microbial load in falafel sandwiches [19]. Similarly, the presence of additional ingredients like tomatoes in pre-packed sandwiches can increase microbial counts [20].

Analytical limitations may be due to microbial community complexity. The complexity of microbial communities in sandwiches poses a challenge for comprehensive analysis. Traditional culturing methods may not capture the full diversity of microorganisms present, leading to an incomplete understanding of the microbiota. Advanced techniques like metagenomics can provide a more detailed picture, but are not always used [180]. Another analytical limitation is pathogen detection. While some studies focus on specific pathogens like L. monocytogenes and Salmonella spp., the absence of these pathogens in samples does not necessarily indicate overall microbial safety. The presence of other harmful microorganisms, such as S. aureus and B. cereus, can still pose significant health risks [20].

11. Conclusions

Sandwich ingredients and their preparation practices were found to diversify their microbiota with the prevalence of pathogens such as S. aureus, Salmonella sp., E. coli, and L. monocytogenes. Moreover, microbiota diversity was found to cause many food poisoning outbreaks globally. Many sandwich-associated pathogens, such as B. cereus, S. aureus, L. monocytogenes, and STEC E. coli, were found to possess virulence factors such as Nhe, sea, InlA and eaeA. Most sandwich-isolated pathogens, which showed antibiotic resistance, included S. aureus, L. monocytogenes, and E. coli. Sandwich-associated pathogens showed significant resistance to commonly used antibiotics, such as penicillin, tetracycline, gentamicin, erythromycin, trimethoprim–sulfamethoxazole, and ciprofloxacin. To minimize the presence of pathogens with antibiotic resistance in sandwiches, the current review proposed certain strategies, such as strengthening surveillance systems, implementing responsible, safe food production practices, raising consumer awareness about the risks associated with ARB in RTE foods, including sandwiches, and promoting safe food handling practices among consumers.

Abbreviations

The following abbreviations are used in this manuscript:

RTE	Ready-to-eat food
EHEC	Enterohemorrhagic E. coli
ETEC	Enterotoxigenic E. coli
EAEC	Enteroaggregative E. coli
EIEC	Enteroinvasive E. coli
DAEC	Diffuse-adherent E. coli
MIC	Minimum inhibitory concentration
ARB	Antibiotic-resistant bacteria
MRSA	Multidrug-resistant S. aureus
VRSA	Vancomycin-resistant S. aureus
MDR	Multidrug-resistant
ESBL	Extended-Spectrum β-Lactamases
AmpC	AmpC β-Lactamases
ARG	Antibiotic resistance genes
ATP	Adenosine triphosphate
PMF	Proton-motive force
MFS	Major facilitator superfamily
SMR	Small multidrug resistance family
HGT	Horizontal gene transfer

Author Contributions

Conceptualization, I.M.A.-B.; writing—original draft preparation, I.M.A.-B. and Z.S.A.-K.; writing—review and editing, I.M.A.-B., Z.S.A.-K., M.K.A.-K., K.N.A.-S. and M.A. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.