Multidrug-resistant Enterococcus faecalis and Enterococcus faecium isolated from oriental meat pies and beef- and chicken-based pizzas

Multidrug-resistant Enterococcus faecalis and Enterococcus faecium isolated from oriental meat pies and beef- and chicken-based pizzas | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multidrug-resistant Enterococcus faecalis and Enterococcus faecium isolated from oriental meat pies and beef- and chicken-based pizzas Amira Mahmoud Elsayeh, Amira Ibrahim Zakaria, Samir Mohammed Abd El-Ghany, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9259480/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract This study was conducted to determine whether ready-to-eat meat products could indirectly threaten consumer health by serving as a reservoir for Enterococcus strains that carry virulence determinants and antimicrobial resistance profiles. Overall, 76.4% (84/110) of the examined RTE meat samples, including oriental meat pies, meat pizza, and chicken pizza, were contaminated with Enterococcus . Polymerase chain reaction (PCR) analysis showed that 65.8% (219/333) of Enterococcus isolates were identified as E . faecalis , while 34.2% (114/333) were E . faecium . The virulence genes gelE (gelatinase) and ace (collagen-binding protein) were detected in 53.2% (177/333) and 43.2% (144/333) of the isolates, respectively. Interestingly, 100% (333/333) of Enterococcus isolates were resistant to cefepime, 97.3% (324/333) to penicillin, 94.89% (316/333) to meropenem, 92.19% (307/333) to kanamycin, 87.99% (293/333) to clindamycin, and 64.86% (216/333) to erythromycin. Remarkably, 98.5% (328/333) of the isolates showed resistance to at least four antibiotics, with an average multiple antibiotic resistance (MAR) index of 0.42. The vanA and vanB genes were not detected in any isolates, while the ermB gene was found in 26.4% (88/333), and tetL in 11.1% (37/333) of the Enterococcus isolates. To our knowledge, this is the first study in Egypt to assess antibiotic resistance and virulence characteristics in Enterococcus spp. recovered from pizza and oriental meat pies. The findings of this study may be valuable for evaluating potential human health risks associated with consuming cooked and processed meat products. Biological sciences/Microbiology Biological sciences/Molecular biology Enterococcus Pizza Ready-to-eat meat products PCR Antimicrobial resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction In recent decades, the demand for fast meals has increased substantially, making them a prominent feature of modern eating habits. Many people rely on these meals due to limited time available for home cooking and the convenience fast food offers. Among the most popular fast meals is pizza, which is enjoyed worldwide for its rich flavor, delicious taste, and easy availability. Approximately 13% of Americans consume pizza on any given day, highlighting its popularity in the USA (Rhodes et al., 2023 ). Although the precise per capita pizza consumption figures for Egypt are not easily accessible, the Egyptian pizza market exhibits robust growth, with a market growing by nearly 18% in 2023 alone, reflecting increased consumption combined with booming online meal delivery, particularly for fast food. Pizza provides various nutrients, including carbohydrates from the crust, proteins from meat, and fats that supply energy. Although pizza provides several essential nutrients, it can also be high in saturated fats, salt, and calories, which may contribute to public health issues. Pizza frequently needs temperature regulation for safety when kept in restaurants (Xu and Schaffner, 2023 ). If not prepared or stored properly, pizza may be contaminated with pathogenic bacteria that cause foodborne illnesses, posing further health risks. According to the US CDC's National Outbreak Reporting System (NORS), most outbreaks in the United States have been associated with pizza (Centers for Disease Control and Prevention, 2021). Enterococcus species are typically inhabitants of the human digestive tract as commensal bacteria. If the commensal relationship between enterococci and their host is broken, enterococci can turn into opportunistic pathogens and cause invasive diseases (Hamza and Kadem, 2018). They can also be found in the environment in water, soil, and animal-based food. The presence of Enterococcus spp. in food is considered an indicator of fecal contamination or poor hygiene during processing or storage. The most prevalent Enterococcus spp are E . faecalis and E . faecium , which are the main cause of human infections, including septicemia, urinary tract infections, endocarditis, neonatal sepsis, meningitis, and wound infections (Kafil and Asgharzadeh, 2014 ). Furthermore, E . faecium and E . faecalis strains are the most predominant nosocomial opportunistic pathogens responsible for approximately 10–15% and 80–90% of infections, respectively (El Zowalaty et al., 2023 ). Testing for Enterococcus species is not part of the routine management of the production and distribution of food of animal origin. Unlike coliform bacteria and E. coli, their number and concentration are not restricted (Milanove et al., 2025). Enterococcus spp comprises virulence genes, such as Gelatinase ( gelE ), which hydrolyze gelatin, and Accessory colonization factors ( ace ), which are capable of colonization by binding to proteins of the extracellular matrix, as well as sharing in binding type I and IV collagen (Wioleta et al., 2016). Globally, antimicrobial resistance has grown to be a serious concern to both human and animal health, making it more difficult to treat some diseases with traditional antibiotics. Antimicrobials are typically needed by food-producing animals as a preventative measure or treatment for a variety of bacterial illnesses. Multidrug-resistant bacterial strains have emerged as a result of the global overuse and abuse of antibiotics. These strains spread through animal-based foods and pose a serious risk to human health. These days, one of the biggest problems in treating infections in humans and animals is the accelerated creation of bacterial diseases that are resistant to drugs (Sallam et al., 2023 ). Enterococci are sentinel bacteria for monitoring antibiotic resistance because of their individuality and their widespread behavior (Smoglica et al., 2022 ). AMR is exacerbated by the direct transfer of resistant bacteria from animals to humans, especially from food-producing animals (Jans et al., 2018 ). Antimicrobial resistance (AMR) is the main and increasing public health concern, both in human and veterinary medicine, affected by reducing antibiotic effectiveness and delaying bacterial infection treatment (Islam et al., 2023). Antimicrobial-resistant Enterococcus strains were elevated throughout time (Carlet et al., 2012); due to the high prevalence of resistance and virulence characteristics, Enterococcal spp danger evaluations are difficult (EFSA, 2008). Because Enterococcal organisms inherently resist several antibiotic classes, including cephalosporins, aminoglycosides, macrolides, and sulfonamides, managing these infections can be difficult (Hollenbeck and Rice, 2012 ). To acknowledge the various components, including human, animal, and environmental factors that contribute to the rising levels of AMR, it is necessary to embrace a comprehensive "One Health" viewpoint (Islam et al., 2023). No research has been conducted on the prevalence of E . faecalis and E . faecium in Oriental meat pies, as well as in meat- and chicken-based pizza in Egypt. Therefore, this study aimed to determine the prevalence of E . faecalis and E . faecium strains in oriental meat pies and various types of pizza from restaurants in Dakahlia Province, Egypt, and to examine the presence of virulence genes ( sodA faecalis , sodA faecium , Ace , and gelE ) and some resistance genes, including tetL , ermB , vanA , and vanB , as well as the antimicrobial resistance profile of E . faecalis and E . faecium strains against 16 antibiotics from different classes. 2. Materials and methods 2.1 Sample collection A total of one hundred ten random samples of locally produced meat products, including 30 meat pizzas, 30 chicken pizzas, and 50 oriental meat pies, were purchased from various restaurants in Mansoura City, Egypt, between February 2024 and November 2024. The samples were individually packed in sterile plastic bags and promptly transported in an ice box under strictly aseptic conditions to the laboratory of Food Hygiene, Safety, & Technology Department, Faculty of Veterinary Medicine, Mansoura University, where bacteriological analysis was conducted immediately to detect the presence of Enterococcus spp. 2.2 Isolation and identification of Enterococcus spp . A total of 25 grams from each sample was aseptically excised using a sterile scalpel and homogenized in a sterile laboratory stomacher (Seward Laboratory, London, UK) containing 225 ml of buffered peptone water (Oxoid CM0509). The homogenized samples were then incubated at 37 °C for 24 h. Following this, 10-fold Serial dilutions were prepared in sterile test tubes. From the selected dilutions (10 −3 and 10 −4 ), 0.1 mL was spread onto Bile Esculin Agar (M493I; HiMedia, India). The inoculated plates were incubated at 37 °C for 24 h to obtain pure culture colonies. Presumptive Enterococcus colonies on Bile Esculin Azide agar plates appear as small, translucent or slightly opaque colonies surrounded by dark brown, indicating esculin hydrolysis. Morphological examination of the suspected colonies was performed using Gram staining, along with biochemical identification of the isolates through catalase activity, oxidase test, carbohydrate fermentation (mannitol and sucrose), bile esculin test, and detection of hemolysis. 2.3. Preparation of Genomic DNA A total of 333 typical Enterococcus colonies were subjected to genomic DNA extraction using the Gene JET Genomic DNA Purification Kits (Roche Applied Science, Germany, Cat. No. 11 796 828 001) following the manufacturer's instructions. Genomic DNA was also isolated from E . faecalis ATCC 29212 and E . faecium ATCC 19434 as positive control strains, while genomic DNA was extracted from the E . coli K-12 DH5α strain as a negative control. 2.4. Molecular confirmation and characterization of Enterococcus spp. 2.4.1. Molecular differentiation of Enterococcus spp. The 333 phenotypically and biochemically verified Enterococcus isolates, recovered from examined samples, were molecularly confirmed using the polymerase chain reaction assays (PCR), based on the presence of sodA ( E. faecalis ) and sodA ( E. faecium ) marker genes using their specific primer sets according to Jackson et al. (2004) at the PCR conditions mentioned in Table 1. 2.4.2. PCR detection of gelE and ace virulent genes in Enterococcus isolates The differentiated E . faecalis and E . faecium isolates obtained from examined RTE meat product samples were tested for the presence of the gelatinase ( gelE ) and the collagen-binding proteins ( ace ) virulent genes, using PCR-specific primer sets for the gelE gene (Eaton and Gasson, 2001) and the ace gene (Creti et al., 2004), following the cycling conditions outlined in Table 1. 2.4.3. PCR detection of vancomycin-resistant genes (vanA, vanB), erythromycin-resistant gene ( ermB), and tetracycline-resistant gene (tetL) among Enterococcus isolates The Enterococcus isolates that showed phenotypic resistance to erythromycin and tetracycline by the disk diffusion method were further examined using PCR assays for the detection of the tetL (Malhotra-Kumar et al., 2005) and the ermB (Knysz et al., 2025)resistance genes. At the same time, the Enterococcus isolates were also tested using PCR for the possible detection of Van A and Van B genes. Primer sets for the detection of antimicrobial-resistant genes are listed in Table 1. PCR was performed using SimpliAmp thermal cycler (Thermo Fisher Scientific Inc.) in a 25 final reaction volume, the amplification of marker, virulence, and resistant genes was performed using the following component: 12.5 μL of DreamTaq TM Green Master Mix (2X) (Fermentas, Inc., Hanover, MD, USA), 1.0 μL from each of sense and antisense primer (10 pmol each) specific for sodA ( E . faecalis ), sodA ( E . faecium ), ermB , tetL , vanA , or vanB (Sigma-Aldrich, Co., St. Louis, MO, USA), 2 μL of the genomic DNA template, and 8.5 μL of DNase/RNase-free water. Each PCR test included both positive and negative controls. The resulting PCR product (8 μL aliquots from each reaction mixture) was separated within the agarose gel (1.5%) electrophoresis at 100 V for 55 min using 1X TBE running buffer and stained with ethidium bromide solution (10 μg/ml) (Sigma-Aldrich, Co., St. Louis, MO, USA). The separated PCR products were then visualized and photographed using an ultraviolet transilluminator. The GeneRuler 100 bp DNA Ladder (Thermo Fisher Scientific Inc.) was utilized as a molecular size marker in order to ascertain the molecular size of the PCR products. 2.5 Antimicrobial resistance profile of isolated Enterococcus spp. The antimicrobial resistance profile of Enterococcus isolates (n = 333) was determined using the Kirby-Bauer disc diffusion technique on Mueller–Hinton agar (MH; CM0337; Oxoid Ltd., Basingstoke, UK) against 16 antimicrobials, which comprised 7 antimicrobial classes. Vancomycin, on the other hand, was tested using the agar dilution method. Enterococci with a MIC breakpoint of 4 µg/mL are identified susceptible, 8–16 µg/mL are identified intermediate, and ≥ 32 µg/mL are identified vancomycin-resistant Enterococcus (VRE). The variable concentration of antibiotics used are Penicillin (10 μg, P 10), Amoxicillin –Clavulanic aid (30 μg, AMC 15), Erythromycin (15 μg, E 15), Gentamicin (10 μg, GM 10), tetracycline (30 μg, TE 30), Ciprofloxacin5 μg, CIP 5), Levofloxacin (15 μg, LEV 15), Vancomycin (30 μg, VA 30), Cefepim (30 μg, FEB 30), Nitrofurantion (300 μg, F 300), Linezolid (30 μg, LNZ 30), Clindamycin (10 μg, DA10), Kanamycin(10 μg, K 10), Rifampin (5 μg, RA 10), Meropenem (10 μg, MEM 10), and Doxycycline (30 μg, DO 30). Results were interpreted according to the standard breakpoints for inhibition zone provided by CLSI (2020). The strains tested were categorized as susceptible, intermediate, and resistant. The multiple antimicrobial resistance (MAR) index for each Enterococcus strain was calculated according to Singh et al. (2010) as the ratio of the number of antibiotics to which the isolate exhibited resistance to the total number of antibiotics tested. 3. Results and discussion 3.1. Prevalence of Enterococcus species in ready-to-eat samples tested Epidemiological studies reported that food of animal origin, particularly undercooked poultry and red meat, are the most common sources of human disease caused by foodborne microorganisms (Khudhair and Kadium, 2025; Sharafi and Nateghi, 2020). The rising global demand for RTE pizza and pies prompts scientists to evaluate their microbiological quality, particularly for foodborne pathogens like Staphylococcus aureus (Elsalkh et al., 2025), Salmonella (Raza et al., 2021), and Escherichia coli O157:H7 (Gieraltowski, 2017). In the present approach, the contamination levels with Enterococcus spp., which serve as important indicators of fecal contamination, were examined in 110 samples of RTE pies and pizza samples. The results indicated that 76.4% (84/110) of ready-to-eat samples, distributed as 84% (42/50) of oriental meat pies, 73.3% (22/30) of meat pizza, and 66.7% (20/30) of chicken pizza were contaminated with Enterococcus (Figure 2). Similar to our findings, Chajęcka‐Wierzchowska et al. (2016) reported an incidence rate of 74.1% (289/390) for Enterococcus spp. in ready-to-eat meat products tested in Poland. In contrast, lower prevalence rates were recorded in other regions; for instance, 61.7 % (129/209) in meat samples from Spain (Martinez-Laorden et al., 2023), 35 % (21/60) in chicken meat samples from Bangladesh (Samad et al., 2022), 60 % (15/25) in chicken samples from Egypt (El-Hamid et al., 2016), and 61.8 % in meat products from Iran (Hosseini et al., 2016). Enterococci are often isolated from food-producing animals that cause hazards to consumers (Tyson et al., 2017), and their presence reflects fecal contamination of meat products during slaughter and carcass preparation. 3.2. Frequency distribution of Enterococcus isolates (n = 333) among Oriental meat pies, meat pizza, and chicken pizza The biochemically identified Enterococcus isolates (n = 333), including 168 from oriental meat pies, 85 from meat pizza, and 80 from chicken pizza, were further confirmed using PCR targeting sodA ( faecalis ) and sodA ( faecium ) specific marker genes to differentiate E . faecalis and E . faecium , which were detected at the expected molecular size of 360 bp (Figure 1A) and 215 bp (Figure 1B), respectively. Individually on the sample bases, 58% (29/50), 56.7% (17/30), and 30% (9/30) of the examined oriental meat pies, meat pizza, and chicken pizza samples, with an overall prevalence rate of 50% (55/110), were positive for E. faecalis (Figure 2). On the Enterococcus isolate bases, 117 (69.6%), 65 (76.5%), and 37 (46.3%) of E. faecalis isolates were recovered from oriental meat pies, meat pizza, and chicken pizza samples, respectively, with an overall incidence of 219 (65.8%) (Figure 3). On the other hand, 26% (13/50), 16.7% (5/30), and 36.7% (11/30) of the examined oriental meat pies, meat pizza, and chicken pizza samples, with an overall prevalence rate of 26.36% (29/110), were positive for E. faecium (Figure 2). At the isolate level, however, 51 (30.4%), 20 (23.5%), and 43 (53.8%) of E. faecium isolates were recovered from Oriental meat pies, meat pizza, and chicken pizza samples, respectively, for an overall prevalence of 114 (34.2%) (Figure 3). E. faecalis is a zoonotic enteric bacterium that may cause severe problems in humans and animals, including GIT infections, urinary tract infections, periodontitis, meningitis, septicemia, peritonitis, and endocarditis (Said, 2022). E . faecium is also a zoonotic enteric bacterium of major clinical and public health importance, and in many circumstances, it is considered more problematic than E . faecalis due to its high level of antimicrobial resistance. In humans and animals, E . faecium is associated with a wide range of infections, particularly in healthcare and foodborne exposure settings (Arias and Murray, 2012; Lebreton et al., 2014). One of the main causes of infections contracted in hospital settings is E . faecalis , which poses serious hazards, especially to those with weakened immune systems (Asfaw, 2019). Poor hygiene is the main way that E . faecalis infections spread from person to person. Food modification and poor cleanliness can allow the germs to infect food (Hanchi et al., 2018). The distribution and predominance of E . faecalis observed in the present study are consistent with findings from various other publications. For instance, Nüesch-Inderbinen et al. (2023) reported that E . faecalis was the most dominant species, found in 28.8% (17/59) of Enterococcus isolates collected from meat samples in Switzerland, followed by E . faecium at 6.78% (4/59). Similarly, Samad et al. (2022) indicated that E . faecalis was the most common species, with a prevalence of 20% (12/60), while E . faecium was present in 10% (6/60) of the isolates obtained from chicken meat tested in Bangladesh, whereas Chajęcka‐Wierzchowska et al. (2016) reported that E . faecalis was the most common species, with a prevalence of 37.7% (147/390), while E . faecium was found in 30.8% (120/390) among the isolates obtained from ready-to-eat meat products in Poland. Also, Milanov et al. (2025) revealed that E . faecalis was present in 52% of beef and 61.9% in poultry samples, while E . faecium was detected 24% of beef and 33.3% of poultry meat samples. Additionally, Martinez-Laorden et al. (2023) indicated that E . faecalis was the most dominant species (46.4%; 147/317), followed by E . faecium (22.7%; 72/317), among Enterococcus isolates collected from various meat samples in Spain. Furthermore, Hosseini et al. (2016) reported that 51 (48.8%) and 44 (41.9%) of meat products examined in Iran were positive for E . faecalis and E . faecium , respectively. The prevalence rate of 65.8% (219/333) determined for E . faecalis among the Enterococcus isolates in the current study is higher than that reported by Barbosa et al. (2009),who indicated that 41.8% (76/182) of the Enterococcus isolates recovered from fermented meat products in Portugal were verified as E . faecalis . A lower prevalence rate of 35% (35/100) was reported for E . faecalis among Enterococci recovered from chicken samples in Zanjan, Iran (Afshar et al, 2023). Enterococcus faecalis also demonstrated a low prevalence rate of 46.4% meat samples in Spain (Martinez-Laorden et al., 2023), as well as a low rate of 44.3% (31/70) from beef samples in Italy (Pesavento et al., 2014). Higher prevalence rates of 69.5% were reported for E . faecalis among Enterococcus isolates recovered from red meat samples in Slovenia (Golob et al., 2019), as well as in 94% and 73% of Enterococcus isolates recovered from poultry meat and beef samples tested in Canada, respectively (Aslam et al., 2012). The predominance rate of E . faecium among identified Enterococcus isolates in this study was 34.2% (114/333), which is nearly consistent with that reported by Pesavento et al. (2014), who documented predominance rates of 35.7% (25/68) in beef samples and 36.7% (25 out of 68) in poultry samples from Italy. A higher predominance rate of 44.8% (14/38) was reported among Enterococcus isolates from fermented sausage tested in Germany (Jahan et al., 2013). Likewise, a higher predominance rate of 65% (65/100) was reported among Enterococcus isolates from chicken samples in Iran (Afshar et al., 2022). Conversely, lower prevalence rates of 22.7% and 24.18% (44/182) have been reported among Enterococcus isolates recovered from meat samples in Spain (Martínez-Laorden et al., 2023) and from traditional fermented meat products in Portugal (Barbosa et al., 2009), respectively. 3.3. Prevalence of virulent genes among Enterococcus species recovered from oriental meat pies and pizza samples The pathogenesis of enterococci is associated with a variety of virulence genes that facilitate the colonization and invasion of host tissues. This process leads to an increase in enterococcal infections, as these genes enable the release of toxins and enzymes outside of the cells, adversely affecting the host's immune response and worsening the condition (Cebeci, 2024). The primary adhesion factors, associated with biofilm formation, and other related virulence components include Ebp (endocarditis and biofilm-associated pili), Asa (aggregation substance), Esp (extracellular surface protein), EfaA ( E . faecalis antigen A), Ace (collagen-binding cell wall protein), cad1 (pheromone cAD1 precursor lipoprotein), cylA (hemolysin), efm ( E . faecium -specific cell wall adhesin), sagA (secreted antigen), (secreted antigen), agg (aggregation substance), gelE (gelatinase), and cpd1 (pheromone cPD1 lipoprotein) (Alzahrani et al., 2022). Furthermore, certain virulent genes and toxins linked to Enterococcus spp increase the severity and pathogenicity of diseases they can spread (Goh et al., 2017). The distribution and prevalence of virulence genes ( sodA , gelE , and ace ) among the 333 Enterococcus isolates recovered from RTE oriental meat pie, meat pizza, and chicken pizza tested in the current study are shown in Figure 5 and Table 2. The sodA gene was detected in all isolates (100%) from the different RTE samples tested. The sodA gene, as a key marker gene that differentiates E . faecalis and E . faecium, was also detected in 100% of E . faecalis and E . faecium in many other studies. PCR was employed to differentiate between the virulence of Enterococcus isolates by targeting gelE, which was detected at 419bp (Figure 4A), and the ace gene, which was detected at 616 bp (Figure 4B). The matrix metalloproteinase gelatinase, encoded by gelE , hydrolyzes collagen and gelatin, which interfere with complement-mediated immunity and may contribute to the development of infectious endocarditis caused by Enterococcus faecalis (Geraldes et al., 2022). The gelE gene was detected at incidence rates of 70.1% (82/117), 47.7% (31/65), 21.6% (8/37) in E . faecalis isolates recovered from oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 55.3% (121/219).On the other hand, 39.2% (20/114), 60% (12/114), 55.8% (24/114) were the prevalence rates for gelE gene in E . faecium isolates recovered from oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 49.1% (56/114), with an overall incidence of 60.7% (102/168), 50.6% (43/85), and 40% (32/80) in Enterococcus isolates recovered from oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 53.2% (177/333) among all Enterococcus isolates. These results are aligned with those reported by Maasjost et al. (2019), who found that 53.6% (15/28) of Enterococci isolates from turkey meat in Germany were positive for the gelE gene; Also, 56% (79/141) of Enterococcus isolates recovered from retail red meat in Slovenia tested positive for gelE (Golob et al., 2019). In contrast, a higher prevalence rate of 79% was found for gelE in Enterococcus isolates recovered from chicken meat in Bangladesh (Samad et al., 2022). Likewise, 75% (75/100) of Enterococcus isolates isolated from beef samples collected from butcher shops and supermarkets in Turkey harbored the gelE gene (Yılmaz et al., 2016), while the gelE gene was determined in a much higher incidence of 95.8% (46/48) in Enterococcus isolates from meat samples from Tunisian markets (Klibi et al., 2013). Furthermore, a high detection rate of 88.1% (155/176) was reported for the gelE gene in Enterococcus isolates recovered from meats in South Korea (Kim et al., 2020), and a higher prevalence rate of 92.3% of gelE gene was reported in E . faecalis isolates from chicken meat collected at food stores in Athens (McGowan-Spicer et al., 2008). Additionally, Ozmen et al. (2010) reported that 100% of Enterococci isolates from naturally fermented sausage samples in Turkey were positive for gelE . On the contrary, the gelE gene was found at lower prevalence rates of 36% in Enterococcus isolates recovered from meat products in Serbia (Valenzuela et al., 2009) and at a detection rate of35% (7/20) in Enterococcus strains isolated from sausages in Portugal (Ribeiro et al., 2011). A much lower detection rate of 12.3% was determined for gelE gene in Enterococcus isolates recovered from chicken salad, chicken burgers, and meat tested in Kansas, USA (Macovei and Zurek, 2007). The ace gene was detected in 59.8% (70/117), 52.3% (34/65), 24.3%(9/37) in E . faecalis isolates recovered from Oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 51.6%(113/219) On the other hand, 52.9% (27/51), 20% (4/20), 0% (0/43) was the prevalence rate for ace gene in E . faecium isolates recovered from Oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 27.2% (31/114). Additionally, The ace gene was detected in Enterococcus isolates recovered from Oriental meat pies, meat pizza, and chicken pizza, tested with an incidence of 57.7% (97/168), 44.7% (38/85), and 11.3% (9/80), respectively, and an overall prevalence of 43.2% (144/333) (Figure 5 & Table 2). Higher prevalence rates of the ace gene in Enterococcus spp. have been reported in other studies. For instance, Golob et al. (2019) found the ace gene in 65.2% (92/141) of Enterococcal isolates collected from retail red meat in Slovenia. In contrast, a lower incidence of 37% (81/219) was observed for the ace gene in Enterococcus isolates from Brazilian food samples (Gomes et al., 2008). Additionally, a much lower detection rate of 12% (30/250) was reported for the ace gene in Enterococcus isolates recovered from various foodstuffs, such as dairy products, ready-to-eat meat products, fruits, and vegetables (Trivedi et al., 2011). Likewise, Guerrero-Ramos et al. (2016) noted that 10.9% (6/55) of Enterococcus isolates recovered from wild game meat in North-Western Spain harbored the ace gene. 3.4. Antibiotic resistance profile of Enterococcus isolates (n = 333) recovered from RTE meat products The emergence of multidrug-resistant Enterococcus from food of animal origins is strongly linked to the misuse of antibiotics in animal-rearing practices (Kim and Ahn, 2022 ). The significance of Enterococcus in veterinary and human medicine arises from its ability to acquire antimicrobial-resistance genes, posing a potential public health risk(Nurrahmat et al., 2025). As meat products are recognized as potential vehicles for the transmission of antibiotic-resistant bacteria, there is a critical need to investigate the antimicrobial resistance characteristics of bacteria recovered from frequently consumed meats, including poultry and beef. The antibiotic resistance patterns of all examined Enterococcus isolates (n = 333) against 16 antibiotics from 7 antimicrobial classes are shown in Table 3. In this study, complete resistance was observed in Enterococcus isolates against several antimicrobial agents from various classes, particularly those linked to β-lactams. Interestingly, 100% (333/333) of the isolates were resistant to at least one β-lactam antibiotic. Additionally, all E . faecalis (219/219) and E . faecium (114/114) isolates demonstrated complete resistance to cefepime. Likewise, 100% (81/81) of E . faecalis isolates from red meat from slaughterhouses in Borujerd city were susceptible to nitrofurantoin and cefotaxime discs (Madanipour et al., 2017). A lower resistance rate of 23.8% (55/231) was recorded towards cephalothin among Enterococcus isolates from beef samples tested in Botswana (Chingwaru et al., 2003). In the present study, a 100% absolute susceptibility rate of Enterococcus spp. was observed against gentamicin, vancomycin, and levofloxacin (Table 3). This finding aligns with data from northern Portugal, where 99.5% (181/182) of Enterococcus isolates from fermented meat products were susceptible to vancomycin (Barbosa et al. 2009). Nonetheless, resistance rates of 32.8% (23/70) and 4.3% (3/70) were recorded for enterococcal isolates recovered from beef against gentamicin and vancomycin tested in Italy, respectively (Pesavento et al. 2014). Moreover, 47.3% and 9.1% of enterococci isolated from wild game meat in northwest Spain were resistant to vancomycin and gentamicin, respectively (Guerrero et al., 2016). Additionally, 7.9% (24/302) and 0.7% (2/302) of enterococcal isolates from ready-to-eat meat products in northeast Poland showed resistance to gentamicin and vancomycin, respectively (Chajęcka-Wierzchowska et al., 2016). In this study, All (100%; 333/333) Enterococcus isolates were resistant to cefepime, present in all isolates, followed by penicillin (97.3%; 324/333), meropenem (94.89%; 316/333), kanamycin (92.19%; 307/333), clindamycin (87.99%; 293/333), and erythromycin (64.86%; 216/333) (Table 3). Unlike our findings, Milanov et al. (2025) reported that the most frequent resistance of Enterococcus isolates was to tetracycline, found in 32 isolates (34.78%), followed by erythromycin resistance in 25 isolates (27.17%), doxycycline in 20 isolates (21.73%), and streptomycin in 12 isolates (13.04%). Numerous innate and acquired mechanisms are implicated in the notable decrease in susceptibility to β-lactam antibiotics exhibited by Enterococcus , mainly E . faecalis and E . faecium . One important aspect that lowers the effectiveness of β-lactam antibiotics is the presence of penicillin-binding proteins (PBPs). Furthermore, some Enterococcus species generate β-lactamase enzymes, which cause β-lactam antibiotics to be broken (Gagetti et al., 2019).Due to the abuse of antimicrobial agents in agricultural, food-producing animals and human medications, the emergence of antibiotic-resistant bacteria has increased, and constitutes a human public health (Manyi-Loh et al., 201 8 ). Many environments have been proven to be essential reservoirs for antibiotic resistance genes found in soil, water, wastewater, and other natural habitats that may transfer these genes to human-associated bacteria (Martínez et al., 2015). The emergence of multidrug-resistant Enterococcus species has substantially limited therapeutic options and made the treatment of enterococcal infections increasingly difficult for clinicians . Because of its administration for several decades, the penicillin aminoglycoside combination is the cornerstone of managing enterococcal infections caused by susceptible strains (Leone et al., 2016). One well-known concern for global health is the creation and spread of bacterial antimicrobial resistance. Because humans, animals, food, and the environment can all serve as potential reservoirs for AMR bacteria and resistance genes, the idea of "One Health" is essential to addressing this problem (Huijberset al., 2015). In the recent survey, 14.41% (48/333) of Enterococcus isolates were resistant to rifampin (Table 3). A higher resistance rate of 60% (109/182) was recorded among Enterococcus isolates recovered from fermented meat products in the North of Portugal toward rifampicin (Barbosa et al., 2009), while 23.8% (35/147) and 15.8% (19/120) of E . faecalis and E . faecium from RTE meat products in Poland were resistant to rifampin, respectively with an overall resistance of 19.2% (58/302) of enterococcal isolates (Chajęcka‐Wierzchowska.et al., 2016). A combination of rifampin and linezolid may be more effective than either medication by itself, particularly in infections caused by bacteria that cause biofilms. Rifampin and linezolid work well together due to their capacity to break through biofilms and prevent the creation of new proteins; this combination may also aid in preventing the development of antibiotic resistance (Baldoni et al., 2009). Vancomycin has long been regarded as the preferred treatment because of its effectiveness in treating a wide range of drug-resistant infectious pathogens. VRE in food of animal origin has been linked to avoparcin as a growth promotor due to avoparcin is a vancomycin equivalent that gives cross-resistance to vancomycin (Bager et al., 1997). This study revealed the highest sensitivity rates of 100% (219/219) and 100% (114/114) towards vancomycin in E . faecalis and E . faecium isolates, respectively, with a total susceptibility rate of 100% (333/333) for all enterococcal isolates (Table 3). Comparable with our results, a susceptibility rate of 100% (302/302) was determined for Enterococcal isolates from ready-to-eat meat products toward vancomycin (Chajęcka‐Wierzchowska et al., 2016). Absolute sensitivity (100%) against vancomycin was reported for enterococcal isolates from beef and poultry meat (Milanov et al., 2025), retail meats in Alberta, Canada (Aslam et al., 2012), chicken breast meat samples in Sleman District, Indonesia (Nurrahmat et al., 2025), and fermented meat products in the North of Portugal (Barbosa et al., 2009). In contrast to our findings, high rates of vancomycin resistance among enterococci have been reported elsewhere, including 47.3% of isolates from wild game meat (Guerrero et al., 2016), 75% (12/16) of Enterococcus faecium from meat products (Pavia et al., 2000), and 83.95% (68/81) of E. faecalis isolated from red meat in slaughterhouses in Borujerd, Iran (Madanipour et al., 2017). Similarly, 85.6% (231/270) of enterococcal isolates recovered from ready-to-eat meat products in Egypt were resistant to vancomycin (Sallam et al., 2026). Tetracycline antibiotics, including tetracycline, oxytetracycline, and chlortetracycline, have been widely used in human and veterinary medicine (Johnson and Adams, 1992). One of the most common classes of antibiotics used in agriculture, veterinary medicine, and human therapy is tetracycline. In the present study, Enterococcus spp isolates showed a resistance rate of 68.5% (228/333) toward tetracycline (Table 3). Conversely, lower resistance rates ranging from 14.9% (14/94) to 36.4% (110/302) were reported for enterococcal isolates recovered from different meat products worldwide (Barbosa et al., 2009; Pesavento et al., 2014; Golob et al., 2019; Chajęcka‐Wierzchowska et al., 2016; Milanov et al., 2025; Holman et al., 2021). In the present study, tetracycline resistance was detected in 72.15% (158/219) of E . faecalis and 61.4% (70/114) of E . faecium isolated from ready-to-eat (RTE) meat products (Table 3). In contrast, lower resistance rates for E . faecalis have been reported from red meat in Borujerd, Iran (41.97%; Madanipour et al., 2017), Turkey (53%; Yılmaz et al., 2016), and raw meat in Athens, Georgia, USA (64.9%; McGowan-Spicer et al., 2008). Similarly, lower tetracycline resistance rates were observed among E . faecium isolates from chicken meat (57.6%) and beef meat (0%) in Alberta, Canada (Aslam et al., 2012), Poland (6.7%; Chajęcka-Wierzchowska et al., 2016), and Italy (16.4%; Pesavento et al., 2014). None of the enterococcal isolates (0%; 0/333) were resistant to the 3rd-generation (Levofloxacin), while a low resistance rate of 4.8% (16/333) was reported for the 2nd- generation ciprofloxacin (Table 3). Comparable resistance rates of ciprofloxacin among enterococcal isolates recovered from raw red meat in Georgia, USA (3.5%; 2/57) (McGowan-Spicer et al., 2008), Borujerd, Iran (3.7%) (Madanipour et al., 2017), and Slovenia 1. 4% (2/141) (Golob et al., 2019). Relatively higher resistance rates of 12% (12/100) and 16.5% (30/182) were reported against ciprofloxacin for enterococcal isolates recovered from meat samples tested in Turkey ( Yılmaz et al., 2016) and the North of Portugal (Barbosa et al., 2009), respectively, although much higher resistance rate of 92.7% was reported for enterococcal isolates recovered wild game meat in Spain (Guerrero et al., 2016). Enterococci are intrinsically sensitive to a wide range of aminoglycoside antibiotics (such as gentamicin, GEN) due to inefficient transport across the cytoplasmic membrane (Leclercq, 1997). Thus, aminoglycosides alone are ineffective in the treatment of enterococcal infections, and therefore they are used in combination therapy with inhibitors of cell wall synthesis (such as ampicillin), which facilitate their uptake (Lefort et al., 2000). For severe enterococcal infections like endocarditis, gentamicin, an aminoglycoside antibiotic, is frequently utilized in combination treatment (Chaves & Tadi, 2020). Notably, all E . faecalis isolates (100%; 219/219) were sensitive to gentamicin (Table 3). Likewise, 100% of E . faecalis isolates recovered from retail ground beef tested in Canada (Aslam et al., 2012; Holman et al., 2021) and Poland (Różańska et al., 2015) were sensitive to gentamicin. Nevertheless, resistance rates of 10.9% (16/147) (Chajęcka‐Wierzchowska et al., 2016), 17.5% (10/57) (McGowan-Spicer et al., 2008), and 21.9% (25/114) (Pesavento et al., 2014) were confirmed by E . faecalis isolates from different meat samples against gentamicin. Similar to E . faecalis tested in the present study, 100 % (114/114) of E . faecium were sensitive to gentamicin (Table 3). A 100% gentamicin-sensitive isolates were found among E . faecium isolates in Canada (Aslam et al., 2012) and in Slovenia (Golob et al., 2019). Relatively higher resistance rates of 5% (6/120) (Chajęcka‐Wierzchowska et al., 2016)and 13. 6% (19/140) (Pesavento et al., 2014)were reported among E . faecium isolates recovered from meat products in Poland and Italy, respectively. In 2000, the U.S. Food and Drug Administration (FDA) approved linezolid for the treatment of infections caused by vancomycin-resistant Enterococcus (VRE) (Wang and Hsueh, 2009; Bi et al., 2018), and because of its effectiveness against Gram-positive pathogens, it can be used to treat complex skin and soft tissue infections, community-acquired pneumonia, and nosocomial pneumonia. Among 333 enterococcal isolates of the current surveys, 26/333 (7.81%) showed resistance to linezolid (Table 3). Nonetheless, no resistance to linezolid (LZD) was detected in Enterococcus isolates from meat products in Serbia (Milanov et al., 2025) and Slovenia (Golob et al., 2019). Among 219 E. faecalis isolates, 11.87% (26/219) were resistant to linezolid. This finding is consistent with the results of Różańska et al. (2015), who reported linezolid resistance in 11.4% (4/35) among E. faecalis isolates. However, a markedly higher resistance rate of 86.4% (70/81) was reported by Madanipour et al. (2017). In contrast, lower resistance rates of 6.1% (9/147) and 2.9% (1/34) have been reported by Chajęcka-Wierzchowska et al. (2016) and Martinez-Laorden et al. (2023), respectively, among E. faecalis isolates from meat samples. On the other hand, no (0%) linezolid resistance was detected among E. faecium isolates in the present study, which is consistent with the findings of Martinez-Laorden et al. (2023), who revealed 0% resistance among E. faecium isolates from meat tested in Spain. A low resistance rate of 2.5% (3/120) was reported by Chajęcka-Wierzchowska et al. (2016). Rifampicin is used for the treatment of enterococcal infections. An acquired resistance to rifampicin has been detected in both E. faecium and E. faecalis related to the mutations in the gene encoding the RNA polymerase subunit ( rpoB ) (Kakoullis et al., 2021). The present study revealed rifampicin-resistance rates of 16.44% (36/219) and 10. 53% (12/114) among E . faecalis and E . faecium , respectively, with a total resistance of 14. 41 % (48/333) among all Enterococcus isolates. Higher resistance rates of 19.2% (58/302) (Chajęcka‐Wierzchowska.et al., 2016), 36.4% (Guerrero-Ramos et al., 2016), and 60% (109/182) (Barbosa et al., 2009) were reported for Enterococcus isolates from various red meats towards rifampicin. 3.5. Multiple antimicrobial resistance (MAR) index and classification of Enterococcus isolates based on their antimicrobial resistance pattern The antimicrobial resistance pattern and multiple antibiotic resistance index (MAR) of Enterococcus spp. recovered from oriental meat pies, meat pizza, chicken pizza revealed that 98.5% (328/333) of Enterococcus isolates, including 214 E . faecalis (117 from oriental meat pies, 65 meat pizza, and 32 from chicken pizza), and 114 from E . faecium (51 from oriental meat pies, 20 meat pizza, and 43 from chicken pizza) were classified as multidrug-resistant (MDR) as they showed resistance to more than 2 antibiotics with a MAR value between 0.442 and 0.0.419, respectively (Table 4). Interestingly, among the MDR enterococci analyzed in the current study, 98.5% (328/333) were resistant to at least 4 antimicrobial agents, while 87.39% (291/333) were resistant to at least 6 antibiotics (Table 4). Lower multidrug-resistant (MDR) profiles have also been verified among Enterococcus isolates recovered from various types of raw meat and meat products worldwide, ranging from 15.14% (48/317) to 90.6% (48/53) in Spain (Martínez-Laorden et al., 2023), Indonesia (Nurrahmat et al., 2025), Serbia (Milanov et al., 2025), Bangladesh (Samad et al., 2022), Italy (Vignaroli et al., 2011), and Turkey (Yılmaz et al., 2016). The average MAR index of the 333 Enterococci isolates tested was 0.43, with 95.2% (317/333) of Enterococcus isolates showing a MAR index higher than 0.3 (Table 4). A MAR index exceeding 0.2 indicates antimicrobial abuse, whereas a value exceeding 0.4 generally indicates human-related fecal contamination (Gessew et al., 2022). 3.6. Prevalence and distribution of vancomycin-resistant genes (vanA, vanB), erythromycin-resistant gene ( ermB), and tetracycline-resistant gene (tetL) among Enterococcus isolates In the present study, vanA , vanB , ermB , and tetL genes were identified in isolates resistant to vancomycin, erythromycin, and tetracycline at the predicted molecular sizes of 559, 467, 229, and 639 bp, respectively (Table 1 and Fig 6). Consistent with the phenotypic results of antimicrobial sensitivity testing for vancomycin, vancomycin-resistant genes were not detected in any of the 333 Enterococcus isolates tested in the current study (Table 5). On the other hand, of the216 phenotypically erythromycin-resistant isolates, 142 (65.7%) were positive for the ermB gene, with 63 isolates from oriental meat pies, 60 isolates from meat pizza, and 19 isolates from chicken pizza. Of the 228 phenotypically tetracycline-resistant isolates, 94 (41.23%) were positive for tetL gene, distributed as 34 isolates from oriental meat pies, 42 from meat pizza, and 18 from chicken pizza. Of the 186 (29.03%) phenotypically erythromycin- and tetracycline-resistant isolates, 54 showed coexistence of ermB and tetL genes distributed as 24 isolates from oriental meat pies, 23 from meat pizza, and 7 from chicken pizza. The ermB gene was the most prevalent at 42.6% (142/333), followed by the tetL gene at 28.2% (94/333). It was observed that 41.9% (49/117), 81.5% (53/65), and 24.3% (9/37) of E . faecalis isolates, and 27.5% (14/51), 35% (7/20), and 23.3% (10/43) of E . faecium isolates from oriental meat pies, meat pizza, and chicken pizza, respectively, were positive for the ermB gene (Table 2 and Fig. 7). On the other hand, 17.95% (21/117), 53.9% (35/65), and 0% (0/37) of E . faecalis isolates and 25.49% (13/51), 35% (7/20), and 41.9% (18/43) of E . faecium isolates from the corresponding products harbored tetL genes (Table 2 and Fig. 7). Overall, ermB genes were detected in 50.7% (111/219) of E . faecalis isolates and in 27.2% (31/114) of E . faecium isolates. On the other hand, tetL genes were present in 25.6% (56/219) of E . faecalis isolates and in 33.3% (38/114) of E . faecium isolates from all examined RTE meat products. Previous surveys reported a higher prevalence of vanA and vanB genes. Hosseini et al. (2016) found that the prevalence of vanA in E . faecalis and E . faecium was 93.3% (14/15) and 38.9% (7/18), respectively, while vanB was found in 26.6% (4/15) and 11.1% (2/18) among these species from raw chicken meat. In raw meat, vanA prevalence was 63.9% (23/36) in E . faecalis and 50% (13/26) in E . faecium , whereas vanB was present in 11.1% (4/36) and 0% among these species, respectively. Additionally, Madanipour et al. (2017) reported that 83.95% (68/81) of E . faecalis isolates from red meat in Borujerd, Iran, harbored vanA , 25.9% (21/81) harbored vanB , and 22.2% (18/81) carried both vanA and vanB . Similarly, Yılmaz et al. (2016) found that 4.95% (5/101) of Enterococcus isolates from chicken meat in Turkey carried vancomycin-resistant genes. Conversely, Chajęcka-Wierzchowska et al. (2016) did not detect vanA or vanB genes in Enterococcus isolated from RTE meat products from northeast Poland, nor were these genes found in other studies of ready-to-eat meat products in Brno, Czech Republic (Trivedi et al., 2011). The rise of vancomycin-resistant Enterococcus in food animals is associated with the use of growth-promoting additives like avoparcin and vancomycin analogs, with vanB emerging more recently despite vanA being responsible for most VRE cases worldwide (Telli et al., 2021). As a whole, E . faecalis examined in the present study harbored a greater number of the ermB gene than the Enterococcus isolates. This finding is in agreement with the results reported by Chajęcka‐Wierzchowska et al. (2016), who indicated that the ermB antimicrobial resistance-encoding gene was detected in 52.4% (77/147) of E . faecalis versus 18.3% (22/120) in E . faecium . Also, Macovei & Zurek (2007) reported that 13.6% (3/22) were positive for ermB in E . faecalis versus 0% (0/26) in E . faecium, with an overall prevalence in Enterococcus spp 6.38% (9/141). Moreover, 22.2% (10/45) and 11.1% (5/45) of E . faecium harbored ermB and tetL , respectively, while 71.4 % (5/7) and 42.9% of E . faecalis harbored ermB and ( tetL and tetM ), respectively (Guerrero-Ramos et al., 2016 ). In our study, 71.7% (157/219) of E . faecalis isolates were morphologically resistant to erythromycin, with 70.7% (111/157) among them carrying the ermB gene. In another research, 44.5% (149/335) of E . faecalis were morphologically resistant to erythromycin, with 96% (143/149) of these erythromycin-resistant isolates harboring the ermB gene (kim et al., 2020). On the other hand, 89.3% (133/ 149) of E. faecalis isolates with simultaneous erythromycin and tetracycline resistance carried the tetL gene, and 96.0% (143/149) carried the ermB gene, while 81.2% (121/149) of the isolates simultaneously possessed ermB , tetL , and tetM genes (kim et al., 2020). Conclusion The current study revealed that 76.4% (84/110) of RTE pizza and oriental meat pies examined in Dakahlia province, Egypt, were contaminated with Enterococcus spp. that harbored various virulence genes, such as sodA , gelE , and ace in 100% (333/333), 53.15% (177/333), and 43.24% (144/333) of the isolates, respectively. Interestingly, 98.5% (328/333) of the isolates were multidrug-resistant, suggesting antibiotic overuse. The study highlights the major public health hazards of Enterococcus and the urgent requirement for reliable monitoring methods to control the overuse of antibiotics in veterinary medicine. More research is required to deepen the global understanding of this pathogen and to improve effective methods to eradicate it from RTE meat products, to ensure the protection of public health and safeguard population safety. Declarations CRediT authorship contribution statement Amira Mahmoud Elsayeh Conceptualization, Formal analysis, Methodology, and Writing – original draft. Amira Ibrahim Zakaria Supervision, Validation and Writing – original draft. Samir Abd-Elghany , Supervision, Data curation, investigation. Khalid Ibrahim Sallam Conceptualization, Supervision, and Writing – review & editing. Funding: Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Open access funding is provided by the Science, Technology & Innovation Funding Authority (STDF) in cooperation with the Egyptian Knowledge Bank (EKB). Data availability All data supporting the findings of this study are included within the article. No datasets were generated or analyzed during the current study. Declaration of Competing Interest The authors have declared that there is no conflict of interest. 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Supplementary Files Table1primers.docx Table2Distributionofvirulenceresistantcegenes.docx Table3Antibioticresistanceprofile.docx Table4classificationofantibioticMARindex.docx table5Associationofvirulentwithresistantgenes.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 19 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 05 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor invited by journal 02 Apr, 2026 Editor assigned by journal 30 Mar, 2026 Submission checks completed at journal 30 Mar, 2026 First submitted to journal 29 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9259480","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":626059575,"identity":"1c0a6d37-7027-4c5b-ae05-b1e9f6c85073","order_by":0,"name":"Amira Mahmoud Elsayeh","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Amira","middleName":"Mahmoud","lastName":"Elsayeh","suffix":""},{"id":626059576,"identity":"0aed4d38-a28a-4d78-812e-161679602a40","order_by":1,"name":"Amira Ibrahim Zakaria","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Amira","middleName":"Ibrahim","lastName":"Zakaria","suffix":""},{"id":626059577,"identity":"31d8842d-eaa4-4290-b2b6-dd42f325e494","order_by":2,"name":"Samir Mohammed Abd El-Ghany","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Samir","middleName":"Mohammed Abd","lastName":"El-Ghany","suffix":""},{"id":626059578,"identity":"fe6f356f-6a97-45eb-aa91-6920f457b9df","order_by":3,"name":"Khalid Ibrahim Sallam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYBACCQkGxgMJbAwMfOxA3geYKAEtDGAtbMwMDIwziNbCANXCzEOMFsnZzQ8OPCirk2djZn662TbHLk++gfngbR4GG7sGHFqkZY4ZHEg4d9iwjZnN7HbutuRigwNsydY8DGnJuLTISSQYHEhsO8DYxswA0sKcuIGBx0yah+FwMi6HyUmkfwBqqbNvY2b/dttyW33i/Ab+b0At/3FqkZbIAdnCDEQ8ZrcZtx1ObDjAwwbUcsAOp/dn5BSA/JIM1FJ2s3fb8cQNh9mMLecYJCfg0iJxI33jwx9ldbb97O3bbvzcVp04v7354Y03FXb2uLRgAcwgwoAhsYEEPRBAii2jYBSMglEwvAEA1yNVswxOPegAAAAASUVORK5CYII=","orcid":"","institution":"Mansoura University","correspondingAuthor":true,"prefix":"","firstName":"Khalid","middleName":"Ibrahim","lastName":"Sallam","suffix":""}],"badges":[],"createdAt":"2026-03-29 14:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9259480/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9259480/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107913674,"identity":"6d8a7ba8-2706-499d-8256-5d872197e7c5","added_by":"auto","created_at":"2026-04-27 13:51:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":641949,"visible":true,"origin":"","legend":"\u003cp\u003eDemonstrative agarose gel electrophoresis for PCR showed the amplified bands of the \u003cem\u003eSodA\u003c/em\u003e (\u003cem\u003eEnterococcus faecalis\u003c/em\u003e) and \u003cem\u003eSodA\u003c/em\u003e (\u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecium\u003c/em\u003e) genes (Lanes 1–17) were detected at the anticipated molecular size of 360 bp specific for \u003cem\u003eEnterococcus\u003c/em\u003efaecalis (A) and 215 bp specific for \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecium\u003c/em\u003eisolates recovered from RTE meat products tested. M stands for DNA marker (1200 bp gene ladder). C+: Control positive [strains of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003eATCC 29212 (A) and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e ATCC 19434 (B)], C–: Control negative (strains of \u003cem\u003eEscherichia\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e K-12 DH5α). Electrophoresis on a 1.5% agarose gel was used to separate eight microliters of the PCR product, which was detectable under a UV lamp.\u003c/p\u003e","description":"","filename":"Figure1OKSodA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/c8495cc42d4c8f8ccd3c1a25.jpg"},{"id":108007280,"identity":"265040f7-623e-48ba-9dc2-44010b1eb523","added_by":"auto","created_at":"2026-04-28 12:59:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":479484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecium\u003c/em\u003e determination in the RTE meat product tested, indicating the proportion of positive samples for each or both bacteria separately.\u003c/p\u003e","description":"","filename":"Figure2PrevalenceofEnterococcus.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/6e1f9d3ed1dd16a9a230b057.jpg"},{"id":108007264,"identity":"c1e45401-26fe-460e-a991-74a0aca9603b","added_by":"auto","created_at":"2026-04-28 12:59:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":586118,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecalis\u003c/em\u003e (n = 219) and \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecium\u003c/em\u003e (n = 114) among RTE meat products tested.\u003c/p\u003e","description":"","filename":"Figure3DistributionofEnterococcusamongmeatproducts.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/d287ff29412e3ed9368f0d0f.jpg"},{"id":107913682,"identity":"dc04a4e5-9b0c-4b54-8c19-477643cbbe25","added_by":"auto","created_at":"2026-04-27 13:51:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":545281,"visible":true,"origin":"","legend":"\u003cp\u003eDemonstrative agarose gel electrophoresis for PCR products showed the amplified bands of the virulent genes \u003cem\u003egelE\u003c/em\u003e (A) and \u003cem\u003eace\u003c/em\u003e(B) in \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecalis\u003c/em\u003e (Lanes 1–8) and \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecium\u003c/em\u003e(Lanes 9–16) isolates from RTE meat products tested. The amplified bands were found at the anticipated molecular sizes of 616 bp for the \u003cem\u003eace\u003c/em\u003e gene (B) and 419 bp for the \u003cem\u003egelE\u003c/em\u003e gene (A). M stands for DNA marker (1200 bp gene ladder). C+*: \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e ATCC 29212 strain; C–: \u003cem\u003eEscherichia\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e K-12 DH5α control negative. Electrophoresis on a 1.5% agarose gel was used to separate eight microliters of the PCR result, which was then visible under a UV lamp.\u003c/p\u003e","description":"","filename":"Figure4gelEAceOk.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/82787a385c179fc03df18f10.jpg"},{"id":109081020,"identity":"47ee65e4-f69d-442e-a0e7-72a87d36df5f","added_by":"auto","created_at":"2026-05-12 11:52:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":499963,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution and frequency of the virulent genes (\u003cem\u003eace\u003c/em\u003e, \u003cem\u003egelE\u003c/em\u003e, and \u003cem\u003esodA\u003c/em\u003e) in the 333 \u003cem\u003eEnterococcus\u003c/em\u003espp. isolates recovered from the RTE meat products tested.\u003c/p\u003e","description":"","filename":"Figure5Virulencegenesdistribution.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/628371da408036ccfd8439d4.jpg"},{"id":108006504,"identity":"ef3b7b0b-6a05-4db5-879d-996230a1e548","added_by":"auto","created_at":"2026-04-28 12:55:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":323378,"visible":true,"origin":"","legend":"\u003cp\u003eThe amplified bands of the tetracycline-resistant \u003cem\u003etetL\u003c/em\u003eand erythromycin-resistant \u003cem\u003eermB\u003c/em\u003e in \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecalis\u003c/em\u003e(Lanes 1–8) and \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecium\u003c/em\u003e (Lanes 9–16) isolates isolated from RTE meat products tested were shown by representative Agarose Gel Electrophoresis for PCR. The amplified bands were found at the anticipated molecular sizes of 229 bp for the \u003cem\u003etetL\u003c/em\u003e and 639 bp for the \u003cem\u003eermB\u003c/em\u003e. M stands for DNA marker (1200 bp gene ladder). C+: \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e ATCC 29212 strain control positive; C–: \u003cem\u003eEscherichia\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e K-12 DH5α control negative. Electrophoresis on a 1.5% agarose gel was used to separate eight microliters of the PCR result, which was then visible under a UV lamp.\u003c/p\u003e","description":"","filename":"Figure6tetLermBOK.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/e22e6ab51fc157cd6c5d4dcb.jpg"},{"id":108181180,"identity":"9cfeb750-7220-4999-85fe-37e2f93fd5d9","added_by":"auto","created_at":"2026-04-30 08:58:15","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":636373,"visible":true,"origin":"","legend":"\u003cp\u003eTetracycline-resistant \u003cem\u003etetL\u003c/em\u003e and erythromycin-resistant \u003cem\u003eermB\u003c/em\u003e prevalence and distribution among \u003cem\u003eEnterococcus\u003c/em\u003espp. isolates (n = 333) recovered from the RTE meat products under investigation.\u003c/p\u003e","description":"","filename":"Figure7ermBtetLdistributiongenes.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/31d7c8527bb109b1afc892bd.jpg"},{"id":109204527,"identity":"8a1c0816-8bd0-491a-b0d6-3639db9f34d5","added_by":"auto","created_at":"2026-05-13 15:00:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4203703,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/f4c0fb4a-8006-499c-8bf5-b1e954ec4505.pdf"},{"id":107913677,"identity":"d62c86a4-859a-4a90-83ab-83bed7d08ac2","added_by":"auto","created_at":"2026-04-27 13:51:11","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19678,"visible":true,"origin":"","legend":"","description":"","filename":"Table1primers.docx","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/93426d6c1a5543c0fb0e820a.docx"},{"id":108006572,"identity":"dc323117-5c28-4313-b201-ba091e8133a7","added_by":"auto","created_at":"2026-04-28 12:56:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18569,"visible":true,"origin":"","legend":"","description":"","filename":"Table2Distributionofvirulenceresistantcegenes.docx","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/6273a8007e9f85f348b1e4f9.docx"},{"id":107913675,"identity":"68bb31d6-0bc2-4f18-9081-c8c0aef31661","added_by":"auto","created_at":"2026-04-27 13:51:11","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28473,"visible":true,"origin":"","legend":"","description":"","filename":"Table3Antibioticresistanceprofile.docx","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/3ed13b7bb5f542b5a4ff1f8e.docx"},{"id":108006796,"identity":"73faad70-96f5-4efb-95fa-6201a7c6cd61","added_by":"auto","created_at":"2026-04-28 12:57:17","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":22153,"visible":true,"origin":"","legend":"","description":"","filename":"Table4classificationofantibioticMARindex.docx","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/534ffd735dde8569fbac6284.docx"},{"id":107913680,"identity":"e987b3cb-a649-4f16-a652-ef49268ff877","added_by":"auto","created_at":"2026-04-27 13:51:11","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":28349,"visible":true,"origin":"","legend":"","description":"","filename":"table5Associationofvirulentwithresistantgenes.docx","url":"https://assets-eu.researchsquare.com/files/rs-9259480/v1/b22a8fdeb7bd2d05ed85c3ad.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multidrug-resistant Enterococcus faecalis and Enterococcus faecium isolated from oriental meat pies and beef- and chicken-based pizzas","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent decades, the demand for fast meals has increased substantially, making them a prominent feature of modern eating habits. Many people rely on these meals due to limited time available for home cooking and the convenience fast food offers. Among the most popular fast meals is pizza, which is enjoyed worldwide for its rich flavor, delicious taste, and easy availability. Approximately 13% of Americans consume pizza on any given day, highlighting its popularity in the USA (Rhodes et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although the precise per capita pizza consumption figures for Egypt are not easily accessible, the Egyptian pizza market exhibits robust growth, with a market growing by nearly 18% in 2023 alone, reflecting increased consumption combined with booming online meal delivery, particularly for fast food. Pizza provides various nutrients, including carbohydrates from the crust, proteins from meat, and fats that supply energy. Although pizza provides several essential nutrients, it can also be high in saturated fats, salt, and calories, which may contribute to public health issues.\u003c/p\u003e \u003cp\u003ePizza frequently needs temperature regulation for safety when kept in restaurants (Xu and Schaffner, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). If not prepared or stored properly, pizza may be contaminated with pathogenic bacteria that cause foodborne illnesses, posing further health risks. According to the US CDC's National Outbreak Reporting System (NORS), most outbreaks in the United States have been associated with pizza (Centers for Disease Control and Prevention, 2021).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnterococcus\u003c/em\u003e species are typically inhabitants of the human digestive tract as commensal bacteria. If the commensal relationship between enterococci and their host is broken, enterococci can turn into opportunistic pathogens and cause invasive diseases (Hamza and Kadem, 2018). They can also be found in the environment in water, soil, and animal-based food. The presence of \u003cem\u003eEnterococcus\u003c/em\u003e spp. in food is considered an indicator of fecal contamination or poor hygiene during processing or storage.\u003c/p\u003e \u003cp\u003eThe most prevalent \u003cem\u003eEnterococcus\u003c/em\u003e spp are \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e, which are the main cause of human infections, including septicemia, urinary tract infections, endocarditis, neonatal sepsis, meningitis, and wound infections (Kafil and Asgharzadeh, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e strains are the most predominant nosocomial opportunistic pathogens responsible for approximately 10\u0026ndash;15% and 80\u0026ndash;90% of infections, respectively (El Zowalaty et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Testing for Enterococcus species is not part of the routine management of the production and distribution of food of animal origin. Unlike coliform bacteria and E. coli, their number and concentration are not restricted (Milanove et al., 2025). \u003cem\u003eEnterococcus\u003c/em\u003e spp comprises virulence genes, such as Gelatinase (\u003cem\u003egelE\u003c/em\u003e), which hydrolyze gelatin, and Accessory colonization factors (\u003cem\u003eace\u003c/em\u003e), which are capable of colonization by binding to proteins of the extracellular matrix, as well as sharing in binding type I and IV collagen (Wioleta et al., 2016).\u003c/p\u003e \u003cp\u003eGlobally, antimicrobial resistance has grown to be a serious concern to both human and animal health, making it more difficult to treat some diseases with traditional antibiotics. Antimicrobials are typically needed by food-producing animals as a preventative measure or treatment for a variety of bacterial illnesses. Multidrug-resistant bacterial strains have emerged as a result of the global overuse and abuse of antibiotics. These strains spread through animal-based foods and pose a serious risk to human health. These days, one of the biggest problems in treating infections in humans and animals is the accelerated creation of bacterial diseases that are resistant to drugs (Sallam et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEnterococci are sentinel bacteria for monitoring antibiotic resistance because of their individuality and their widespread behavior (Smoglica et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). AMR is exacerbated by the direct transfer of resistant bacteria from animals to humans, especially from food-producing animals (Jans et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Antimicrobial resistance (AMR) is the main and increasing public health concern, both in human and veterinary medicine, affected by reducing antibiotic effectiveness and delaying bacterial infection treatment (Islam et al., 2023). Antimicrobial-resistant \u003cem\u003eEnterococcus\u003c/em\u003e strains were elevated throughout time (Carlet et al., 2012); due to the high prevalence of resistance and virulence characteristics, \u003cem\u003eEnterococcal\u003c/em\u003e spp danger evaluations are difficult (EFSA, 2008). Because \u003cem\u003eEnterococcal\u003c/em\u003e organisms inherently resist several antibiotic classes, including cephalosporins, aminoglycosides, macrolides, and sulfonamides, managing these infections can be difficult (Hollenbeck and Rice, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To acknowledge the various components, including human, animal, and environmental factors that contribute to the rising levels of AMR, it is necessary to embrace a comprehensive \"One Health\" viewpoint (Islam et al., 2023).\u003c/p\u003e \u003cp\u003eNo research has been conducted on the prevalence of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e in Oriental meat pies, as well as in meat- and chicken-based pizza in Egypt. Therefore, this study aimed to determine the prevalence of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e strains in oriental meat pies and various types of pizza from restaurants in Dakahlia Province, Egypt, and to examine the presence of virulence genes (\u003cem\u003esodA faecalis\u003c/em\u003e, \u003cem\u003esodA faecium\u003c/em\u003e, \u003cem\u003eAce\u003c/em\u003e, and \u003cem\u003egelE\u003c/em\u003e) and some resistance genes, including \u003cem\u003etetL\u003c/em\u003e, \u003cem\u003eermB\u003c/em\u003e, \u003cem\u003evanA\u003c/em\u003e, and \u003cem\u003evanB\u003c/em\u003e, as well as the antimicrobial resistance profile of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e strains against 16 antibiotics from different classes.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cem\u003e2.1 Sample collection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of one hundred ten random samples of locally produced meat products, including 30 meat pizzas, 30 chicken pizzas, and 50 oriental meat pies, were purchased from various restaurants in Mansoura City, Egypt, between February 2024 and November 2024. The samples were individually packed in sterile plastic bags and promptly transported in an ice box under strictly aseptic conditions to the laboratory of Food Hygiene, Safety, \u0026amp; Technology Department, Faculty of Veterinary Medicine, Mansoura University, where bacteriological analysis was conducted immediately to detect the presence of \u003cem\u003eEnterococcus\u003c/em\u003e spp.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2 Isolation and identification of Enterococcus spp\u003c/em\u003e\u003cem\u003e\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 25 grams from each sample was aseptically excised using a sterile scalpel and homogenized in a sterile laboratory stomacher (Seward Laboratory, London, UK) containing 225 ml of buffered peptone water (Oxoid CM0509). The homogenized samples were then incubated at 37 \u0026deg;C for 24 h. Following this, 10-fold Serial dilutions were prepared in sterile test tubes. From the selected dilutions (10\u003csup\u003e\u0026minus;3\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;4\u003c/sup\u003e), 0.1 mL was spread onto Bile Esculin Agar (M493I; HiMedia, India). The inoculated plates were incubated at 37 \u0026deg;C for 24 h to obtain pure culture colonies. Presumptive Enterococcus colonies on Bile Esculin Azide agar plates appear as small, translucent or slightly opaque colonies surrounded by dark brown, indicating esculin hydrolysis. Morphological examination of the suspected colonies was performed using Gram staining, along with biochemical identification of the isolates through catalase activity, oxidase test, carbohydrate fermentation (mannitol and sucrose), bile esculin test, and detection of hemolysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3. Preparation of Genomic\u003c/em\u003e\u003cem\u003e DNA\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 333 typical \u003cem\u003eEnterococcus\u003c/em\u003e colonies were subjected to genomic DNA extraction using the Gene JET Genomic DNA Purification Kits (Roche Applied Science, Germany, Cat. No. 11 796 828 001) following the manufacturer\u0026apos;s instructions. Genomic DNA was also isolated from \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e ATCC 29212 and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e ATCC 19434 as positive control strains, while genomic DNA was extracted from the \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e K-12 DH5\u0026alpha; strain as a negative control. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4. Molecular confirmation and characterization of Enterococcus spp.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4.1. Molecular differentiation of Enterococcus spp.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 333 phenotypically and biochemically verified \u003cem\u003eEnterococcus\u003c/em\u003e isolates, recovered from examined samples, were molecularly confirmed using the polymerase chain reaction assays (PCR), based on the presence of \u003cem\u003esodA \u003c/em\u003e(\u003cem\u003eE. faecalis\u003c/em\u003e) and \u003cem\u003esodA \u003c/em\u003e(\u003cem\u003eE. faecium\u003c/em\u003e) marker genes using their specific primer sets according to Jackson et al. (2004) at the PCR conditions mentioned in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4.2. PCR detection of gelE and ace virulent genes in Enterococcus isolates\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe differentiated \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates obtained from examined RTE meat product samples were tested for the presence of the gelatinase (\u003cem\u003egelE\u003c/em\u003e) and the collagen-binding proteins (\u003cem\u003eace\u003c/em\u003e) virulent genes, using PCR-specific primer sets for the \u003cem\u003egelE \u003c/em\u003egene (Eaton and Gasson, 2001) and the \u003cem\u003eace\u003c/em\u003e gene (Creti et al., 2004), following the cycling conditions outlined in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4.3. PCR detection of \u003c/em\u003e\u003cem\u003evancomycin-resistant genes (vanA, vanB), erythromycin-resistant gene (\u003c/em\u003e\u003cem\u003eermB), and tetracycline-resistant gene (tetL) among Enterococcus isolates\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eEnterococcus\u003c/em\u003e isolates that showed phenotypic resistance to erythromycin and tetracycline by the disk diffusion method were further examined using PCR assays for the detection of the \u003cem\u003etetL\u003c/em\u003e (Malhotra-Kumar et al., 2005) and the \u003cem\u003eermB\u003c/em\u003e (Knysz et al., 2025)resistance genes. At the same time, the \u003cem\u003eEnterococcus\u003c/em\u003e isolates were also tested using PCR for the possible detection of \u003cem\u003eVan A\u003c/em\u003e and \u003cem\u003eVan B \u003c/em\u003egenes. Primer sets for the detection of antimicrobial-resistant genes are listed in Table 1.\u003c/p\u003e\n\u003cp\u003ePCR was performed using SimpliAmp thermal cycler (Thermo Fisher Scientific Inc.) in a 25 final reaction volume, the amplification of marker, virulence, and resistant genes was performed using the following component: 12.5 \u0026mu;L of DreamTaq TM Green Master Mix (2X) (Fermentas, Inc., Hanover, MD, USA), 1.0 \u0026mu;L from each of sense and antisense primer (10 pmol each) specific for \u003cem\u003esodA\u003c/em\u003e (\u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e), \u003cem\u003esodA\u003c/em\u003e (\u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e), \u003cem\u003eermB\u003c/em\u003e, \u003cem\u003etetL\u003c/em\u003e, \u003cem\u003evanA\u003c/em\u003e, or \u003cem\u003evanB \u003c/em\u003e (Sigma-Aldrich, Co., St. Louis, MO, USA), 2 \u0026mu;L of the genomic DNA template, and 8.5 \u0026mu;L of DNase/RNase-free water. Each PCR test included both positive and negative controls.\u003c/p\u003e\n\u003cp\u003eThe resulting PCR product (8 \u0026mu;L aliquots from each reaction mixture) was separated within the agarose gel (1.5%) electrophoresis at 100 V for 55 min using 1X TBE running buffer and stained with ethidium bromide solution (10 \u0026mu;g/ml) (Sigma-Aldrich, Co., St. Louis, MO, USA). The separated PCR products were then visualized and photographed using an ultraviolet transilluminator. The GeneRuler 100 bp DNA Ladder (Thermo Fisher Scientific Inc.) was utilized as a molecular size marker in order to ascertain the molecular size of the PCR products.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5 Antimicrobial \u003c/em\u003e\u003cem\u003eresistance profile of isolated Enterococcus spp.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial resistance profile of \u003cem\u003eEnterococcus\u003c/em\u003e isolates (n = 333) was determined using the Kirby-Bauer disc diffusion technique on Mueller\u0026ndash;Hinton agar (MH; CM0337; Oxoid Ltd., Basingstoke, UK) against 16 antimicrobials, which comprised 7 antimicrobial classes. Vancomycin, on the other hand, was tested using the agar dilution method. Enterococci with a MIC breakpoint of 4 \u0026micro;g/mL are identified susceptible, 8\u0026ndash;16 \u0026micro;g/mL are identified intermediate, and \u0026ge; 32 \u0026micro;g/mL are identified vancomycin-resistant \u003cem\u003eEnterococcus\u003c/em\u003e (VRE). The variable concentration of antibiotics used are Penicillin (10 \u0026mu;g, P 10), Amoxicillin \u0026ndash;Clavulanic aid (30 \u0026mu;g, AMC 15), Erythromycin (15 \u0026mu;g, E 15), Gentamicin (10 \u0026mu;g, GM 10), tetracycline (30 \u0026mu;g, TE 30), Ciprofloxacin5 \u0026mu;g, CIP 5), Levofloxacin (15 \u0026mu;g, LEV 15), Vancomycin (30 \u0026mu;g, VA 30), Cefepim (30 \u0026mu;g, FEB 30), Nitrofurantion (300 \u0026mu;g, F 300), Linezolid (30 \u0026mu;g, LNZ 30), Clindamycin (10 \u0026mu;g, DA10), Kanamycin(10 \u0026mu;g, K 10), Rifampin (5 \u0026mu;g, RA 10), Meropenem (10 \u0026mu;g, MEM 10), and Doxycycline (30 \u0026mu;g, DO 30). Results were interpreted according to the standard breakpoints for inhibition zone provided by CLSI (2020). The strains tested were categorized as susceptible, intermediate, and resistant. The multiple antimicrobial resistance (MAR) index for each \u003cem\u003eEnterococcus\u003c/em\u003e strain was calculated according to Singh et al. (2010) as the ratio of the number of antibiotics to which the isolate exhibited resistance to the total number of antibiotics tested.\u003c/p\u003e"},{"header":"3. Results and discussion ","content":"\u003cp\u003e\u003cem\u003e3.1. \u003c/em\u003e\u003cem\u003ePrevalence of Enterococcus \u003c/em\u003e\u003cem\u003especies \u003c/em\u003e\u003cem\u003ein \u003c/em\u003e\u003cem\u003eready-to-eat \u003c/em\u003e\u003cem\u003esamples tested\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEpidemiological studies reported that food of animal origin, particularly undercooked poultry and red meat, are the most common sources of human disease caused by foodborne microorganisms (Khudhair and Kadium, 2025; Sharafi and Nateghi, 2020). The rising global demand for RTE pizza and pies prompts scientists to evaluate their microbiological quality, particularly for foodborne pathogens like \u003cem\u003eStaphylococcus aureus \u003c/em\u003e(Elsalkh et al., 2025), \u003cem\u003eSalmonella \u003c/em\u003e(Raza et al., 2021), and \u003cem\u003eEscherichia coli\u003c/em\u003e O157:H7 (Gieraltowski, 2017). In the present approach, the contamination levels with \u003cem\u003eEnterococcus\u003c/em\u003e spp., which serve as important indicators of fecal contamination, were examined in 110 samples of RTE pies and pizza samples. The results indicated that 76.4% (84/110) of ready-to-eat samples, distributed as 84% (42/50) of oriental meat pies, 73.3% (22/30) of meat pizza, and 66.7% (20/30) of chicken pizza were contaminated with \u003cem\u003eEnterococcus \u003c/em\u003e(Figure 2). \u003c/p\u003e\n\u003cp\u003eSimilar to our findings, Chajęcka‐Wierzchowska et al. (2016) reported an incidence rate of 74.1% (289/390) for \u003cem\u003eEnterococcus\u003c/em\u003e spp. in ready-to-eat meat products tested in Poland. In contrast, lower prevalence rates were recorded in other regions; for instance, 61.7 % (129/209) in meat samples from Spain (Martinez-Laorden et al., 2023), 35 % (21/60) in chicken meat samples from Bangladesh (Samad et al., 2022), 60 % (15/25) in chicken samples from Egypt (El-Hamid et al., 2016), and 61.8 % in meat products from Iran (Hosseini et al., 2016).\u003cem\u003e Enterococci\u003c/em\u003e are often isolated from food-producing animals that cause hazards to consumers (Tyson et al., 2017), and their presence reflects fecal contamination of meat products during slaughter and carcass preparation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2. Frequency distribution of Enterococcus isolates (n = 333) among Oriental meat pies, meat pizza, and chicken pizza \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe biochemically identified \u003cem\u003eEnterococcus\u003c/em\u003e isolates (n = 333), including 168 from oriental meat pies, 85 from meat pizza, and 80 from chicken pizza, were further confirmed using PCR targeting \u003cem\u003esodA \u003c/em\u003e(\u003cem\u003efaecalis\u003c/em\u003e)\u003cem\u003e and sodA \u003c/em\u003e(\u003cem\u003efaecium\u003c/em\u003e)\u003cem\u003e \u003c/em\u003especific marker genes to differentiate \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e, which were detected at the expected molecular size of 360 bp (Figure 1A) and 215 bp (Figure 1B), respectively. \u003c/p\u003e\n\u003cp\u003eIndividually on the sample bases, 58% (29/50), 56.7% (17/30), and 30% (9/30) of the examined oriental meat pies, meat pizza, and chicken pizza samples, with an overall prevalence rate of 50% (55/110), were positive for\u003cem\u003e E. faecalis \u003c/em\u003e(Figure 2). On the \u003cem\u003eEnterococcus\u003c/em\u003e isolate bases, 117 (69.6%), 65 (76.5%), and 37 (46.3%) of \u003cem\u003eE. faecalis\u003c/em\u003e isolates were recovered from oriental meat pies, meat pizza, and chicken pizza samples, respectively, with an overall incidence of 219 (65.8%) (Figure 3). \u003c/p\u003e\n\u003cp\u003eOn the other hand, 26% (13/50), 16.7% (5/30), and 36.7% (11/30) of the examined oriental meat pies, meat pizza, and chicken pizza samples, with an overall prevalence rate of 26.36% (29/110), were positive for \u003cem\u003eE. faecium \u003c/em\u003e(Figure 2). At the isolate level, however, 51 (30.4%), 20 (23.5%), and 43 (53.8%) of \u003cem\u003eE. faecium\u003c/em\u003e isolates were recovered from Oriental meat pies, meat pizza, and chicken pizza samples, respectively, for an overall prevalence of 114 (34.2%) (Figure 3). \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. faecalis \u003c/em\u003eis a zoonotic enteric bacterium that may cause severe problems in humans and animals, including GIT infections, urinary tract infections, periodontitis, meningitis, septicemia, peritonitis, and endocarditis (Said, 2022). \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e is also a zoonotic enteric bacterium of major clinical and public health importance, and in many circumstances, it is considered more problematic than \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e due to its high level of antimicrobial resistance. In humans and animals, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e is associated with a wide range of infections, particularly in healthcare and foodborne exposure settings (Arias and Murray, 2012; Lebreton et al., 2014).\u003c/p\u003e\n\u003cp\u003eOne of the main causes of infections contracted in hospital settings is \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e, which poses serious hazards, especially to those with weakened immune systems (Asfaw, 2019). Poor hygiene is the main way that \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e infections spread from person to person. Food modification and poor cleanliness can allow the germs to infect food (Hanchi et al., 2018).\u003c/p\u003e\n\u003cp\u003eThe distribution and predominance of\u003cem\u003e E\u003c/em\u003e. \u003cem\u003efaecalis \u003c/em\u003eobserved in the present study are consistent with findings from various other publications. For instance, N\u0026uuml;esch-Inderbinen et al. (2023) reported that \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis \u003c/em\u003ewas the most dominant species, found in 28.8% (17/59) of \u003cem\u003eEnterococcus\u003c/em\u003e isolates collected from meat samples in Switzerland, followed by \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e at 6.78% (4/59). Similarly, Samad et al. (2022) indicated that \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e was the most common species, with a prevalence of 20% (12/60), while \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium \u003c/em\u003ewas present in 10% (6/60) of the isolates obtained from chicken meat tested in Bangladesh, whereas\u003cem\u003e \u003c/em\u003eChajęcka‐Wierzchowska et al. (2016) reported that \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e was the most common species, with a prevalence of 37.7% (147/390), while \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium \u003c/em\u003ewas found in 30.8% (120/390) among the isolates obtained from ready-to-eat meat products in Poland. Also, Milanov et al. (2025) revealed that \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e was present in 52% of beef and 61.9% in poultry samples, while \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e was detected 24% of beef and 33.3% of poultry meat samples. Additionally, Martinez-Laorden et al. (2023) indicated that \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e was the most dominant species (46.4%; 147/317), followed by \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e (22.7%; 72/317), among \u003cem\u003eEnterococcus\u003c/em\u003e isolates collected from various meat samples in Spain. Furthermore, Hosseini et al. (2016) reported that 51 (48.8%) and 44 (41.9%) of meat products examined in Iran were positive for \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e, respectively. \u003c/p\u003e\n\u003cp\u003eThe prevalence rate of 65.8% (219/333) determined for \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e among the \u003cem\u003eEnterococcus\u003c/em\u003e isolates in the current study is higher than that reported by Barbosa et al. (2009),who indicated that\u003cem\u003e \u003c/em\u003e41.8% (76/182) of the\u003cem\u003e Enterococcus \u003c/em\u003eisolates recovered from fermented meat products in Portugal were verified as \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e. A lower prevalence rate of 35% (35/100) was reported for \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e among \u003cem\u003eEnterococci \u003c/em\u003erecovered from chicken samples in Zanjan, Iran (Afshar et al, 2023). \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecalis \u003c/em\u003ealso\u003cem\u003e \u003c/em\u003edemonstrated a low prevalence rate of 46.4% meat samples in Spain (Martinez-Laorden et al., 2023), as well as a low rate of 44.3% (31/70) from beef samples in Italy (Pesavento et al., 2014). Higher prevalence rates of 69.5% were reported for \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e among \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from red meat samples in Slovenia (Golob et al., 2019), as well as in 94% and 73% of \u003cem\u003eEnterococcus \u003c/em\u003eisolates recovered from poultry meat and beef samples tested in Canada, respectively (Aslam et al., 2012). \u003c/p\u003e\n\u003cp\u003eThe predominance rate of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium \u003c/em\u003eamong\u003cem\u003e \u003c/em\u003eidentified \u003cem\u003eEnterococcus\u003c/em\u003e isolates in this study was 34.2% (114/333), which is nearly consistent with that reported by Pesavento et al. (2014), who documented predominance rates of 35.7% (25/68) in beef samples and 36.7% (25 out of 68) in poultry samples from Italy. A higher predominance rate of 44.8% (14/38) was reported among \u003cem\u003eEnterococcus\u003c/em\u003e isolates from fermented sausage tested in Germany (Jahan et al., 2013). Likewise, a higher predominance rate of 65% (65/100) was reported among \u003cem\u003eEnterococcus\u003c/em\u003e isolates from chicken samples in Iran (Afshar et al., 2022). Conversely, lower prevalence rates of 22.7% and 24.18% (44/182) have been reported among \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from meat samples in Spain (Mart\u0026iacute;nez-Laorden et al., 2023) and from traditional fermented meat products in Portugal (Barbosa et al., 2009), respectively.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e3.3. Prevalence of virulent genes among Enterococcus species recovered from oriental meat pies and pizza samples\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe pathogenesis of enterococci is associated with a variety of virulence genes that facilitate the colonization and invasion of host tissues. This process leads to an increase in enterococcal infections, as these genes enable the release of toxins and enzymes outside of the cells, adversely affecting the host\u0026apos;s immune response and worsening the condition (Cebeci, 2024). The primary adhesion factors, associated with biofilm formation, and other related virulence components include \u003cem\u003eEbp\u003c/em\u003e (endocarditis and biofilm-associated pili), \u003cem\u003eAsa \u003c/em\u003e(aggregation substance), \u003cem\u003eEsp\u003c/em\u003e (extracellular surface protein), \u003cem\u003eEfaA\u003c/em\u003e (\u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e antigen A), \u003cem\u003eAce\u003c/em\u003e (collagen-binding cell wall protein), \u003cem\u003ecad1\u003c/em\u003e (pheromone cAD1 precursor lipoprotein), \u003cem\u003ecylA\u003c/em\u003e (hemolysin), \u003cem\u003eefm\u003c/em\u003e (\u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e-specific cell wall adhesin), \u003cem\u003esagA\u003c/em\u003e (secreted antigen), (secreted antigen), \u003cem\u003eagg\u003c/em\u003e (aggregation substance), \u003cem\u003egelE\u003c/em\u003e (gelatinase), and \u003cem\u003ecpd1\u003c/em\u003e (pheromone cPD1 lipoprotein) (Alzahrani et al., 2022). Furthermore, certain virulent genes and toxins linked to \u003cem\u003eEnterococcus\u003c/em\u003e spp increase the severity and pathogenicity of diseases they can spread (Goh et al., 2017). The distribution and prevalence of virulence genes (\u003cem\u003esodA\u003c/em\u003e, \u003cem\u003egelE\u003c/em\u003e, and \u003cem\u003eace\u003c/em\u003e) among the 333 \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from RTE oriental meat pie, meat pizza, and chicken pizza tested in the current study are shown in Figure 5 and Table 2. The \u003cem\u003esodA\u003c/em\u003e gene was detected in all isolates (100%) from the different RTE samples tested. The \u003cem\u003esodA\u003c/em\u003e gene, as a key marker gene that differentiates \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium,\u003c/em\u003e was also detected in 100% of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e in many other studies. PCR was employed to differentiate between the virulence of \u003cem\u003eEnterococcus\u003c/em\u003e isolates by targeting \u003cem\u003egelE,\u003c/em\u003e which was detected at 419bp (Figure 4A), and the \u003cem\u003eace\u003c/em\u003e gene, which was detected at 616 bp (Figure 4B).\u003c/p\u003e\n\u003cp\u003eThe matrix metalloproteinase gelatinase, encoded by \u003cem\u003egelE\u003c/em\u003e, hydrolyzes collagen and gelatin, which interfere with complement-mediated immunity and may contribute to the development of infectious endocarditis caused by \u003cem\u003eEnterococcus\u003c/em\u003e \u003cem\u003efaecalis\u003c/em\u003e (Geraldes et al., 2022). The \u003cem\u003egelE\u003c/em\u003e gene was detected at incidence rates of 70.1% (82/117), 47.7% (31/65), 21.6% (8/37) in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates recovered from oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 55.3% (121/219).On the other hand, 39.2% (20/114), 60% (12/114), 55.8% (24/114) were the prevalence rates for \u003cem\u003egelE\u003c/em\u003e gene in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates recovered from oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 49.1% (56/114), with an overall incidence of 60.7% (102/168), 50.6% (43/85), and 40% (32/80) in \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 53.2% (177/333) among all \u003cem\u003eEnterococcus\u003c/em\u003e isolates. These results are aligned with those reported by Maasjost et al. (2019), who found that 53.6% (15/28) of \u003cem\u003eEnterococci\u003c/em\u003e isolates from turkey meat in Germany were positive for the \u003cem\u003egelE\u003c/em\u003e gene; Also, 56% (79/141) of \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from retail red meat in Slovenia tested positive for \u003cem\u003egelE\u003c/em\u003e (Golob et al., 2019).\u003c/p\u003e\n\u003cp\u003eIn contrast, a higher prevalence rate of 79% was found for \u003cem\u003egelE\u003c/em\u003e in \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from chicken meat in Bangladesh (Samad et al., 2022). Likewise, 75% (75/100) of \u003cem\u003eEnterococcus\u003c/em\u003e isolates isolated from beef samples collected from butcher shops and supermarkets in Turkey harbored the \u003cem\u003egelE \u003c/em\u003egene (Yılmaz et al., 2016), while the \u003cem\u003egelE\u003c/em\u003e gene was determined in a much higher incidence of 95.8% (46/48) in \u003cem\u003eEnterococcus\u003c/em\u003e isolates from meat samples from Tunisian markets (Klibi et al., 2013). Furthermore, a high detection rate of 88.1% (155/176) was reported for the \u003cem\u003egelE \u003c/em\u003egene in \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from meats in South Korea (Kim et al., 2020), and a higher prevalence rate of 92.3% of \u003cem\u003egelE\u003c/em\u003e gene was reported in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis \u003c/em\u003eisolates from chicken meat collected at food stores in Athens (McGowan-Spicer et al., 2008). Additionally, Ozmen et al. (2010) reported that 100% of \u003cem\u003eEnterococci\u003c/em\u003e isolates from naturally fermented sausage samples in Turkey were positive for \u003cem\u003egelE\u003c/em\u003e. \u003c/p\u003e\n\u003cp\u003eOn the contrary, the \u003cem\u003egelE\u003c/em\u003e gene was found at lower prevalence rates of 36% in \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from meat products in Serbia (Valenzuela et al., 2009) and at a detection rate of35% (7/20) in \u003cem\u003eEnterococcus\u003c/em\u003e strains isolated from sausages in Portugal (Ribeiro et al., 2011). A much lower detection rate of 12.3% was determined for \u003cem\u003egelE \u003c/em\u003egene in \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from chicken salad, chicken burgers, and meat tested in Kansas, USA (Macovei and Zurek, 2007). \u003c/p\u003e\n\u003cp\u003eThe ace gene was detected in 59.8% (70/117), 52.3% (34/65), 24.3%(9/37) in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates recovered from Oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 51.6%(113/219) On the other hand, 52.9% (27/51), 20% (4/20), 0% (0/43) was the prevalence rate for \u003cem\u003eace\u003c/em\u003e gene in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates recovered from Oriental meat pies, meat pizza, and chicken pizza, respectively, with an overall prevalence of 27.2% (31/114). Additionally, The \u003cem\u003eace\u003c/em\u003e gene was detected in \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from Oriental meat pies, meat pizza, and chicken pizza, tested with an incidence of 57.7% (97/168), 44.7% (38/85), and 11.3% (9/80), respectively, and an overall prevalence of 43.2% (144/333) (Figure 5 \u0026amp; Table 2). Higher prevalence rates of the \u003cem\u003eace\u003c/em\u003e gene in \u003cem\u003eEnterococcus\u003c/em\u003e spp. have been reported in other studies. For instance, Golob et al. (2019) found the \u003cem\u003eace \u003c/em\u003egene in 65.2% (92/141) of \u003cem\u003eEnterococcal\u003c/em\u003e isolates collected from retail red meat in Slovenia. In contrast, a lower incidence of 37% (81/219) was observed for the \u003cem\u003eace\u003c/em\u003e gene in \u003cem\u003eEnterococcus\u003c/em\u003e isolates from Brazilian food samples (Gomes et al., 2008). Additionally, a much lower detection rate of 12% (30/250) was reported for the \u003cem\u003eace\u003c/em\u003e gene in\u003cem\u003e Enterococcus\u003c/em\u003e isolates recovered from various foodstuffs, such as dairy products, ready-to-eat meat products, fruits, and vegetables (Trivedi et al., 2011). Likewise, Guerrero-Ramos et al. (2016) noted that 10.9% (6/55) of \u003cem\u003eEnterococcus \u003c/em\u003eisolates recovered from wild game meat in North-Western Spain harbored the \u003cem\u003eace\u003c/em\u003e gene. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4.\u003c/em\u003e \u003cem\u003eAntibiotic resistance\u003c/em\u003e\u003cem\u003e profile of Enterococcus isolates (n = 333) recovered from RTE meat products \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe emergence of multidrug-resistant \u003cem\u003eEnterococcus\u003c/em\u003e from food of animal origins is strongly linked to the misuse of antibiotics in animal-rearing practices (Kim and Ahn, 2022\u003cem\u003e). \u003c/em\u003eThe significance of \u003cem\u003eEnterococcus\u003c/em\u003e in veterinary and human medicine arises from its ability to acquire antimicrobial-resistance genes, posing a potential public health risk(Nurrahmat et al., 2025). As meat products are recognized as potential vehicles for the transmission of antibiotic-resistant bacteria, there is a critical need to investigate the antimicrobial resistance characteristics of bacteria recovered from frequently consumed meats, including poultry and beef.\u003c/p\u003e\n\u003cp\u003eThe antibiotic resistance patterns of all examined \u003cem\u003eEnterococcus\u003c/em\u003e isolates (n = 333) against 16 antibiotics from 7 antimicrobial classes are shown in Table 3. In this study, complete resistance was observed in \u003cem\u003eEnterococcus\u003c/em\u003e isolates against several antimicrobial agents from various classes, particularly those linked to \u0026beta;-lactams. Interestingly, 100% (333/333) of the isolates were resistant to at least one \u0026beta;-lactam antibiotic. Additionally, all \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e (219/219) and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e (114/114) isolates demonstrated complete resistance to cefepime. Likewise, 100% (81/81) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates from red meat from slaughterhouses in Borujerd city were susceptible to nitrofurantoin and cefotaxime discs (Madanipour et al., 2017). A lower resistance rate of 23.8% (55/231) was recorded towards cephalothin among \u003cem\u003eEnterococcus\u003c/em\u003e isolates from beef samples tested in Botswana (Chingwaru et al., 2003). \u003c/p\u003e\n\u003cp\u003eIn the present study, a 100% absolute susceptibility rate of Enterococcus spp. was observed against gentamicin, vancomycin, and levofloxacin (Table 3). This finding aligns with data from northern Portugal, where 99.5% (181/182) of \u003cem\u003eEnterococcus\u003c/em\u003e isolates from fermented meat products were susceptible to vancomycin (Barbosa et al. 2009). Nonetheless, resistance rates of 32.8% (23/70) and 4.3% (3/70) were recorded for enterococcal isolates recovered from beef against gentamicin and vancomycin tested in Italy, respectively (Pesavento et al. 2014). Moreover, 47.3% and 9.1% of enterococci isolated from wild game meat in northwest Spain were resistant to vancomycin and gentamicin, respectively (Guerrero et al., 2016). Additionally, 7.9% (24/302) and 0.7% (2/302) of enterococcal isolates from ready-to-eat meat products in northeast Poland showed resistance to gentamicin and vancomycin, respectively (Chajęcka-Wierzchowska et al., 2016). \u003c/p\u003e\n\u003cp\u003eIn this study, All (100%; 333/333) \u003cem\u003eEnterococcus\u003c/em\u003e isolates were resistant to cefepime, present in all isolates, followed by penicillin (97.3%; 324/333), meropenem (94.89%; 316/333), kanamycin (92.19%; 307/333), clindamycin (87.99%; 293/333), and erythromycin (64.86%; 216/333) (Table 3). Unlike our findings, Milanov et al. (2025) reported that the most frequent resistance of \u003cem\u003eEnterococcus\u003c/em\u003e isolates was to tetracycline, found in 32 isolates (34.78%), followed by erythromycin resistance in 25 isolates (27.17%), doxycycline in 20 isolates (21.73%), and streptomycin in 12 isolates (13.04%).\u003c/p\u003e\n\u003cp\u003eNumerous innate and acquired mechanisms are implicated in the notable decrease in susceptibility to \u0026beta;-lactam antibiotics exhibited by \u003cem\u003eEnterococcus\u003c/em\u003e, mainly \u003cem\u003eE\u003c/em\u003e.\u003cem\u003e faecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e. One important aspect that lowers the effectiveness of \u0026beta;-lactam antibiotics is the presence of penicillin-binding proteins (PBPs). Furthermore, some \u003cem\u003eEnterococcus\u003c/em\u003e species generate \u0026beta;-lactamase enzymes, which cause \u0026beta;-lactam antibiotics to be broken (Gagetti et al., 2019).Due to the abuse of antimicrobial agents in agricultural, food-producing animals and human medications, the emergence of antibiotic-resistant bacteria has increased, and constitutes a human public health (Manyi-Loh et al., 201\u003cspan dir=\"RTL\"\u003e8\u003c/span\u003e). Many environments have been proven to be \u003cstrong\u003eessential reservoirs for antibiotic resistance genes\u003c/strong\u003e found in soil, water, wastewater, and other natural habitats that may transfer these genes to human-associated bacteria (Mart\u0026iacute;nez et al., 2015).\u003c/p\u003e\n\u003cp\u003eThe emergence of multidrug-resistant \u003cem\u003eEnterococcus\u003c/em\u003e species has substantially limited therapeutic options and made the treatment of enterococcal infections increasingly difficult for clinicians\u003cem\u003e.\u003c/em\u003e Because of its administration for several decades, the penicillin aminoglycoside combination is the cornerstone of managing \u003cem\u003eenterococcal\u003c/em\u003e infections caused by susceptible strains (Leone et al., 2016). One well-known concern for global health is the creation and spread of bacterial antimicrobial resistance. Because humans, animals, food, and the environment can all serve as potential reservoirs for AMR bacteria and resistance genes, the idea of \u0026quot;One Health\u0026quot; is essential to addressing this problem (Huijberset al., 2015).\u003c/p\u003e\n\u003cp\u003eIn the recent survey, 14.41% (48/333) of \u003cem\u003eEnterococcus \u003c/em\u003eisolates were resistant to rifampin (Table 3). A higher resistance rate of 60% (109/182) was recorded among \u003cem\u003eEnterococcus\u003c/em\u003e isolates recovered from fermented meat products in the North of Portugal toward rifampicin (Barbosa et al., 2009), while 23.8% (35/147) and 15.8% (19/120) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e from RTE meat products in Poland were resistant to rifampin, respectively with an overall resistance of 19.2% (58/302) of enterococcal isolates (Chajęcka‐Wierzchowska.et al., 2016). A combination of rifampin and linezolid may be more effective than either medication by itself, particularly in infections caused by bacteria that cause biofilms. Rifampin and linezolid work well together due to their capacity to break through biofilms and prevent the creation of new proteins; this combination may also aid in preventing the development of antibiotic resistance (Baldoni et al., 2009). \u003c/p\u003e\n\u003cp\u003eVancomycin has long been regarded as the preferred treatment because of its effectiveness in treating a wide range of drug-resistant infectious pathogens. VRE in food of animal origin has been linked to avoparcin as a growth promotor due to avoparcin is a vancomycin equivalent that gives cross-resistance to vancomycin (Bager et al., 1997). \u003c/p\u003e\n\u003cp\u003eThis study revealed the highest sensitivity rates of 100% (219/219) and 100% (114/114) towards vancomycin in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates, respectively, with a total susceptibility rate of 100% (333/333) for all \u003cem\u003eenterococcal\u003c/em\u003e isolates (Table 3). Comparable with our results, a susceptibility rate of 100% (302/302) was determined for \u003cem\u003eEnterococcal \u003c/em\u003eisolates from ready-to-eat\u003cem\u003e \u003c/em\u003emeat products toward vancomycin (Chajęcka‐Wierzchowska et al., 2016). Absolute sensitivity (100%) against vancomycin was reported for \u003cem\u003eenterococcal\u003c/em\u003e isolates from beef and poultry meat (Milanov et al., 2025), retail meats in Alberta, Canada (Aslam et al., 2012), chicken breast meat samples in Sleman District, Indonesia (Nurrahmat et al., 2025), and fermented meat products in the North of Portugal (Barbosa et al., 2009).\u003c/p\u003e\n\u003cp\u003eIn contrast to our findings, high rates of vancomycin resistance among enterococci have been reported elsewhere, including 47.3% of isolates from wild game meat (Guerrero et al., 2016), 75% (12/16) of \u003cem\u003eEnterococcus faecium\u003c/em\u003e from meat products (Pavia et al., 2000), and 83.95% (68/81) of \u003cem\u003eE. faecalis\u003c/em\u003e isolated from red meat in slaughterhouses in Borujerd, Iran (Madanipour et al., 2017). Similarly, 85.6% (231/270) of enterococcal isolates recovered from ready-to-eat meat products in Egypt were resistant to vancomycin (Sallam et al., 2026).\u003c/p\u003e\n\u003cp\u003eTetracycline antibiotics, including tetracycline, oxytetracycline, and chlortetracycline, have been widely used in human and veterinary medicine (Johnson and Adams, 1992). One of the most common classes of antibiotics used in agriculture, veterinary medicine, and human therapy is tetracycline. In the present study, \u003cem\u003eEnterococcus spp\u003c/em\u003e isolates showed a resistance rate of 68.5% (228/333) toward tetracycline (Table 3). Conversely, lower resistance rates ranging from 14.9% (14/94) to 36.4% (110/302) were reported for enterococcal isolates recovered from different meat products worldwide (Barbosa et al., 2009; Pesavento et al., 2014; Golob et al., 2019; Chajęcka‐Wierzchowska et al., 2016; Milanov et al., 2025; Holman et al., 2021).\u003c/p\u003e\n\u003cp\u003eIn the present study, tetracycline resistance was detected in 72.15% (158/219) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and 61.4% (70/114) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolated from ready-to-eat (RTE) meat products (Table 3). In contrast, lower resistance rates for \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e have been reported from red meat in Borujerd, Iran (41.97%; Madanipour et al., 2017), Turkey (53%; Yılmaz et al., 2016), and raw meat in Athens, Georgia, USA (64.9%; McGowan-Spicer et al., 2008). Similarly, lower tetracycline resistance rates were observed among \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates from chicken meat (57.6%) and beef meat (0%) in Alberta, Canada (Aslam et al., 2012), Poland (6.7%; Chajęcka-Wierzchowska et al., 2016), and Italy (16.4%; Pesavento et al., 2014).\u003c/p\u003e\n\u003cp\u003eNone of the enterococcal isolates (0%; 0/333) were resistant to the 3rd-generation (Levofloxacin), while a low resistance rate of 4.8% (16/333) was reported for the 2nd- generation ciprofloxacin (Table 3). Comparable resistance rates of ciprofloxacin among enterococcal isolates recovered from raw red meat in Georgia, USA (3.5%; 2/57) (McGowan-Spicer et al., 2008), Borujerd, Iran (3.7%) (Madanipour et al., 2017), and Slovenia 1. 4% (2/141) (Golob et al., 2019). Relatively higher resistance rates of 12% (12/100) and 16.5% (30/182) were reported against ciprofloxacin for enterococcal isolates recovered from meat samples tested in Turkey \u003cstrong\u003e(\u003c/strong\u003eYılmaz et al., 2016) and the North of Portugal (Barbosa et al., 2009), respectively, although much higher resistance rate of 92.7% was reported for enterococcal isolates recovered wild game meat in Spain (Guerrero et al., 2016). \u003c/p\u003e\n\u003cp\u003eEnterococci are intrinsically sensitive to a wide range of aminoglycoside antibiotics (such as gentamicin, GEN) due to inefficient transport across the cytoplasmic membrane (Leclercq, 1997). Thus, aminoglycosides alone are ineffective in the treatment of \u003cem\u003eenterococcal\u003c/em\u003e infections, and therefore they are used in combination therapy with inhibitors of cell wall synthesis (such as ampicillin), which facilitate their uptake (Lefort et al., 2000). For severe \u003cem\u003eenterococcal\u003c/em\u003e infections like endocarditis, gentamicin, an aminoglycoside antibiotic, is frequently utilized in combination treatment (Chaves \u0026amp; Tadi, 2020). \u003c/p\u003e\n\u003cp\u003eNotably, all \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates (100%; 219/219) were sensitive to gentamicin (Table 3). Likewise, 100% of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates recovered from retail ground beef tested in Canada (Aslam et al., 2012; Holman et al., 2021) and Poland (R\u0026oacute;żańska et al., 2015) were sensitive to gentamicin. Nevertheless, resistance rates of 10.9% (16/147) (Chajęcka‐Wierzchowska et al., 2016), 17.5% (10/57) (McGowan-Spicer et al., 2008), and 21.9% (25/114) (Pesavento et al., 2014) were confirmed by \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates from different meat samples against gentamicin. Similar to \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e tested in the present study, 100 % (114/114) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e were sensitive to gentamicin (Table 3). A 100% gentamicin-sensitive isolates were found among \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates in Canada (Aslam et al., 2012) and in Slovenia (Golob et al., 2019). Relatively higher resistance rates of 5% (6/120) (Chajęcka‐Wierzchowska et al., 2016)and 13. 6% (19/140) (Pesavento et al., 2014)were reported among \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates recovered from meat products in Poland and Italy, respectively.\u003c/p\u003e\n\u003cp\u003eIn 2000, the U.S. Food and Drug Administration (FDA) approved linezolid for the treatment of infections caused by vancomycin-resistant Enterococcus (VRE) (Wang and Hsueh, 2009; Bi et al., 2018), and because of its effectiveness against Gram-positive pathogens, it can be used to treat complex skin and soft tissue infections, community-acquired pneumonia, and nosocomial pneumonia. \u003c/p\u003e\n\u003cp\u003eAmong 333 enterococcal isolates of the current surveys, 26/333 (7.81%) showed resistance to linezolid (Table 3). Nonetheless, no resistance to linezolid (LZD) was detected in \u003cem\u003eEnterococcus\u003c/em\u003e isolates from meat products in Serbia (Milanov et al., 2025) and Slovenia (Golob et al., 2019). Among 219 \u003cem\u003eE. faecalis\u003c/em\u003e isolates, 11.87% (26/219) were resistant to linezolid. This finding is consistent with the results of R\u0026oacute;żańska et al. (2015), who reported linezolid resistance in 11.4% (4/35) among \u003cem\u003eE. faecalis\u003c/em\u003e isolates. However, a markedly higher resistance rate of 86.4% (70/81) was reported by Madanipour et al. (2017). In contrast, lower resistance rates of 6.1% (9/147) and 2.9% (1/34) have been reported by Chajęcka-Wierzchowska et al. (2016) and Martinez-Laorden et al. (2023), respectively, among \u003cem\u003eE. faecalis\u003c/em\u003e isolates from meat samples. On the other hand, no (0%) linezolid resistance was detected among \u003cem\u003eE. faecium\u003c/em\u003e isolates in the present study, which is consistent with the findings of Martinez-Laorden et al. (2023), who revealed 0% resistance among \u003cem\u003eE. faecium\u003c/em\u003e isolates from meat tested in Spain. A low resistance rate of 2.5% (3/120) was reported by Chajęcka-Wierzchowska et al. (2016).\u003c/p\u003e\n\u003cp\u003eRifampicin is used for the treatment of enterococcal infections. An acquired resistance to rifampicin has been detected in both \u003cem\u003eE. faecium\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e related to the mutations in the gene encoding the RNA polymerase subunit (\u003cem\u003erpoB\u003c/em\u003e) (Kakoullis et al., 2021). The present study revealed rifampicin-resistance rates of 16.44% (36/219) and 10. 53% (12/114) among \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e, respectively, with a total resistance of 14. 41 % (48/333) among all \u003cem\u003eEnterococcus\u003c/em\u003e isolates. Higher resistance rates of 19.2% (58/302) (Chajęcka‐Wierzchowska.et al., 2016), 36.4% (Guerrero-Ramos et al., 2016), and 60% (109/182) (Barbosa et al., 2009) were reported for \u003cem\u003eEnterococcus \u003c/em\u003eisolates from various red meats towards rifampicin.\u003c/p\u003e\n\u003cp\u003e3.5. \u003cem\u003eMultiple antimicrobial resistance (MAR) index and classification of Enterococcus isolates based on their antimicrobial resistance pattern\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial resistance pattern and multiple antibiotic resistance index (MAR) of \u003cem\u003eEnterococcus \u003c/em\u003espp. recovered from oriental meat pies, meat pizza, chicken pizza revealed that 98.5% (328/333) of\u003cem\u003e Enterococcus \u003c/em\u003eisolates, including 214 \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis \u003c/em\u003e(117 from oriental meat pies, 65 meat pizza, and 32 from chicken pizza), and 114 from \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e (51 from oriental meat pies, 20 meat pizza, and 43 from chicken pizza) were classified as multidrug-resistant (MDR) as they showed resistance to more than 2 antibiotics with a MAR value between 0.442 and 0.0.419, respectively (Table 4). Interestingly, among the MDR enterococci analyzed in the current study, 98.5% (328/333) were resistant to at least 4 antimicrobial agents, while 87.39% (291/333) were resistant to at least 6 antibiotics (Table 4). Lower multidrug-resistant (MDR) profiles have also been verified among \u003cem\u003eEnterococcus\u003c/em\u003e\u003cem\u003e \u003c/em\u003eisolates recovered from various types of raw meat and meat products worldwide, ranging from 15.14% (48/317) to 90.6% (48/53) in Spain (Mart\u0026iacute;nez-Laorden et al., 2023), Indonesia (Nurrahmat et al., 2025), Serbia (Milanov et al., 2025), Bangladesh (Samad et al., 2022), Italy (Vignaroli et al., 2011), and Turkey (Yılmaz et al., 2016).\u003c/p\u003e\n\n\u003cp\u003eThe average MAR index of the 333 \u003cem\u003eEnterococci \u003c/em\u003eisolates tested was 0.43, with 95.2% (317/333) of \u003cem\u003eEnterococcus \u003c/em\u003eisolates showing a MAR index higher than 0.3 (Table 4). A MAR index exceeding 0.2 indicates antimicrobial abuse, whereas a value exceeding 0.4 generally indicates human-related fecal contamination (Gessew et al., 2022).\u003c/p\u003e\n\n\u003cp\u003e3.6. \u003cem\u003ePrevalence and distribution \u003c/em\u003e\u003cem\u003eof \u003c/em\u003e\u003cem\u003evancomycin-resistant genes (vanA, vanB), erythromycin-resistant gene (\u003c/em\u003e\u003cem\u003eermB), and tetracycline-resistant gene (tetL) among Enterococcus isolates\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, \u003cem\u003evanA\u003c/em\u003e, \u003cem\u003evanB\u003c/em\u003e, \u003cem\u003eermB\u003c/em\u003e, and \u003cem\u003etetL\u003c/em\u003e genes were identified in isolates resistant to vancomycin, erythromycin, and tetracycline at the predicted molecular sizes of 559, 467, 229, and 639 bp, respectively (Table 1 and Fig 6). Consistent with the phenotypic results of antimicrobial sensitivity testing for vancomycin, vancomycin-resistant genes were not detected in any of the 333 \u003cem\u003eEnterococcus\u003c/em\u003e isolates tested in the current study (Table 5). On the other hand, of the216 phenotypically erythromycin-resistant isolates, 142 (65.7%) were positive for the \u003cem\u003eermB\u003c/em\u003e gene, with 63 isolates from oriental meat pies, 60 isolates from meat pizza, and 19 isolates from chicken pizza. Of the 228 phenotypically tetracycline-resistant isolates, 94 (41.23%) were positive for \u003cem\u003etetL \u003c/em\u003egene, distributed as 34 isolates from oriental meat pies, 42 from meat pizza, and 18 from chicken pizza.\u003c/p\u003e\n\u003cp\u003eOf the 186 (29.03%) phenotypically erythromycin- and tetracycline-resistant isolates, 54 showed coexistence of \u003cem\u003eermB\u003c/em\u003e and \u003cem\u003etetL\u003c/em\u003e genes distributed as 24 isolates from oriental meat pies, 23 from meat pizza, and 7 from chicken pizza. The \u003cem\u003eermB\u003c/em\u003e gene was the most prevalent at 42.6% (142/333), followed by the \u003cem\u003etetL\u003c/em\u003e gene at 28.2% (94/333). It was observed that 41.9% (49/117), 81.5% (53/65), and 24.3% (9/37) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates, and 27.5% (14/51), 35% (7/20), and 23.3% (10/43) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates from oriental meat pies, meat pizza, and chicken pizza, respectively, were positive for the \u003cem\u003eermB\u003c/em\u003e gene (Table 2 and Fig. 7). On the other hand, 17.95% (21/117), 53.9% (35/65), and 0% (0/37) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates and 25.49% (13/51), 35% (7/20), and 41.9% (18/43) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates from the corresponding products harbored \u003cem\u003etetL\u003c/em\u003e genes (Table 2 and Fig. 7). Overall, \u003cem\u003eermB\u003c/em\u003e genes were detected in 50.7% (111/219) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates and in 27.2% (31/114) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates. On the other hand, \u003cem\u003etetL\u003c/em\u003e genes were present in 25.6% (56/219) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates and in 33.3% (38/114) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e isolates from all examined RTE meat products. \u003c/p\u003e\n\u003cp\u003ePrevious surveys reported a higher prevalence of \u003cem\u003evanA\u003c/em\u003e and \u003cem\u003evanB\u003c/em\u003e genes. Hosseini et al. (2016) found that the prevalence of \u003cem\u003evanA\u003c/em\u003e in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e was 93.3% (14/15) and 38.9% (7/18), respectively, while \u003cem\u003evanB\u003c/em\u003e was found in 26.6% (4/15) and 11.1% (2/18) among these species from raw chicken meat. In raw meat, \u003cem\u003evanA\u003c/em\u003e prevalence was 63.9% (23/36) in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e and 50% (13/26) in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e, whereas \u003cem\u003evanB\u003c/em\u003e was present in 11.1% (4/36) and 0% among these species, respectively. Additionally, Madanipour et al. (2017) reported that 83.95% (68/81) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates from red meat in Borujerd, Iran, harbored \u003cem\u003evanA\u003c/em\u003e, 25.9% (21/81) harbored \u003cem\u003evanB\u003c/em\u003e, and 22.2% (18/81) carried both \u003cem\u003evanA\u003c/em\u003e and \u003cem\u003evanB\u003c/em\u003e. Similarly, Yılmaz et al. (2016) found that 4.95% (5/101) of \u003cem\u003eEnterococcus\u003c/em\u003e isolates from chicken meat in Turkey carried vancomycin-resistant genes. Conversely, Chajęcka-Wierzchowska et al. (2016) did not detect \u003cem\u003evanA\u003c/em\u003e or \u003cem\u003evanB\u003c/em\u003e genes in \u003cem\u003eEnterococcus\u003c/em\u003e isolated from RTE meat products from northeast Poland, nor were these genes found in other studies of ready-to-eat meat products in\u003cstrong\u003eBrno, Czech Republic\u003c/strong\u003e (Trivedi et al., 2011).\u003c/p\u003e\n\u003cp\u003eThe rise of vancomycin-resistant \u003cem\u003eEnterococcus\u003c/em\u003e in food animals is associated with the use of growth-promoting additives like avoparcin and vancomycin analogs, with \u003cem\u003evanB\u003c/em\u003e emerging more recently despite \u003cem\u003evanA\u003c/em\u003e being responsible for most VRE cases worldwide (Telli et al., 2021).\u003c/p\u003e\n\u003cp\u003eAs a whole, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e examined in the present study harbored a greater number of the \u003cem\u003eermB\u003c/em\u003e gene than the \u003cem\u003eEnterococcus\u003c/em\u003e isolates. This finding is in agreement with the results reported by Chajęcka‐Wierzchowska et al. (2016), who indicated that the \u003cem\u003eermB\u003c/em\u003e antimicrobial resistance-encoding gene was detected in 52.4% (77/147) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e versus 18.3% (22/120) in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e. Also, Macovei \u0026amp; Zurek (2007) reported that 13.6% (3/22) were positive for \u003cem\u003eermB\u003c/em\u003e in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e versus 0% (0/26) in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium,\u003c/em\u003e with an overall prevalence in \u003cem\u003eEnterococcus\u003c/em\u003e spp 6.38% (9/141). Moreover, 22.2% (10/45) and 11.1% (5/45) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e harbored \u003cem\u003eermB\u003c/em\u003e and \u003cem\u003etetL\u003c/em\u003e, respectively, while 71.4 % (5/7) and 42.9% of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e harbored \u003cem\u003eermB\u003c/em\u003e and (\u003cem\u003etetL\u003c/em\u003e and \u003cem\u003etetM\u003c/em\u003e), respectively (Guerrero-Ramos et al., \u003cspan dir=\"RTL\"\u003e2016\u003c/span\u003e). In our study, 71.7% (157/219) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e isolates were morphologically resistant to erythromycin, with 70.7% (111/157) among them carrying the \u003cem\u003eermB \u003c/em\u003egene. In another research, 44.5% (149/335) of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e were morphologically resistant to erythromycin, with 96% (143/149) of these erythromycin-resistant isolates harboring the \u003cem\u003eermB \u003c/em\u003egene (kim et al., 2020). On the other hand, \u003cstrong\u003e89.3% (133/\u003c/strong\u003e149) of \u003cem\u003eE. faecalis\u003c/em\u003e isolates with simultaneous erythromycin and tetracycline resistance carried the \u003cstrong\u003e\u003cem\u003etetL\u003c/em\u003e\u003c/strong\u003e gene, and \u003cstrong\u003e96.0%\u003c/strong\u003e (143/149) carried the \u003cstrong\u003e\u003cem\u003eermB \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003egene, while\u003c/strong\u003e\u003cstrong\u003e81.2% (121/149) of the isolates simultaneously possessed \u003cem\u003eermB\u003c/em\u003e, \u003cem\u003etetL\u003c/em\u003e, and \u003cem\u003etetM\u003c/em\u003e genes\u003c/strong\u003e(kim et al., 2020).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current study revealed that 76.4% (84/110) of RTE pizza and oriental meat pies examined in Dakahlia province, Egypt, were contaminated with \u003cem\u003eEnterococcus spp.\u003c/em\u003e that harbored various virulence genes, such as \u003cem\u003esodA\u003c/em\u003e, \u003cem\u003egelE\u003c/em\u003e, and \u003cem\u003eace\u003c/em\u003e in 100% (333/333), 53.15% (177/333), and 43.24% (144/333) of the isolates, respectively. Interestingly, 98.5% (328/333) of the isolates were multidrug-resistant, suggesting antibiotic overuse. The study highlights the major public health hazards of \u003cem\u003eEnterococcus\u003c/em\u003e and the urgent requirement for reliable monitoring methods to control the overuse of antibiotics in veterinary medicine. More research is required to deepen the global understanding of this pathogen and to improve effective methods to eradicate it from RTE meat products, to ensure the protection of public health and safeguard population safety.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmira Mahmoud Elsayeh\u0026nbsp;\u003c/strong\u003eConceptualization,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFormal analysis, Methodology, and Writing \u0026ndash; original draft.\u0026nbsp;\u003cstrong\u003eAmira Ibrahim Zakaria\u0026nbsp;\u003c/strong\u003eSupervision, Validation and Writing \u0026ndash; original draft. \u003cstrong\u003eSamir Abd-Elghany\u003c/strong\u003e, Supervision, Data curation, investigation. \u003cstrong\u003eKhalid Ibrahim Sallam\u0026nbsp;\u003c/strong\u003eConceptualization, Supervision, and Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eOpen access funding provided by The Science, Technology \u0026amp; Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Open access funding is provided by the Science, Technology \u0026amp; Innovation Funding Authority (STDF) in cooperation with the Egyptian Knowledge Bank (EKB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are included within the article. No datasets were generated or analyzed during the current study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that there is no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAfshari, A., Taheri, S., Hashemi, M., Norouzy, A., Nematy, M., Mohamadi, S., 2022. Methicillin- and vancomycin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and vancomycin-resistant enterococci isolated from hospital foods: Prevalence and antimicrobial resistance patterns. Curr. 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Extensively drug-resistant and heat-resistant \u003cem\u003eEnterococcus faecalis\u003c/em\u003e and \u003cem\u003eEnterococcus faecium\u003c/em\u003e in ready-to-eat meat products. npj Science of Food. Article in Press. DOI: https://doi.org/10.1038/s41538-026-00790-y\u003c/li\u003e\n\u003cli\u003eSharafi, S., Nateghi, L., 2020. Optimization of gamma-aminobutyric acid production by probiotic bacteria through response surface methodology. \u003cem\u003eIran. J. Microbiol.\u003c/em\u003e 12, 584. https://doi.org/10.18502/ijm.v12i6.5033\u003c/li\u003e\n\u003cli\u003eSingh, S., Yadav, A.S., Singh, S.M., Bharti, P., 2010. Prevalence of \u003cem\u003eSalmonella\u003c/em\u003e in chicken eggs collected from poultry farms and marketing channels and their antimicrobial resistance. Food Res. 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Soc.\u003c/em\u003e 72 (4), 3345\u0026ndash;3354. https://doi.org/10.12681/jhvms.29373\u003c/li\u003e\n\u003cli\u003eTrivedi, K., Cupakova, S., Karpiskova, R., 2011. Virulence factors and antibiotic resistance in enterococci isolated from food-stuffs. \u003cem\u003eVet. Med. (Praha)\u003c/em\u003e 56(7), 352\u0026ndash;357. https://doi.org/10.17221/1584-VETMED\u003c/li\u003e\n\u003cli\u003eTyson, G.H., Nyirabahizi, E., Crarey, E., Kabera, C., Lam, C., Rice-Trujillo, C., Tate, H., 2018. Prevalence and antimicrobial resistance of enterococci isolated from retail meats in the United States, 2002 to 2014. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e 84, e01902-17. https://doi.org/10.1128/AEM.01902-17\u003c/li\u003e\n\u003cli\u003eValenzuela, A.S., ben Omar, N., Abriouel, H., L\u0026oacute;pez, R.L., Veljovic, K., Ca\u0026ntilde;amero, M.M., G\u0026aacute;lvez, A., 2009. Virulence factors, antibiotic resistance, and bacteriocins in enterococci from artisan foods of animal origin. \u003cem\u003eFood Control\u003c/em\u003e 20, 381\u0026ndash;385. https://doi.org/10.1016/j.foodcont.2008.06.004\u003c/li\u003e\n\u003cli\u003eVignaroli, C., Zandri, G., Aquilanti, L., Pasquaroli, S., Biavasco, F., 2011. Multidrug-resistant enterococci in animal meat and faeces and co-transfer of resistance from an Enterococcus durans to a human Enterococcus faecium. \u003cem\u003eCurr. Microbiol.\u003c/em\u003e 62 (5), 1438\u0026ndash;1447. https://doi.org/10.1007/s00284-011-9880-x\u003c/li\u003e\n\u003cli\u003eWang, J.L., Hsueh, P.R., 2009. Therapeutic options for infections due to vancomycin-resistant enterococci. \u003cem\u003eExpert Opin. Pharmacother.\u003c/em\u003e 10 (5), 785\u0026ndash;796. https://doi.org/10.1517/14656560902811811\u003c/li\u003e\n\u003cli\u003eWioleta, C., Wierzchowska, C., Zadernowska, A., Łaniewska-Trokenheim, Ł., 2017. Virulence factors of \u003cem\u003eEnterococcus\u003c/em\u003e spp. presented in food. \u003cem\u003eLWT Food Sci. Technol.\u003c/em\u003e 75, 670\u0026ndash;676. https://doi.org/10.1016/j.lwt.2016.10.026\u003c/li\u003e\n\u003cli\u003eXu, Y., Schaffner, D.W., 2023. Microbiological quality and safety of pizza held out of temperature control in university dining halls. \u003cem\u003eJ. Food Prot.\u003c/em\u003e 86, 100111. https://doi.org/10.1016/j.jfp.2023.100111\u003c/li\u003e\n\u003cli\u003eYılmaz, E.Ş., Aslantaş, \u0026Ouml;., \u0026Ouml;nen, S.P., T\u0026uuml;rkyılmaz, S., K\u0026uuml;rekci, C., 2016. Prevalence, antimicrobial resistance and virulence traits in enterococci from food of animal origin in Turkey. \u003cem\u003eLWT Food Sci. Technol.\u003c/em\u003e 66, 20\u0026ndash;26. https://doi.org/10.1016/j.lwt.2015.10.009\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Enterococcus, Pizza, Ready-to-eat meat products, PCR, Antimicrobial resistance","lastPublishedDoi":"10.21203/rs.3.rs-9259480/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9259480/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study was conducted to determine whether ready-to-eat meat products could indirectly threaten consumer health by serving as a reservoir for \u003cem\u003eEnterococcus\u003c/em\u003e strains that carry virulence determinants and antimicrobial resistance profiles. Overall, 76.4% (84/110) of the examined RTE meat samples, including oriental meat pies, meat pizza, and chicken pizza, were contaminated with \u003cem\u003eEnterococcus\u003c/em\u003e. Polymerase chain reaction (PCR) analysis showed that 65.8% (219/333) of \u003cem\u003eEnterococcus\u003c/em\u003e isolates were identified as \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecalis\u003c/em\u003e, while 34.2% (114/333) were \u003cem\u003eE\u003c/em\u003e. \u003cem\u003efaecium\u003c/em\u003e. The virulence genes \u003cem\u003egelE\u003c/em\u003e (gelatinase) and \u003cem\u003eace\u003c/em\u003e (collagen-binding protein) were detected in 53.2% (177/333) and 43.2% (144/333) of the isolates, respectively. Interestingly, 100% (333/333) of \u003cem\u003eEnterococcus\u003c/em\u003e isolates were resistant to cefepime, 97.3% (324/333) to penicillin, 94.89% (316/333) to meropenem, 92.19% (307/333) to kanamycin, 87.99% (293/333) to clindamycin, and 64.86% (216/333) to erythromycin. Remarkably, 98.5% (328/333) of the isolates showed resistance to at least four antibiotics, with an average multiple antibiotic resistance (MAR) index of 0.42. The \u003cem\u003evanA\u003c/em\u003e and \u003cem\u003evanB\u003c/em\u003e genes were not detected in any isolates, while the \u003cem\u003eermB\u003c/em\u003e gene was found in 26.4% (88/333), and \u003cem\u003etetL\u003c/em\u003e in 11.1% (37/333) of the \u003cem\u003eEnterococcus\u003c/em\u003e isolates. To our knowledge, this is the first study in Egypt to assess antibiotic resistance and virulence characteristics in \u003cem\u003eEnterococcus\u003c/em\u003e spp. recovered from pizza and oriental meat pies. The findings of this study may be valuable for evaluating potential human health risks associated with consuming cooked and processed meat products.\u003c/p\u003e","manuscriptTitle":"Multidrug-resistant Enterococcus faecalis and Enterococcus faecium isolated from oriental meat pies and beef- and chicken-based pizzas","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 13:51:00","doi":"10.21203/rs.3.rs-9259480/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-19T10:37:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332903228909343549042885002266235240248","date":"2026-05-07T12:47:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161308296548979181858203527054346057981","date":"2026-05-05T12:19:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197125497422347314470056142867764031490","date":"2026-04-30T06:43:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T16:42:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-02T11:14:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-30T05:49:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-30T05:49:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-29T14:25:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"301855c0-b6f5-47e7-947d-15193d5763f0","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-19T10:37:37+00:00","index":77,"fulltext":""},{"type":"reviewerAgreed","content":"332903228909343549042885002266235240248","date":"2026-05-07T12:47:27+00:00","index":75,"fulltext":""},{"type":"reviewerAgreed","content":"161308296548979181858203527054346057981","date":"2026-05-05T12:19:36+00:00","index":74,"fulltext":""},{"type":"reviewerAgreed","content":"197125497422347314470056142867764031490","date":"2026-04-30T06:43:01+00:00","index":68,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66627251,"name":"Biological sciences/Microbiology"},{"id":66627252,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-27T13:51:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 13:51:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9259480","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9259480","identity":"rs-9259480","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}