Prevalence, Antibiogram and Genetic Characterization of Listeria monocytogenes from Food Products in Egypt

World Health Organization classified Listeria monocytogenes as a major notable foodborne pathogen associated with high mortality and hospitalization. The study reports the prevalence, antibiogram, virulence determination and genetic characterization of L. monocytogenes from different food products. A total of 250 food samples, fifty samples each from raw milk, ice cream, minced meat, fish fillet and sausage were collected from the Menoufiya governorate in Egypt. L. monocytogenes was detected in 17 (6.8%) of the tested food samples including minced meat (14%), fish fillet (8%), sausage (6%) and raw milk (6%). The antimicrobial susceptibility assay of 17 L. monocytogenes isolates against seventeen antibiotics belonging to eight antibiotics classes revealed a high susceptibility to norfloxacin (82.3%), amoxicillin-clavulanic acid (76.4%), cefotaxime (70.5%), erythromycin (64.6%), amoxicillin (64.6%), gentamicin (58.7%) and vancomycin (58.7%). While, high resistance was observed against oxytetracycline (76.4%), trimethoprim-sulfamethoxazole (76.4%), chloramphenicol (70.5%), doxycycline (64.6%), levofloxacin (41.2%) and azithromycin (41.2%). Of note, all L. monocytogenes isolates were multidrug-resistant. The multiplex PCR successfully amplified L. monocytogenes in all tested isolates. Screening of the five virulence-related genes revealed the hlyA and iap as the most prevalent genes followed by actA gene, however, the inlA and prfA genes were not detected in any of the studied isolates. The partial 16S rRNA gene sequencing of three L. monocytogenes isolates showed a high nucleotide similarity (99.1–99.8%) between the study isolates and various global clones, and phylogenetic analysis clustered these L. monocytogenes strains with other Listeria species including L. welshimeri, L. seeligeri and L. innocua. This study demonstrates the impact of L. monocytogenes as a major contaminant of various food products and suggests more attention to the awareness and hygienic measures in the food industry.


Introduction
Listeria monocytogenes is a ubiquitous foodborne pathogen associated with high hospitalization and outbreaks of food-borne illness worldwide [1]. L. monocytogenes causes listeriosis in humans and animals and can be found in varieties of foods and dairy products [2]. The bacteria is also considered as a potential contaminant source for chilled and marine food products [3] and unpasteurized dairy products and has been detected in recent outbreaks and sporadic cases of listeriosis [4]. In a dairy herd, contaminated teat cups often serve as a potential source in the transmission of L. monocytogenes [2]. In line with this, three L. monocytogenes variant clones, the hypervirulent clones Clonal Complexes 1 (CC1) and hypovirulent clones (CC9 and CC121), were reported in humans that were closely associated with the dairy and meat products, respectively [5].
The pathogenicity of L. monocytogenes is largely determined by a group of virulence genes. L. monocytogenes strains from dairy herds carried a number of virulence markers including the Listeria pathogenicity islands (LIPI) 3 and LIPI-4 that were associated with severe human infections [4]. Virulence factors are associated with crucial stages of adhesion, invasion, reproduction, motility and intercellular spread into host cells and therefore play a key role in virulence and resistance against host immunity [6]. The major determinant virulence genes include internalins encoded by inlA, inlC and inlJ genes, listeriolysin encoded by hlyA gene, actin encoded by actA gene and the invasion associated protein encoded by iap gene [7]. The hlyA, plcB and prfA virulence genes have been found in L. monocytogenes strains recovered from blood and milk isolates [2]. Furthermore, the prfA virulence gene is a transcriptional activator identified among pathogenic L. monocytogenes [8]. Moreover, biofilm activity is commonly associated with L. monocytogenes of milk origin that carry a variety of virulence and antibiotics resistance genes [2]. Traditionally, Listeria diagnosis is primarily based on isolation and biochemical characterization, which is time consuming. Molecular approaches such as PCR and multiplex PCR (mPCR) provide rapid and specific techniques for the diagnosis of L. monocytogenes targeting specific genes [9].
Antimicrobial resistance is an important public health issue and one of the highest priorities of the World Health Organization (WHO). The growing level of antimicrobial resistance has led to higher patient morbidity and mortality rates and increased healthcare expenditure over the last decades [10,11]. The multidrug-resistant L. monocytogenes particularly in ready-to-eat foods is being considered as a public health indicator especially among the high-risk groups. It is highly recommended to build awareness about the importance of food safety regulations as well as drugs used in humans and animals [12]. The nucleotide sequence analysis of L. monocytogenes is an effective modern tool for genotyping and analysis of the relatedness of Listeria species with other local or global lineages. A surveillance study conducted in the United States reported a substantial genetic similarity between L. monocytogenes strains from milk tanks with virulent strains isolated from dairy products, which were associated with the outbreaks of food-borne illness in humans [4].
The Menoufiya governorate of Egypt is a densely populated rural governorate where people consume many famous and popular ready-to-eat foods such as minced meat meal, fish dishes, milk products and ice cream. Such raw uncooked or undercooked foods can be a potential source of pathogenic L. monocytogenes in humans. This study describes the virulence and antibiotic resistance profiles of L. monocytogenes detected in various readyto-eat food products from Egypt. In addition, partial nucleotide sequence analysis of the 16S rRNA gene of three L. monocytogenes isolates was performed to correlate the genetic similarities between the study isolates and various global clones having potential public health implications.

Ethics Statement
This study followed the guidelines of the Ethics Committee and current legislation on research and ethical approval of the Faculty of Veterinary Medicine (Local ethical approval), University of Sadat City, Egypt.

Study Area
This study was carried out in the Monufiya governorate in Lower Egypt. The majority of residents in this governorate live in rural areas, with an urbanization rate of only 20.6% [13]. The area has a high population density and people consume a lot of ready-toeat popular foods such as minced meat, fish dishes, raw milk products and ice cream.

Samples Collection and Processing
A total of 250 food products (50 samples each from raw milk, ice cream, minced meat from beef, fish fillet and sausage) were collected from different local markets at Menoufiya governorate in Egypt from January to August 2020. Each sample was collected separately in a sterile plastic bag and transferred to the laboratory in cooled condition.

Phenotypic Isolation and Identification of Listeria from Food Products
Twenty-five grams of food sample was homogenized with 9 mL of nutrient broth using a blender. The harvested homogenate was firstly pre-enriched in 225 mL of Buffered Listeria Enrichment Broth with pyruvate and incubated at 30 • C for 48 h, then cultured in specific Oxford medium, CHROM agar and sheep blood agar (Himedia, India) for 48 h at 35 • C as described by FDA BAM and ISO 11290 method [14]. Characteristic colonies of L. monocytogenes were identified on different agar media. Morphological and biochemical characteristics of the bacteria were analyzed using Gram staining, catalase test, sugar fermentation test and motility test according to FDA BAM and ISO 11290 method [14]. The CAMP (Christie-Atkins-Munch-Peterson) test was performed as described previously [15] using standard hemolytic Staphylococcus aureus strain (MT211620), which was streaked on blood agar in a straight line across the center of the plate, then the L. monocytogenes strain was streaked in a direction perpendicular or vertical to S. aureus without touching the S. aureus culture. Then the plates were incubated at 37 • C for 18-24 h and checked for β-hemolysis which appeared as an arrowhead, circle or rectangle shape in CAMPpositive species.

Antibiogram Profile of L. monocytogenes Isolates Recovered from Food Products
The Kirby-Bauer disk diffusion method was used to analyze the antibiogram profile of L. monocytogenes isolates. The bacterial suspension was adjusted to a 0.  [16]. The strains displaying resistance to at least three antibiotic classes were considered multidrug-resistant (MDR) [17]. The L. monocytogenes strain LMEGY1 was used as a quality control organism in antimicrobial susceptibility determination. All samples were tested twice.

Molecular Detection of L. monocytogenes from Food Products
DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The list of primers used in the study is provided in Table 1. Taq PCR Master Mix Kit (Qiagen, Hilden, Germany) was used in the PCR. A 50 µL reaction was prepared to contain 25 µL PCR Master Mix, 1 µL (10 pmol/µL) of each primer, 2 µL (50 ng/µL) DNA and the remaining volume needed to reach 50 µL was adjusted with deionized water. The following thermal profile was used: initial denaturation at 95 • C for 3 min; 35 cycles each consisting of denaturation at 94 • C for 30 s, annealing at 53 • C for 15 s and extension at 72 • C for 90 s; and final extension at 72 • C for 7 min. PCR products (15 µL) were analyzed by agarose gel (1.5%) electrophoresis and visualized under UV light in a gel documentation system. The L. monocytogenes LMEGY1 strain was used as a positive control in the PCR.

Sequencing and Phylogenetic Analysis of L. monocytogenes from Food Products
Three PCR amplicon of the 16S rRNA gene of L. monocytogenes, each from raw milk, fish fillet and minced meat were purified using Gene JET PCR Purification kit (Thermo Scientific, Waltham, MA, USA). The purified PCR products were sequenced from a commercial laboratory (GATC Biotech Company, Konstanz, Germany) in both directions. Nucleotide and amino acid sequence homology analysis among studied isolates and global strains was performed using BLAST 2.0 and PSI-BLAST search programs (http://www.ncbi.nlm.nih.gov/, accessed on 20 March 2021), respectively. Multiple alignments with reference strains as well as the deduction of amino acid sequences were performed using the BioEdit [19], CLUSTALX [20], ClustalW [21], ClustalV [22] and MegAlign software (DNASTAR, Lasergene ® , Version 7.1.0, Madison, WI, USA) [23]. A neighborjoining phylogenetic tree was built using MegAlign software. A random bootstrapping value of 111 was applied [21]. The partial nucleotide sequences of the three L. monocytogenes strains from the fish fillet, minced meat and raw milk were submitted in the GenBank with accession number MW090062, MW090063 and MW090064, respectively.

Statistical Analysis
To show the multidrug resistance profile of L. monocytogenes isolates, an UpSetR plot was prepared using an online platform (https://gehlenborglab.shinyapps.io/upsetr/, accessed on 21 April 2021).

Prevalence of Listeria Species and L. monocytogenes in Different Food Products
The overall prevalence of L. monocytogenes was 6.8% (n = 17), while other Listeria species were as followed: L. innoca 3.2% (n = 8), L. grayi 2.4% (n = 6), L. ivanovii 0.4% (n = 1) and L. welshimeri 0.8% (n = 2). Among the different food samples, L. monocytogenes was most commonly detected in minced meat (14%) followed by fish fillet (8%), sausage (6%) and milk (6%) ( Table 2). Analysis of the ice cream samples yielded no L. monocytogenes. Typical colony characters of L. monocytogenes on Oxford medium appeared as gray to black color colonies bounded by a black halo. A green to bluish colony was observed in CHROM agar medium. On the 5% sheep blood agar medium, L. monocytogenes showed a clear narrow β-hemolysis. A series of biochemical tests were conducted to confirm and differentiate L. monocytogenes and other Listeria species (Supplementary Table S1). L. monocytogenes appeared as Gram positive coccobacilli, catalase and CAMP test positive as well as fermented rhamnose with acid production, while negative for fermentation of mannitol and xylose (Supplementary Table S1).

Antibiogram of L. monocytogenes Isolates from Food Products
Antimicrobial resistance profiles of L. monocytogenes isolates were tested against seventeen antibiotics belonging to eight different antibiotic classes using the disc diffusion method (Table 3). L. monocytogenes isolates showed high susceptibility to β-lactams (amoxicillinclavulanic acid, cefotaxime and amoxicillin), norfloxacin, erythromycin, gentamicin and vancomycin. While, high resistance was observed against tetracycline (oxytetracycline and doxycycline), trimethoprim-sulfamethoxazole and chloramphenicol (Table 3). An overall similar antimicrobial resistance profile was observed in L. monocytogenes isolates from different sources such as milk, fish fillet, sausage and minced meat (Supplementary Table S2). Next, multidrug resistance profiles of the 17 L. monocytogenes isolates were tested. All L. monocytogenes isolates were multidrug-resistant against 3 to 11 different antibiotics belonging to 3 to 7 antibiotic classes in 17 different combinations ( Figure 1). Vancomycin (VA) 30 µg Glycopeptides 5 29.5 2 11.8 10 58.7 % was estimated according to the total number of L. monocytogenes isolates (n = 17).
Next, multidrug resistance profiles of the 17 L. monocytogenes isolates were tested. A L. monocytogenes isolates were multidrug-resistant against 3 to 11 different antibiotic belonging to 3 to 7 antibiotic classes in 17 different combinations (Figure 1).

Screening of Different Virulence Genes in L. monocytogenes from Food Products
Next, we applied multiplex PCR for molecular detection of the L. monocytogenes an screening five virulence-associated genes of the bacteria. Amplification of 938 bp fragmen of the 16S rRNA gene was found in all of the 17 tested isolates, confirming the L monocytogenes species (Figure 2). While, the screening of five virulence-associated gene in L. monocytogenes revealed the hlyA (n = 12, 70.6%) and iap (n = 12, 70.6%) as the mos prevalent genes followed by the actA gene (n = 9, 52.9%) (Figures 2 and 3). However, inlA and prfA genes were absent in all of the tested isolates. Of note, the simultaneous presenc of three virulence genes (hlyA, iap and actA) in six isolates, two of the three virulence gene (hlyA, iap or actA) in six isolates was also detected. Taken together, L. monocytogene isolates from food products carried multiple virulence genes, indicating their pathogeni potential.

Screening of Different Virulence Genes in L. monocytogenes from Food Products
Next, we applied multiplex PCR for molecular detection of the L. monocytogenes and screening five virulence-associated genes of the bacteria. Amplification of 938 bp fragment of the 16S rRNA gene was found in all of the 17 tested isolates, confirming the L. monocytogenes species (Figure 2). While, the screening of five virulence-associated genes in L. monocytogenes revealed the hlyA (n = 12, 70.6%) and iap (n = 12, 70.6%) as the most prevalent genes followed by the actA gene (n = 9, 52.9%) (Figures 2 and 3). However, inlA and prfA genes were absent in all of the tested isolates. Of note, the simultaneous presence of three virulence genes (hlyA, iap and actA) in six isolates, two of the three virulence genes (hlyA, iap or actA) in six isolates was also detected. Taken together, L. monocytogenes isolates from food products carried multiple virulence genes, indicating their pathogenic potential.

Sequence Analysis of L. monocytogenes Isolates from Food Products
The 16S rRNA sequence analysis was used for evaluating the genetic similarity of L. monocytogenes isolates from fish fillet, minced meat and raw milk samples with global strains. The partial sequence data were submitted to the GenBank with accession number MW090062, MW090063 and MW090064. The study isolates showed a very high nucleotide similarity (99.1-99.8%) with L. monocytogenes strains isolated in Turkey and Germany from minced meat and food origin with accession number MT633107.1 (strain: ka89-2), and CP054846 (strain: BfR-LI-00752), respectively ( Figure 4). Additionally, the near identity was also noticed with L. monocytogenes strain identified from the Massachusetts listeriosis outbreak in the USA (accession number CP023862, strain: ScottA). As well as one strain from rabbit tissue from United Kingdom (accession number CP023861, strain: EGD-e) had identical nucleotides similarity (99.6-100%) with our strains from food samples.

Sequence Analysis of L. monocytogenes Isolates from Food Products
The 16S rRNA sequence analysis was used for evaluating the genetic similarity of L. monocytogenes isolates from fish fillet, minced meat and raw milk samples with global strains. The partial sequence data were submitted to the GenBank with accession number MW090062, MW090063 and MW090064. The study isolates showed a very high nucleotide similarity (99.1-99.8%) with L. monocytogenes strains isolated in Turkey and Germany from minced meat and food origin with accession number MT633107.1 (strain: ka89-2), and CP054846 (strain: BfR-LI-00752), respectively ( Figure 4). Additionally, the near identity was also noticed with L. monocytogenes strain identified from the Massachusetts listeriosis outbreak in the USA (accession number CP023862, strain: ScottA). As well as one strain from rabbit tissue from United Kingdom (accession number CP023861, strain: EGD-e) had identical nucleotides similarity (99.6-100%) with our strains from food samples.

Sequence Analysis of L. monocytogenes Isolates from Food Products
The 16S rRNA sequence analysis was used for evaluating the genetic similarity of L. monocytogenes isolates from fish fillet, minced meat and raw milk samples with global strains. The partial sequence data were submitted to the GenBank with accession number MW090062, MW090063 and MW090064. The study isolates showed a very high nucleotide similarity (99.1-99.8%) with L. monocytogenes strains isolated in Turkey and Germany from minced meat and food origin with accession number MT633107.1 (strain: ka89-2), and CP054846 (strain: BfR-LI-00752), respectively ( Figure 4). Additionally, the near identity was also noticed with L. monocytogenes strain identified from the Massachusetts listeriosis outbreak in the USA (accession number CP023862, strain: ScottA). As well as one strain from rabbit tissue from United Kingdom (accession number CP023861, strain: EGD-e) had identical nucleotides similarity (99.6-100%) with our strains from food samples.        the most detected virulence genes with the prevalence rate of 70.6%, 70.6% and 52.9%, respectively. Of note, simultaneous detection of the three virulence genes (hlyA, iap and actA) was found in six L. monocytogenes isolates. Our findings support a recent study in Egypt that detected four virulence genes (inlA, actA, prfA and hlyA) in L. monocytogenes isolates from animal food products [25]. Harb and colleagues showed that the mPCR targeting the 16S rRNA and hlyA genes can effectively detect L. monocytogenes in food samples [33]. In a comparative study in Nigeria, Usman and colleagues used the mPCR to detect multiple virulence-related genes (prf A, inlA, hlyA, actA and iap) in L. monocytogenes from milk and milk products where 25% of them carried one or two of the virulence genes [18]. In Egypt, Abdellrazeq and colleagues examined five virulence genes (prfA, hlyA, actA, inlA and prs) in L. monocytogenes from various fish types by mPCR and detected prs gene in all tested isolates while only seven isolates carried other virulence genes [34]. In India, Kaur and colleagues tested 335 food samples (chicken, pork and fish) from various retail outlets and found that all L. monocytogenes isolates possess the prfA, plcA, actA, hlyA and iap virulence-related genes [35]. Furthermore, Haj Hosseini and colleagues demonstrated the prfA gene in all L. monocytogenes from contaminated foods in Iran [8]. Several virulent genes were also detected in L. monocytogenes from fish and fish products in India [30]. Of note, the two virulence-related genes, prfA and inlA were not found in any of the tested isolates. Our observations might be attributed to complete absence or presence of sequence variations in sites targeted by applied primers within genes found in L. monocytogenes isolates of our study. Since we detected no hemolytic activity differences on sheep blood agar between our all isolates and the reference strain used L. monocytogenes strain LMEGY1 with an intact prfA gene, the lack of prfA gene among our strains can however be ruled out.
The partial sequencing of the 16S rRNA gene was performed to assess the genetic homology among L. monocytogenes isolates from milk, fish fillet and minced meat as well as among related global sequences. Our findings showed a high nucleotide similarity (99.1-99.8%) between L. monocytogenes strains from various global clones. The phylogenetic tree clustered the three L. monocytogenes isolates from this study with other Listeria species including L. welshimeri, L. seeligeri and L. innocua strains. In addition, an apparent diversity was found in the phylogenetic tree with some other strains. These findings corroborate with other studies [36,37] which recorded a close association (more than 99% nucleotide similarity) between the members of Listeria species and this highlighted the significance of the 16S rRNA gene in differentiating Listeria species. Nucleotide sequencing and multilocus sequence typing (MLST) techniques are suggested to be more precise techniques for recognizing the clonal complexes (CC) of L. monocytogenes strains and phylogenetic characters among different Listeria strains in a population [38]. The sequencing analysis of the 16S rRNA gene of L. monocytogenes recovered from bulk tank milk in the USA described a high genetic diversity with many strains encoding virulence markers that were linked with serious human infections [4]. The comparison with global clones grouped the L. monocytogenes isolates into two distinct clusters, linage A, which are typically associated with epidemic listeriosis, and lineage B, which are mostly associated with sporadic cases of listeriosis [18]. Thus, our study emphasizes that these local isolates may have a potentials public health concern for humans through the food chain.

Conclusions
The study demonstrates the impact of L. monocytogenes as a major contaminant of various food products and the need for more attention about the awareness and hygienic measures in the food industry. Most of the L. monocytogenes from food products were multidrug-resistant, adding further burden to the existing global antimicrobial resistance problems. The sequencing analysis reported a high nucleotide sequence similarity of the study isolates with many global clones, indicating the widespread circulation of such strains between different countries via the food trading industry. Therefore, evidence-based recommendations and continuous education for workers particularly in the food industry are necessary to prevent food contamination and the emergence of resistant strains.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/foods10061381/s1, Table S1: Biochemical identification of Listeria monocytogenes and other Listeria species, Table S2: Antimicrobial resistance profiles of Listeria monocytogenes isolates from food products.

Informed Consent Statement: Not applicable.
Data Availability Statement: All authors agree that the data presented in this study are openly available through MDPI publisher platform or others without any restriction.