New Insights into Listeria monocytogenes Antimicrobial Resistance, Virulence Attributes and Their Prospective Correlation

Listeriosis is one of the most common foodborne diseases caused by Listeria monocytogenes (L. monocytogenes). A poor prognosis has been recorded for the invasive listeriosis, especially neurolisteriosis. In several countries throughout the world, foodborne infections with L. monocytogenes exceeded the legal safety limits in animal sourced foods. Therefore, we decided to investigate the variability, virulence and antimicrobial resistance profiles of this pathogen. Both phenotypic and genotypic methods were used for identifying L. monocytogenes isolates and confirming their virulence profiles. The antimicrobial resistances and their correlation analysis with the existence of virulence genes were detected. Additionally, sequencing and phylogenetic analysis based on L. monocytogenes inlA and inlB genes were undertaken. The prevalence rate (11.9%) and the resistance profiles of L. monocytogenes were shocking. The multi-drug resistance (MDR) phenotypes were common among our isolates (64.9%). Fortunately, the resistance phenotypes were always associated with low virulence arrays and the MDR strains possessed low virulence fitness. Herein, the high genotypic and phenotypic diversity of L. monocytogenes isolates and their weak clonality and adaptability highlighted the difficulty in controlling and managing this pathogen. Therefore, it is important to add more restriction guidelines from national authorities on the consumption of ready to eat foods.


Introduction
Listeria monocytogenes is an animal and human pathogen, which is responsible for causing intracellular foodborne diseases as well as invasive listeriosis, encephalitis, endocarditis, perinatal infections, gastroenteritis, septicemia, meningitis, ophthalmitis and abortion. Life threatening listeriosis occurs among people with weakened immune systems, neonates, the elderly, and pregnant women [1]. The fatality rate among infected individuals is low; meanwhile, it may increase in the high-risk patients [2]. Regarding the animal infections, there monocytogenes isolates. The distribution of L. monocytogenes from different sources is illustrated in Figure 1. Regarding the animal origin, the majority of our isolates (26.7%, 8/30) were recovered from liver samples of sheep suffering from septicemia. Moreover, the high proportion of L. monocytogenes isolates from human sources was from placental samples (12%, 3/25), and only one isolate was obtained from 30 animal feed samples (3.3%). Notably, Sharkia had the highest prevalence rate of L. monocytogenes isolates (13.8%, 18/130), followed by Giza (12.2%, 11/90) and then Qaliubia (8.9%, 8/90) Governorates.

Antibiogram Typing and Virulence Gene Profiles of L. monocytogenes
According to the antimicrobial susceptibility results, L. monocytogenes isolates showed high resistance rates to penicillin, cephalexin and cefotaxime (100% each), followed by ampicillin (97.3%); meanwhile, the highest sensitivity levels were recorded to sulfamethoxazole-trimethoprim (78.4%) and amoxicillin-clavulanic acid (64.9%), as shown in Figure 2. Unfortunately, the majority of our isolates (24/37; 64.9%) showed the MDR pattern being resistant to three or more antimicrobial drugs from different classes. Moreover, MAR indices of the recovered isolates ranged from 0.17 to 0.42, indicating that they were originated from high-risk contamination. Regarding the distribution of the tested virulence genes among the recovered isolates, our results documented that all L. monocytogenes isolates possessed both inlA and inlB genes; meanwhile, 73% (27/37) and 64.9% (24/37) of the examined isolates harbored prfA and hlyA genes, respectively.

Antibiogram Typing and Virulence Gene Profiles of L. monocytogenes
According to the antimicrobial susceptibility results, L. monocytogenes isolates showed high resistance rates to penicillin, cephalexin and cefotaxime (100% each), followed by ampicillin (97.3%); meanwhile, the highest sensitivity levels were recorded to sulfamethoxazoletrimethoprim (78.4%) and amoxicillin-clavulanic acid (64.9%), as shown in Figure 2. Unfortunately, the majority of our isolates (24/37; 64.9%) showed the MDR pattern being resistant to three or more antimicrobial drugs from different classes. Moreover, MAR indices of the recovered isolates ranged from 0.17 to 0.42, indicating that they were originated from high-risk contamination. Regarding the distribution of the tested virulence genes among the recovered isolates, our results documented that all L. monocytogenes isolates possessed both inlA and inlB genes; meanwhile, 73% (27/37) and 64.9% (24/37) of the examined isolates harbored prfA and hlyA genes, respectively.

Phenotypic and Genetic Diversity of L. monocytogenes
Based on both antibiogram typing and virulence gene profiles, all of our recovered L. monocytogenes isolates were cloned into 28 clusters (Figure 3), which reflected the high degree of heterogeneity among these isolates. With the exception of six clusters, each isolate belonged to a unique lineage with a specific phenotypic and genotypic profile. The

Phenotypic and Genetic Diversity of L. monocytogenes
Based on both antibiogram typing and virulence gene profiles, all of our recovered L. monocytogenes isolates were cloned into 28 clusters (Figure 3), which reflected the high degree of heterogeneity among these isolates. With the exception of six clusters, each isolate belonged to a unique lineage with a specific phenotypic and genotypic profile. The discriminatory power for combined phenotypic (antibiogram typing) and genotypic (virulence genes' profiles) methods was 0.98, indicating the high variability of the tested isolates. Out of the six clusters, one contained five isolates; but 10 isolates belonged to the other five clusters (two isolates for each cluster) as shown in Figure 3.

Correlation Analysis
The correlation analysis among and between resistances to the investigated antimicrobials and the presence of tested virulence genes are illustrated in Figure 4. The results

Correlation Analysis
The correlation analysis among and between resistances to the investigated antimicrobials and the presence of tested virulence genes are illustrated in Figure 4. The results revealed that there were negative correlations between the existence of prfA or hlyA gene and the resistance to all tested antimicrobial drugs, with the exception of the very weak positive correlations between the presence of prfA gene and the resistances to ciprofloxacin and norfloxacin. Of note, strong positive correlations were detected between resistances to ampicillin and norfloxacin (r-value = 0.7) and resistances to ciprofloxacin and tylosin (r-value = 0.62). On the other hand, strong negative correlations between the existence of hlyA gene and the resistances to each of chloramphenicol and gentamycin (r-value = −0.36 each) is presented in Figure 4. revealed that there were negative correlations between the existence of prfA or hlyA gene and the resistance to all tested antimicrobial drugs, with the exception of the very weak positive correlations between the presence of prfA gene and the resistances to ciprofloxacin and norfloxacin. Of note, strong positive correlations were detected between resistances to ampicillin and norfloxacin (r-value = 0.7) and resistances to ciprofloxacin and tylosin (r-value = 0.62). On the other hand, strong negative correlations between the existence of hlyA gene and the resistances to each of chloramphenicol and gentamycin (r-value = −0.36 each) is presented in Figure 4.

Phylogenetic Analyses of inlA and inlB Genes of L. monocytogenes
Phylogenetic and sequence analyses of inlA and inlB genes' sequences among three representative L. monocytogenes isolates showing the highest resistance rates revealed that the examined isolates exhibited a remarkable genetic identity to other L. monocytogenes strains from different origins, which were deposited on the GenBank database, as

Phylogenetic Analyses of inlA and inlB Genes of L. monocytogenes
Phylogenetic and sequence analyses of inlA and inlB genes' sequences among three representative L. monocytogenes isolates showing the highest resistance rates revealed that the examined isolates exhibited a remarkable genetic identity to other L. monocytogenes strains from different origins, which were deposited on the GenBank database, as demonstrated in Figures 5 and 6. These sequences were deposited in the GenBank under the accession numbers of OM854784, OM854785 and OM854786 for inlA gene and OM854787, OM854788 and OM854789 for inlB gene. The final alignments of inlA gene consisted of 800 bp; 769 conserved and 31 variable sites; meanwhile, that of inlB gene comprised 343 bp; 304 conserved and 39 variable sites.
Based on the sequence analyses of both inlA and inlB genes, we did not find any new or unique sequences, and there were no new or unique mutations in these genes. Phylogenetic and sequence analyses of inlA genes revealed that our tested isolates (Accession No. OM854784, OM854785 and OM854786) displayed a complete identity (100%) to another food L. monocytogenes strain in Japan (Accession No. LC005925). Additionally, they showed high similarity percentages with L. monocytogenes food isolate in France (Accession No. FM178794, 99.9%), L. monocytogenes strain isolated from pig in Spain (Accession No. HQ111554, 99.7%), L. monocytogenes strain isolated from seafood in Japan (Accession No. AB276379, 99.5%), and L. monocytogenes strain isolated from watershed samples in the USA (Accession No. KF728273, 99.0%), as demonstrated in Figure 5. On the other hand, our inlB gene sequences (Accession No. OM854787, OM854788 and OM854789) displayed a remarkable genetic identity to other L. monocytogenes strains from different origins such as the food L. monocytogenes strain in South Korea (Accession No. CP029175, 100%), the L. monocytogenes strain isolated from cold smoked salmon in Denmark (Accession No. GU079625, 99.7%), the L. monocytogenes strain isolated from maternofetal interface in Denmark (Accession No. GU079621, 99.4%), the L. monocytogenes strain isolated from fish smoke house equipment in Denmark (Accession No. GU079614, 99.4%) and the L. monocytogenes strain isolated from spreadable sausage in Denmark (Accession No. GU079619, 93.6%), as demonstrated in Figure 6.

Discussion
Recently, infections from L. monocytogenes increased worldwide, and high death rates associated with this pathogen were recorded. Listeriosis is a serious disease that affects a wide range of animals and people, especially those with weakened immune systems, pregnant women and newborns. The prevalence rates of L. monocytogenes were variable Figure 6. Phylogenetic analysis of inlB gene of the three investigated L. monocytogenes isolates (OM854787; S1, OM854788; S2 and OM854789; S3) illustrating the genetic relationships between the investigated isolates and those have been deposited in the GenBank database, as shown in the phylogenetic tree. Based on the sequence analyses of both inlA and inlB genes, we did not find any new or unique sequences, and there were no new or unique mutations in these genes. Phylogenetic and sequence analyses of inlA genes revealed that our tested isolates (Accession No. OM854784, OM854785 and OM854786) displayed a complete identity (100%) to another food L. monocytogenes strain in Japan (Accession No. LC005925). Additionally, they showed high similarity percentages with L. monocytogenes food isolate in France (Acces-

Discussion
Recently, infections from L. monocytogenes increased worldwide, and high death rates associated with this pathogen were recorded. Listeriosis is a serious disease that affects a wide range of animals and people, especially those with weakened immune systems, pregnant women and newborns. The prevalence rates of L. monocytogenes were variable among different sources in this study, with an overall percentage of 11.9%. The animal subjects were the most common source of L. monocytogenes isolates. This prevalence rate was higher than those recorded in previous studies conducted on different ecological niches in the same geographic area [19,20] reflecting the decreasing in awareness, carefulness and good practices in the Egyptian community. This pathogen can cause a zoonotic disease (listeriosis) in both humans and animals and it can be transmitted via several food chains such as meat, poultry and seafood products. Additionally, L. monocytogenes is widely spread and contaminates a wide range of foods and animal products such as raw milk, cheese, raw meat products and salads due to poor hygienic conditions [21]. Listeria monocytogenes can multiply in food to dangerous levels, even at refrigeration temperatures during distribution and storage [22]. Therefore, there is an urgent need to control L. monocytogenes at all stages in the food chains and manage its infection in animals. We can overcome this challenge by strengthening the good manufacturing and hygienic practices in all sectors of the food chain, avoiding the animal infections and rapid diagnosis and treatment of both animal and human infections.
Another crisis in L. monocytogenes infections is the wide spread of antimicrobial resistance. The MDR phenotypes are common among our isolates, which lead to treatment failure. The acquired resistance to antimicrobial drugs were rarely developed among L. monocytogenes isolates [23,24]; however, and in accordance with our findings, several recent reports have found increased rates of antimicrobial resistance among clinical foodborne pathogens [25][26][27]. Overall, L. monocytogenes isolates in this study exhibited MDR patterns to four classes of antibiotics: penicillins, quinolones, aminoglycoside, and macrolides. This is a great issue for public health, as it may pose a challenge in the treatment of listeriosis. Of note, L. monocytogenes exhibit varying degrees of resistances to the most common antibiotics. High levels of resistance to amoxicillin, cefotaxime, norfloxacin, tetracyclines and gentamicin were recorded in this study and in other studies [28,29]; meanwhile, the high susceptibility of our L. monocytogenes against trimethoprim/sulfamethoxazole was announced and is consistent with an earlier study [30]. The antimicrobial resistance in L. monocytogenes may be attributed to the acquisition of antibiotic resistance genes from other pathogens [27,31]. Moreover, the acquisition of conjugative transposons [32], active efflux associated genes [33], and ribosomal and chromosomal mutations [34] are the major mechanisms of L. monocytogenes resistance. Fortunately, sulfamethoxazole-trimethoprim and amoxicillin-clavulanic acid are still the alternative therapies in the case of unresponsiveness to the treatment with beta lactams' antibiotics and intolerance to the first line therapies, as observed in our study and other reports [35,36].
Many factors affect the pathogenicity of L. monocytogenes involving numerous key virulence arrays such as internalins and haemolysin [37,38]. Notably, L. monocytogenes clinical isolates are multi-virulent; this was announced in several previous publications [39,40] as well as in our study. Previously, all clinical isolates possessed InlA and InlB proteins [41,42], which are essential for the recognition of several receptors and the invasion of different cell types. Furthermore, majority of our isolates showed hemolytic activities and harbored hlyA and prfA genes, which contribute to L. monocytogenes virulence and transmission. As expected from the correlation analysis, negative correlations between the existence of virulence genes (hlyA and prfA) and the antimicrobial resistances were detected.
Of note, the antimicrobial resistance genes were distributed between plasmid and chromosome [43]; however, the resistance genes can be acquired from the environment through plasmids and transposons [34]. Antimicrobial resistance, which enhances bacterial performance in vivo, may carry fitness costs, prompting a renewed interest in understanding the complex relationships between resistance and virulence. When both virulence and antimicrobial resistance genes are on the same mobile genetic element, a direct link can be observed [44]. The prediction of these correlations is highly challenging, as the bacterial genome is context-dependent and highly dynamic. Therefore, the correlation studies between the resistance to antimicrobials with each other, the existence of virulence genes with each other, in addition to the association between resistances to antimicrobials and the existence of virulence genes are very necessary owing to the continuous evolution in the genetic background. The costs of acquisition of antimicrobial resistance genes (increasing the resistance abilities) can be outweighed by benefits such as loss of virulence genes (decreasing in the virulence fitness) [14]. Fortunately, the high virulence arrays were associated with the sensitive phenotypes, and the MDR strains possess low virulence fitness [45]. This finding is accepted depending on the fixed genetic capacity of the bacterial genome. Moreover, the acquisition of resistance genes occurs in parallel with the loss of other genes such as virulence ones [46,47].
In this study, all inlA and inlB genes' sequences were identical to the previous sequence deposited in the database of GeneBank considering different sources and geographic areas. We did not observe any new mutations among our genes sequences. This observation confirmed the slow evolution in the virulence genes compared to antimicrobial resistance profiles. Notably, most evolution in the virulence genes may lead to negative selection [48].
The control of listeriosis becomes more tedious due to the high degree of heterogeneity among L. monocytogenes isolates. Our report recorded the high variability level and the low host specificity among the tested L. monocytogenes isolates. In this context, several authors announced the weak clonality and adaptability of L. monocytogenes due to the high phenotypic and genotypic diversity, which is associated with different evolutionary processes within different hosts or in an ecological niche [49,50]. Therefore, specific control and prevention recommendations and more restricted isolation guidelines are urgently needed.

Ethics Statement
All study procedures were conducted in accordance with the guidelines of the Animal Ethics Review Committee of Suez Canal University (AERC-SCU201856), Egypt. Moreover, written informed consent was obtained from all patients.

Sampling
The study was conducted from April 2018 to November 2021. Three hundred and ten samples were collected from various sources at Sharkia (n = 130), Giza (n = 90) and Qaliubia (n = 90) Governorates, Egypt. The samples were collected from animal (n = 230), human (n = 50), and animal feed (n = 30) sources. The animal samples comprised the brain of sheep suffering from nervous manifestations (n = 30), placenta (n = 30), and aborted foeti (n= 30) of sheep suffering from abortion at the third trimester stage of pregnancy, lung (n= 30), liver (n = 30) and spleen (n = 30) of sheep suffering from septicemia, and finally mastitis milk (n = 50) of sheep suffering from mastitis. Human placental samples (n = 25) and uterine biopsies (n = 25) were collected from febrile and aborted women attending tropical hospitals. The samples were aseptically placed into sterile containers, kept in an ice box and transferred as soon as possible to the laboratory of the Bacteriology Unit at Animal Health Research Institute, Dokki, Giza for further bacteriological examination and the isolation of L. monocytogenes.

Isolation and Characterization of L. monocytogenes
Listeria monocytogenes isolates were phenotypically identified based on characteristic culture, morphological and biochemical features [51,52]. Furthermore, the recovered isolates were further identified using the Microbact™ Listeria 12L system (Oxoid, UK). The genotypic identification was used to confirm all L. monocytogenes isolates via a 16S rRNA gene-based PCR assay [53].

Anton's Test
The test was performed by inserting 0.1 mL of L. monocytogenes suspension (10 9 colony forming units) into the conjunctiva of one eye of rabbits, and the other eye was used as a control. Anton's test positive results were recorded as purulent conjunctivitis within 24-48 h, followed by keratitis [54].

CAMP (Christie-Atkins-Munch-Peterson) Test
The test was conducted, as described previously [55], using a standard beta-hemolytic Staphylococcus aureus (ATCC 25923) strain streaked in a straight line across the center of blood agar (Oxoid, UK) plates, followed by the streaking of identified L. monocytogenes strains in a direction perpendicular or vertical to the S. aureus culture without touching. The plates were subsequently incubated at 37 • C for 18-24 h and examined for the presence of hemolysis, which appeared as an arrowhead, circle, or rectangle in CAMP positive strains.

Antibiogram Patterns of L. monocytogenes Isolates
The Kirby-Bauer disc diffusion method was used to determine the antimicrobial susceptibility profiles of the retrieved L. monocytogenes isolates on Mueller-Hinton agar (Oxoid, UK) supplemented with 5% fresh defibrinated sheep blood using twelve different antimicrobial disks (Oxoid, UK): penicillin (P), ampicillin (AMP), amoxicillin-clavulanic acid (AMC), cefotaxime (CTX), cephalexin (CFX), ciprofloxacin (CIP), norfloxacin (NOR), sulfamethoxazole-trimethoprim (SXT), gentamycin (CN), oxytetracycline (OTC), tylosin (TYL) and chloramphenicol (C). Inhibition zones were measured and interpreted adopting EUCAST guidelines for L. monocytogenes [58]. To ensure the accuracy of the disc diffusion method, the MIC values for the investigated antimicrobials were detected by broth microdilution method according to EUCAST guidelines. Briefly, selected L. monocytogenes strains were streaked onto brain heart infusion agar plates and the plates were incubated at Antibiotics 2022, 11, 1447 10 of 13 37 • C for 24 h. Three to five colonies were picked and incubated in brain heart infusion broth for 6 h, and this culture was adjusted to 5 × 10 6 CFU /mL using 0.9% NaCl solution. Ten microliters of this solution were used to inoculate 96-well microtiter plates containing Mueller-Hinton broth with different antibiotic concentrations. The L. monocytogenes strain LMEGY1 was utilized as a quality control organism. The isolates exhibiting resistance to at least one agent in three or more antimicrobial classes were categorized as MDR [59]. Multiple antibiotic resistance (MAR) indices were then calculated following the previously standardized formula [60]. The chance of a high risk source of contamination is always observed with high MAR indices.

Molecular Detection of L. monocytogenes Virulence Genes
The PCR techniques were used to determine some virulence genes: prfA, inlA, inlB and hlyA in the recovered L. monocytogenes isolates [61][62][63]. The primers' pairs used in PCR protocols and their predicted amplicons' sizes are listed in Table 1. For genomic DNA extraction, the QIAamp DNA Mini Kit (Qiagen, Germantown, MD, USA, catalog no. 51326) was used. All PCR assays were carried out in triplicates and included positive control strains provided by the Animal Health Research Institute, Dokki, Giza, Egypt, with a DNA-free reaction as a negative control. The amplified PCR products were separated on electrophoresis gel (1.5% agarose stained with 0.5 µg/mL of ethidium bromide) and photographed.

Sequencing and Phylogenetic Analysis of L. monocytogenes inlA and inlB Genes
Direct sequencing of inlA and inlB genes among MDR L. monocytogenes isolates were carried out in both directions following purification with the QIAquick PCR Purification Kit (Qiagen, Germantown, MD, USA) using a Bigdye™ Terminator V3.1 cycle sequencing kit (Thermo Fisher Scientific, Waltham, MA, USA). The sequence similarity to GenBank accessions was determined using a Basic Local Alignment Search Tool. Phylogenetic trees were then created by the MegAlign module of Laser gene DNASTAR version 12.1 utilizing the neighbor-joining method in MEGA6.

Statistical Analysis
Hierarchical clustering and the correlation coefficient (r-value) between resistances to various antimicrobials in addition to the existence of virulence genes were evaluated using the R package corrplot, heatmaply, and GraphPad Prism (version 6; GraphPad Software Inc.; San Diego, CA, USA). Moreover, the discriminatory power of the typing methods used in the current study was assessed using Simpson's index of diversity [64].

Conclusions
Several ready to eat foods, especially those of an animal source, are always contaminated with L. monocytogenes, which is associated with serious public health implications worldwide. As observed among our tested L. monocytogenes isolates, this problem is compounded by the high prevalence rate, the wide spread of antimicrobial resistance, the heterogeneity and diversity of this pathogen. In contrast to the evolution in the virulence fitness, we observed acceleration in the acquisition of antimicrobial resistance among L. monocytogenes isolates, which increase the possibility of treatment failure with the available drugs. Therefore, we are in urgent need for new and alternative therapies. Our study spotlighted and predicted the serious health problems in the near future due to the evolution in L. monocytogenes strains. Therefore, we encourage the health organizations in all countries to take more stringent actions to hinder the spread of this resistant pathogen.  Informed Consent Statement: Informed consent was obtained from all patiants involved in the study.
Data Availability Statement: All generated data in this report are available in the submitted manuscript.