Virulence genotyping and antimicrobial resistance profiles of Yersinia enterocolitica isolated from meat and meat products in Egypt

Pathogenic Yersinia enterocolitica (Y. enterocolitica) is one of the food-borne entero-pathogen responsible for yersiniosis in humans. The purpose of this research was to survey the prevalence, virulence-associated genes, and antimicrobial resistance of Y. enterocolitica isolated from meat and meat product samples in Egypt. Forty-one (5.9%) out of 700samples of chicken meat, beef, ground beef, and sausage were positive Y. enterocolitica with a high prevalence in chicken meat (12%). Five virulence genes (ail, inv, ystA, ystB, and yadA) were characterized among 41 Y. enterocolitica isolates with variable frequencies. Among the strains tested, the ystB gene was detected with a high percentage (78.1%), followed by inv gene (70.7%), ail gene (14.6%), ystA gene (12.2%), and yadA gene (2.4%). A high resistance rate was estimated to amoxicillin-clavulanic acid (100%), followed by cefazolin (95%), ampicillin (65.9%), and doxycycline (51.2%), whilst a high sensitivity rate was observed to gentamicin and ciprofloxacin (97.6% each). Interestingly, the multidrug resistance was specified in the 70.7% of strains and showing 13 resistance patterns. Based on nucleotide sequence analysis of the 16s rRNA gene, the phylogenetic tree showed the genetic relatedness amongst Y. enterocolitica isolates. These findings highlighted the emergence of virulent and multidrug-resistant pathogenic Y. entrocolitica in retailed meat and meat products in Egypt.


Introduction
Yersinia genus belongs to the Enterobacteriaceae family and encompasses three well-recognized human pathogens which are Y. enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis (Carniel, 2006). Y. enterocolitica is one of the most important pathogens responsible for foodborne gastroenteritis (Yersiniosis) in Western and Northern Europe (EFSA, 2018). Other clinical syndromes associated with Y. enterocolitica are enterocolitis, mimicking appendicitis, acute mesenteric lymphadenitis, post-infectious arthritis, and systemic infections, so occasion fatal sepsis (Neubauer et al., 2001). Y. enterocolitica is often isolated from humans, a variety of animals, food and the environment (Falcão et al., 2006). Pigs are carriers of Y. enterocolitica without clinical signs in their oral cavity, on tongues, and then excrete these bacteria in their feces (Paixão et al., 2013).
The presence of virulence genes and virulence plasmids were applied for the estimation of pathogenic Y. enterocolitica strains (Platt-Samoraj et al., 2006;Peruzy et al., 2017). Virulence genes, ail, ystA, and ystB are located on the bacterial chromosome (Thong et al., 2018). The Ail protein is encoded by the ail gene and only occurs in pathogenic Y. enterocolitica, it contributes to bacterial adhesion to the host cell as well as strengthens resistance to the bactericidal effects of complement (Thoerner et al., 2003). Moreover, the yst gene, which encodes the thermostable enterotoxin Yst protein, advanced the invasion of the Y. enterocolitica into host cells (Atkinson and Williams, 2016). The ystA and ystB are produced by pathogenic and non-pathogenic Y. enterocolitica, respectively (Howard et al., 2006). The yadA gene is one of the most important virulence plasmids of Y. enterocolitica, its product is implicated in auto-agglutination, serum resistance, in addition to adhesion (Atkinson and Williams, 2016). Also, virF (lcrF) codes transcriptional activators of the yop regulon (Cornelis et al., 1989).
The estimation of Yersinia species is commonly occurred by an examination of the biochemical profile. In contrast, biochemical identification is laborious and restricted, as biochemically atypical strains might be hard to assign to a species (Nanni et al., 1991). Thus, trials were applied to improve the capability to study this bacterium in samples through the development of sensitive and specific PCR assays for the recognition of Yersinia species. The Y. enterocolitica is popular reason in cases of food poisoning and is involved in a wide range of gastrointestinal diseases, the evolution of a PCR assay that could be employed to designate Y. enterocolitica positive sample to a particular bio-group has considerable implications for microbiological research, and epidemiological research (Havens et al., 2003). The sequence analysis of small subunit ribosomal RNA (16S rRNA) or the correlated genes (16S rDNA), then comparing the sequence with the other bacterium is more specialized analysis to appoint genus of bacterium (Woese, 1987;Schmidt and Relman, 1994). The sequence determinants have been simplified by the practice of PCR assay to produce the targets, then direct sequence analysis of the products, consequently terminate the requirement for cloning (Bottger, 1989).
Over several years, many antibiotics have been synthesized, resulting in satisfaction with the risk of bacterial resistance. Resistant of microorganisms to antimicrobial agents as a consequence of chromosomal changes or the interchange of genetic material via plasmids and transposons (Neu, 1992). Determination of drug resistance and the detection of virulence genes have influenced a clinical investigation (Li and Fanning, 2017). Y. enterocolitica was previously detailed to be extremely susceptible to many antimicrobial agents exclude penicillin, ampicillin, amoxicillin-clavulanic acid, and the first-generation cephalosporins (Bolton et al., 2013). Though, the high prevalence of drug-resistant Y. enterocolitica strains in food and the environment have been stated in recent years, because of excessive use of antibiotics in animal farms and antibiotic-resistance bacteria/gene transmission amongst dissimilar species (Ye et al., 2016).
To the best of our data, a limited investigation is available on the estimate of Y. enterocolitica isolated from meat and meat products in Egypt. Therefore, in such investigation, Y. enterocolitica isolated from meat and meat products were tested for their virulence genes, antibiotic susceptibility as well as 16s rRNA gene sequence analysis in Egypt.

Samples collection
A total of 700 random representative samples of chicken meat, beef, ground beef, and sausage samples (175 samples, for each) were purchased from 100 different supermarkets and retail outlets in different localities at Dakahlia Governorate, Egypt, from October 2017 to April 2019. Each sample was weighed, marked clearly, put in a separate sterile plastic bag and kept in icebox during transportation to the laboratory. Each sample was estimated to bacteriological examination.

Isolation and Identification of Y. enterocolitica
A 25 g aliquot of each sample was put into sterile bags containing 225 mL of phosphate-buffered saline pH 7.6 added with 1% sorbitol and 0.15% bile salts and homogenized by bag mixer for 2 min. These diluted samples were incubated at 25 °C for 2-3 d in a shaker incubator. Subsequently, 0.5 mL of the enriched samples was mixed with 4.5 ml of potassium hydroxide (KOH) 0.25% and inoculated onto Yersinia-selective agar (Cefsulodin-Irgasan-Novobiocin (CIN) [Oxoid, UK] agar) plates which then were incubated at 30 °C for 1-2 d. The suspected small colonies with a deep red center and sharp border surrounded by a clear colorless zone with the entire edge in the CIN agar plates were selected. Such characteristic colonies for Yersinia were biochemically confirmed by catalase, oxidase, triple sugar iron, and urease tests (Wang et al., 2009;Liang et al., 2012) Y. enterocolitica were oxidase and H 2 S-negative, catalase-and urease positive, glucose fermenter, and non-lactose-fermenter. Moreover, the determination of virulence plasmid of Y. enterocolitica was performed by auto-agglutination test and Congo red absorption by Congo red magnesium oxalate agar (Oxoid) (Mastrodonato et al., 2018) and were examined for its capability of biofilms production using the tube method (Hassan et al., 2011).

Molecular determination of Y. enterocolitica and virulence encoded genes
PCR assays were completely performed to detect specific 16s rRNA gene for Y. enterocolitica, chromosomal-encoded virulence genes (ail, inv, ystA, and ystB) and plasmid-coded virulence genes (yadA). The extraction of bacterial genome DNA from purified suspected colonies was occurred by the conventional boiling method (Zeinali et al., 2015). The amplification of extracted of DNA was done in Applied Biosystem, 2720 Thermal Cycler (USA) in a total volume of 25 μL consisted of 12.5 μL of 2× PCR master mix (Promega, Madison, USA), 1 μL of individual primer (Metabion, Germany), 4.5 μL PCR-grade water, and 6 μL DNA template. The specific primers used and the PCR conditions were summarized in Table 1. The amplified PCR products were arranged on a 1.5% agarose gel which was stained by 1% ethidium bromide and photo-documented under UV illumination. Y. enterocolitica (ATCC 9610) was used as a model of positive control.

Sequencing of the 16s rRNA in Y. enterocolitica
The purification of amplified product was formed from one representative strain by a QIAquick PCR product extraction kit (Qiagen, Valencia, CA) and was sequenced with Bigdye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) using an Applied Biosystems 3130 Genetic Analyzer (Hitachi, Tokyo, Japan) according to the instructions of the manufacturer.

Sequence analysis
The comparison of sequences of this strain was achieved with other strains on GenBank using BLAST 2.0 and PSI-BLAST research programs, (NCBI). A comparative analysis of sequences was made by the CLUSTAL W multiple sequence alignment program, version 1.83 of the MegAlign module of Lasergene DNAStar software Pairwise, which was intended by Thompson et al. (1994). The phylogenetic analyses were performed using maximum likelihood, neighbor-joining process, and maximum parsimony in MEGA6 (Tamura et al., 2013).

Nucleotide accession number
In this research, the nucleotide sequence of the Yersinia enterocolitica strain YEDH88, comprising the 16s rRNA gene was deposited in GenBank under accession no. MK910030.

Determination of virulence genes in Y. enterocolitica strains
Y. enterocolitica isolates were screened for the existence of chromosomal virulence genes (ail, inv, ystA, and ystB) and plasmid virulence gene (yadA) (Figures 2-6). From all samples, the ystB gene was detected with a high percentage of 78.1% (32/41), followed by inv gene with    Figure 7).

Results of antibiotic susceptibility testing
The phenotypic resistance of Y. enterocolitica isolates using the disk diffusion assay displayed a high resistance to AMC (100%), followed by cefazolin (95%), ampicillin (65.9%), and doxycycline (51.2%). On the other hand, the highest susceptibility rate to gentamicin and ciprofloxacin (97.6% each) was observed, followed by SXT and chloramphenicol (92.7% each), kanamycin and cephalotin (85.4% each), and fosfomycin (70.7%) ( Table 4). Furthermore, thirteen resistance patterns were identified in the examined isolates ( Table 2). Among    Virulence genes of Y. enterocolitica strains were distributed as shown in Table 2. The chromosomal (ail, inv, ystA, and ystB) encoded virulent genes were demonstrated in the several categories of Y. enterocolitica strains in meat and meat products. Of note, plasmid (yadA) encoded virulence gene was determined only in one isolate (No. 1), obtained from ground beef. Twelve isolates from chicken meat, two isolates from beef, nine isolates from ground beef and sausage carried the gene ystB. Ten isolates from chicken meat, two isolates from beef, nine isolates from ground beef, and eight from sausage had the inv gene. Two isolates from chicken meat and ground beef, one isolate from beef and sausage contain the ail gene.

Identification of the sequence of the 16s rRNA gene in Y. enterocolitica strain
Sequencing of the 16s rRNA gene from one purified PCR product of one Y. enterocolitica strain YEDH88 isolated from ground beef was achieved. This Y. enterocolitica strain these resistance patterns, the most common pattern was AMC/CFZ represented by 12 (29.3%) strains followed by AMC/CFZ/AM/DOX displayed by 10 (24.3%) strains. Remarkably, multidrug resistance (MDR) to more than two antimicrobial classes was demonstrated in 29 out of 41 (70.7%) strains to show 13 resistance patterns. Of note, the presence of virulence determinants (ail, inv, ystA, ystB, and yadA) in different Y. enterocolitica strains recovered from meat and meat product samples showed different antimicrobial resistance patterns as illustrated in Table 2. The detailed analysis exhibited relations of resistance phenotypes with potential virulence genes.

Discussion
Y. enterocolitica is one of the most common Gramnegative bacteria causing food poisoning, widespread in The environment, water, meats, and dairy products. Meat and meat products had been suggested as the main source of Y. enterocolitica for humans. In the United States and Canada, the high incidence of Y. enterocolitica was reported, even though this might be a result of the improvement of investigation and detection methods (Wesley et al., 2008). In this work, Y. enterocolitica overall prevalence was 5.9% in the meat and meat products. Y. enterocolitica prevalence in chicken meat (12%) was slightly lesser than previous investigations: 16.7% in Turkey  Moreover, the prevalence in ground beef (5.1%) was consistent with Ozdemir and Arslan (2015), while this is lower than those detected by other authors (Sırıken, 2004). Also, the occurrence of Y. enterocolitica in beef (1.1%) was compatible with previous studies (Dzomir, 2006), whereas it is lower than that reported in previous investigations as 27.9% in Turkey (Sırıken, 2004) and 5.5% in Poland (Zadernowska and Chajęcka-Wierzchowska, 2016). Furthermore, the low frequency (5.1%) of sausage contamination found is expected and following other reports (Ramirez et al., 2000) since it is a cooked and vacuum-packed product. It is well known that the risk of contamination is difficult to be eliminated in this type of product (Logue et al.. 1996). The contamination of meat and other meat products with bacteria from a slaughterhouse as well as from many equipments during processing, workers, air, and water could have occurred (Ray, 2004). In this study, chicken meat had the highest prevalence of Y. enterocolitica due to slaughter the poultry outside the slaughterhouse under unhygienic condition, also high levels of bacterial contamination occur especially during defeathering and water chilling. further, elevate the contamination levels during evisceration of the carcasses, washing, and processing due to contamination by workers (Pieniz et al., 2019). On the other hand, the beef has the lowest percentage of isolates where the slaughter is done in a slaughterhouse of Mansoura city at Dakahlia Governorate, Egypt, showed lower contamination due to absence of section to slaughter pigs which are the carrier of Y. enterocolitica and excrete this bacterium in their feces (Paixão et al., 2013).
The key role in the Y. enterocolitica pathogenicity is the virulence genes (chromosome and plasmid) (Zheng et al., 2008). In the current research, the presence of chromosomal virulence genes (ail, inv, ystA, and ystB) and plasmid virulence genes (yadA) in Y. enterocolitica was occurred by PCR assay. The high occurrence of the ystB (78.1%) and inv (70.7%) gene in the examined strains was detected. In accordance, Bhagat and Virdi (2007) found a high prevalence of inv (100%) and ystB (79%) genes in pathogenic isolates of Y. enterocolitica. In contrast, Kot et al. (2007) determined the low occurrence of inv (13.75%) and ystB (4.35%) genes. Also, Ozdemir and Arslan (2015) recorded ystB in 20% of the isolates. Furthermore, the low percentage of other examined genes (ail, 14.6% and ystA, 12.2%) detected by this investigation. There was a lot of study that had a low or negative incidence of such virulence genes (ail and ystA) in pathogenic strains of Y. enterocolitica isolated from meat and other meat products (Falcão et al., 2006;Kot et al., 2007;Bhagat and Virdi, 2007;Ozdemir and Arslan, 2015). On the other hand, previous reports have a high prevalence of such virulence genes isolated from humans (Zheng et al., 2008;Frazão and Falcão, 2015). Besides, The yadA gene was identified in 2.4% of Y. enterocolitica strains in this research, while Tadesse et al. (2013) detected yadA gene in 12.8% of Y. enterocolitica strains isolated from porcine. There was no detection of the yadA gene in any samples isolates from chicken meat (Shabana et al., 2015).
Generally, pathogenic strains should have whole virulence genes (ail, inv, ystA, and yadA) such virulence genes were perhaps cooperating to cause a public health hazard (Zheng et al., 2008). However, in this work, only one isolate containing all tested virulence genes. A lot of the examined isolates in this research were positive for ystB and inv. On the other hand, other isolates were positive for only some of them, which could still be representative of public health hazards. In contrast to methods of identifying bacteria using the phenotype, the genetic-based approach stands out for its consistency. The small subunit ribosomal RNA (16S rRNA), highly preserved and seldom variable within species, is one desirable candidate and is becoming a principal method for phylogeny study and species classification (Woese, 1987). Therefore, in this study, DNA sequence analysis of the 16s rRNA gene of Y. enterocolitica isolate YEDH88 showed the genetic relatedness amongst 26 Y. enterocolitica strains isolated from many countries as shown in the phylogenetic tree (Figure 7). Similar 16s rRNA genes were previously specified in Y. enterocolitica as Y. enterocolitica T51A14.1 (KM888064.1) (Murros et al., 2016), Y. enterocolitica FE81536 (HE803738.1) (Sihvonen et al., 2012), Y. enterocolitica DSM 13030 (NR_116786.1) (Murros-Kontiainen et al., 2011) Y. enterocolitica FE81198 (HE803743.1) (Wortberg et al., 2012), Y. enterocolitica 2516-87 (CP009838.1) (Johnson et al., 2015).
Antibiotic resistance in pathogenic strains has been increasingly developed around the world in particular Y. enterocolitica (Ozdemir and Arslan, 2015). In this study, assay results from the antimicrobial sensitivity of Y. enterocolitica isolates provided high resistance to AMC followed by cefazolin, ampicillin, and doxycycline that were frequently reported (Bucher et al., 2008;Fredriksson-Ahomaa et al., 2010;Frazão et al., 2017). Such high resistance to AMC and ampicillin is due to the wide distribution of β-lactamases amongst Y. enterocolitica isolates (Fredriksson-Ahomaa et al., 2011). In contrast, the sensitivity of Y. enterocolitica to gentamicin, ciprofloxacin, SXT, chloramphenicol, kanamycin, and cephalotin was noticed in previous studies (Hadef et al., 2015;Frazão et al., 2017). Gentamicin and ciprofloxacin, the most clinically important antimicrobial, has been used very successfully in Y. enterocolitica osteomyelitis and septic arthritis (Carniel, 2006).
Interestingly, MDR pathogenic bacteria cause stiffness in the treatment of diseases affecting humans and animals and strains MDR of Y. enterocolitica were associated with the rise of the the morbidity, compared to the susceptible bacterium (Drozdov et al., 1992;Jean and Hsueh, 2011). Unfortunately, the results obtained in this research detected MDR against more than two antibiotics in 70.7% of isolates with 13 resistance patterns. However, there is little studies on the multidrug-resistant strains of Y. enterocolitica in meat and meat products in Egypt compared to other countries. Similar results in MDR Y. enterocolitica isolates were observed by many investigators (Bonardi et al., 2010;Thong et al., 2018). Younis et al. (2019) identified a low prevalence of MDR Y. enterocolitica isolates (23.33%) from retail and processed meat in Egypt, while Ye et al. (2015) and Peng et al. (2018) demonstrated the high occurrence of MDR Y. enterocolitica strains (94.3%, 92.3%) in China, respectively. There are many reasons for the elevation of percentages of multidrug-resistant pathogenic bacterium including the illegal and inaccurate prescription of antibiotics, the long term use and even abuse of feed antibiotics (Dehkordi et al., 2014). While the global use of antibiotics in modern animal husbandry plays an important role in improving the prevention and control of animal diseases, the elevation of animal growth and high feed utilization rate, frequent use of veterinary antibiotics much more than the essential treatment of animal disease, and most of these antibiotics are used to improve the feed conversion and feed additives (Tsubakishita et al., 2010;Silva et al., 2011). Elevation of the microbial resistance problem and expectations for future use of antimicrobial drugs remains uncertain. Consequently, measures must be taken to reduce this problem, for example, to control the antibiotics used, to develop the research to better understand the genetic mechanisms of resistance, and to continue studies to improve new drugs. The ultimate goal is to provide the patient with appropriate and effective antimicrobial drugs (Höfling et al., 2010). Whatever, diversity in the use of many antibiotics in the treatment of humans and animals causes elevation of the microbial resistant bacterium to the human beings. The transmission of antibiotic-resistant bacteria to humans may be caused by the means of food, so the antimicrobial resistance of isolates in human and animal foods should be monitored continuously to avoid public health hazards (McDermott et al., 2002).
Generally, the acquirement of the antimicrobial resistance in the bacteria influences their virulence according to two alternative ways; elevated resistance is followed by elevated virulence (a positive effect) or raising antimicrobial resistance decreases a bacterium virulence (seemingly negative effect) (Beceiro et al., 2013). The presence of virulence determinants (ail, inv, ystA, ystB, and yadA) in different Y. enterocolitica isolates displayed various antimicrobial resistance patterns in this investigation. This research verified the dissemination of antimicrobial resistance patterns and virulence factors in the examined isolates. These results are important concerning public health and had been formerly reported (Sacchini et al., 2018). The antimicrobial resistance of bacterium is regularly developing and horizontal gene transmission by plasmids plays the main role (Rozwandowicz et al., 2018).

Conclusion
Meat and meat products might be a source of virulent and multi-drug resistant strains of Y. enterocolitica that might have a potential public-health hazard in Egypt. Accordingly, strict hygienic measures should be applied to minimize Y. enterocolitica contamination in meat and meat products. BECEIRO, A., TOMÁS, M. and BOU, G., 2013. Antimicrobial resistance and virulence: a successful or deleterious association in the bacterial world? Clinical Microbiology Reviews, vol. 26, no. 2, pp. 185-230. http://dx.doi.org/10.1128/CMR.00059-12. PMid:23554414. BHAGAT, N. andVIRDI, J.S., 2007. Distribution of virulenceassociated genes in Yersinia enterocoliticabiovar 1A correlates with clonal groups and not the source of isolation. FEMS Microbiology Letters,vol. 266,no. 2,