Molecular Identication of Five Multidrug-resistant Salmonella Enterica Serovars Isolated from Egyptian Poultry Farms

Salmonella spp is the main cause of foodborne salmonellosis that is considered a public health threat all over the world. The robust usage of antibiotics in Egyptian poultry farms resulted in increasing the prevalence of multi-drug resistant Salmonella enterica. In this study, the authors identify ve multi-drug resistant Salmonella enterica serovars. Multidrug-resistant characteristics of these isolates were detected. The taxonomic evidence of these isolates was investigated based on 16S rRNA gene sequence and pairwise sequence alignment between the isolates' sequence and the nearest sequences in the database. In silico restriction maps and phylogenetic trees were also constructed. The obtained sequences were deposited in the database under accession numbers MW311328.1, MW311371.1, MN820824.1, MN822653.1 and MW310702.1 for Salmonella enterica subsp. enterica serovar Enteritidis strains EG.SmE1, EG.SmE2 and Salmonella enterica subsp. enterica serovar Typhimurium strains EG.SmT1, EG.SmT2, EG.SmT3, respectively. The ve Salmonella isolates in this study showed multi-drug resistant characteristics. Salmonella Typhimurium isolate EG.SmT3 revealed resistance to more than ve antibiotics. Results of pairwise sequence alignment, restriction maps and phylogenetic tree conrmed the close relationship between S. enteritidis isolates (EG.SmE1, EG. SmE2) and S. typhimurium isolates (EG.SmT1, EG.SmT2). However, the other S. typhimurium isolate (EG.SmT3) revealed the lowest identity ratio (98.6%) with the nearest sequence. The relative divergence of this isolate could be attributed to proposed mutations as a result of the vigorous use of antibiotics in Egyptian poultry farms.


Introduction
Salmonella is a Gram-negative bacilliform bacterium, which belongs to the family of Enterobacteriaceae. It is a considerable public health threat responsible for one of the most common food-borne illnesses known as Salmonellosis (García-Soto et al. 2020). Globally, it is estimated that Salmonella spp. outbreaks are responsible annually for nearly about 85% (80.3 million cases) of diseases associated with foodborne diseases (93.8 million cases), resulting in over 100,000 deaths (Majowicz et al. 2010). Salmonellosis symptoms are abdominal cramps, fever, vomiting, in ammatory diarrhea, and nausea occurring within 12-72 h of infection and last from 2-7 days. Severe invasive Salmonella infections such as bacteremia and septicemia, often arise in immunocompromised people, lead to hospitalization and death (Musyoka et al. 2018).
More than 2500 serotypes of Salmonella have been identi ed to date date (Grimont and Weill, 2007;Lamas et al. 2018). A great number of these serotypes (1531) are found in subspecies enterica, among which S. Typhimurium and S. Enteritidis stand out as they are principally responsible for more than 99% of human salmonellosis and therefore, they were widely studied. (Lamas et al. 2018). The adaptability of such subspecies in different environmental stresses augmented the encounters to eradicate them from the food chain (Humphrey, 2004;Spector and Kenyon, 2012). Conventional intervention strategies to control Salmonella spp. were predominantly carried out using antibiotics that were considered as an effective method to reduce Salmonella propagation. However, later this was showing to lead to the emergence of multidrug-resistant During the last years, antimicrobial resistance has emerged quickly amongst Salmonella spp from different animal sources, creating a serious health hazard worldwide. Increasing prevalence and dissemination of different multidrug-resistant (MDR) subspecies enterica in broiler populations resulted in Salmonella spreading in the food chain and via poultry products to humans (García-Soto et al. 2020). In Egypt, the excessive uncontrolled use of antibiotics in poultry farms to treat microbial infections led to expanding of antibiotic resistance subspecies including Salmonella enterica serovar Typhi (S. Typhi) and other diarrhoeagenic strains strains (Wasfy et al. 2002;Aouf et al. 2011). However, with no national Salmonella surveillance center to provide reliable statistical data, little is known about food-borne salmonellosis in Egypt (Abdel-Maksoud et al. 2015).
Identi cation of multidrug-resistant bacteria is the rst step to understand its nature, structure and characteristics to control its spread in the food chain. Molecular techniques based on PCR ampli cation targeting conserved regions inside the 16S rRNA gene sequence has proved to be a useful tool for identifying and exploring phylogenetic relationships among bacterial isolates. The analysis of the 16S rRNA gene sequence provides information about proposed mutation and mobile genetic elements that result in the acquisition of antibiotic resistance in Salmonella spp (Abdel-Maksoud et al. 2015).
In this study, the authors identi ed ve Salmonella enterica serovars isolates from poultry farms with history of diarrhea in Benha city, Qalubiya governorate, Egypt. Multidrug-resistant characteristics of these isolates were detected applying the antibiotics susceptibility test. Based on 16S rRNA gene sequence, the taxonomic evidence of these isolates was investigated.
Single nucleotide polymorphisms (SNPs) between the obtained sequences and the nearest sequences in database were determined. The construction of the phylogenetic trees was also performed.

Materials And Methods
Bacterial strains and growth conditions Samples were collected from a wide range of broiler farms with a history of diarrhea in Qalubiya governorate, Egypt. Fecal samples were collected and incubated overnight in buffered peptone water at 37°C, 100 µl of the overnight pre-enrichment broth was then inoculate a 10 ml Tetrathionate broth (Müller-Kaufmann) and again was incubated overnight at 37°C. Finally, 10 µl loop from the inoculated Tetrathionate broth was spread on Xylose Lysine Deoxycholate (XLD) agar plates and on Brilliant Green (BGA) agar plates, plates were then incubated overnight at 37°C. Salmonella like colonies were selected, subcultured and maintained for further identi cation. On XLD agar, a typical Salmonella colony has a slightly red transparent halo with black centers. On BGA agar, a typical Salmonella colony appear red in a red/pink color agar.
Isolated Salmonella serotypes were determined by identifying the outermost portion of the lipopolysaccharide "O" antigen and the agellar protein "H" antigen. Five isolates of Salmonella enterica serovars were puri ed and stored at -80°C in Brain-Heart-Infusion broth supplemented with 20% (v/v) glycerol.

DNA Extraction and PCR ampli cation
Bacterial genomic DNA from pure isolates was extracted using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. DNA integrity was checked using 1% agarose gel electrophoresis and the image was captured using gel documentation system (Gel Doc. BioRad). Concentration and purity of puri ed DNA were measured by BioTek Epoch2 Microplate reader (Thermo Scienti c, USA). For all samples, DNA purity was >1.8 ± 0.20 under absorbance ratio A260/A280. for 50 s, 55 °C as annealing temperature for 1 min with an extension of 72 °C for 1 min followed by nal extension temperature at 72 °C for 10 min. Ampli ed PCR products were stored at −20 °C for further puri cation and downstream application, then 5 μl of PCR amplicons was loaded on 2 % agarose gel electrophoresis stained with Ethidium bromide using GeneRuler™ 1 kb DNA ladder, then visualized using gel documentation system (Gel Doc. BioRad

Bacteria and Antibiotic sensitivity test
Five isolates of Salmonella enterica serovars were obtained. Among them, three isolates were detected as Salmonella enterica serovar Typhimurium and two isolates were identi ed as Salmonella enterica serovar Enteritidis.
Antibiotic susceptibility patterns of the ve obtained isolates demonstrated a susceptibility and resistant pro le characteristic (Table 1). Qualitative results from the antibiograms showed that all isolates were susceptible to Cipro oxacin. Although Salmonella Typhimurium isolates were resistant to Amoxicillin + Clavulanic Acid 20, Salmonella Enteritidis isolates were susceptible to it. On another hand, all isolates were susceptible to Tetracycline except S. Typhimurium EG.SmT3 that was resistant to Tetracycline. This isolate revealed the highest resistant pattern as it was resistant to ve antibiotics (Ampicillin, Amoxicillin + Clavulanic Acid, Ceftriaxone, Chloramphenicol, Tetracycline). Also, it was categorized as intermediate resistant to Amikacin and Gentamycin. While, the other four isolates were more susceptible to the tested antibiotics. As shown in Table 2  The restriction map of the obtained sequences provided predicted data of restriction sites that found in the ve isolates ( Fig. 1). Totally, 46 restriction enzyme site were observed in both isolates EG.SmE2, EG.SmE1 with similar restriction sites pattern. However, there was one speci c restriction site for each isolate that was ECOP15I in EG.SmE2 and BSrG1 in EG.SmE1. Additionally, the isolates EG.SmT2 and EG.SmT1 displayed relatively similar restriction sites pattern with total number of 48 and 46 restriction enzyme site in both isolates, respectively. On another hand, A completely different restriction sites pattern was observed in isolate EG.SmT3 re ecting the divergence of this isolates compared to other isolates. The GC, AT contents of all isolates was 55% GC and 45% AT.

Phylogenetic construction
From the phylogenetic tree (Fig. 2), it can be inferred that the isolates EG.SmE2, EG.SmE1 were clustered together in the same clade as similar the isolates EG.SmT2 and EG.SmT1 were clustered together. While the isolate EG.SmT3 diverged from them and clustered with the nearest deposited sequence in the database. However, all obtained isolates and nearest sequences in database shared the same ancestor of Salmonella sp that diverged from E-coli ancestor.

Discussion
Antimicrobial-resistant strains of Salmonella spp are now widespread all over the world constituting a major threat to human health. In Egypt, some reports on Salmonella serovars dispersion on poultry farms were documented by ( isolates. The divergence of this isolate among others con rmed not only by the relatively low identity ratio but also by the unique pattern of SNPs and GAPs position. These molecular changes in the conserved sequence of 16S rRNA gene could be proposed mutations that affect the multi-drug resistant characters of this isolate.
From restriction maps, the speci c restriction enzymes ECOP15I and BSrG1 could be used to differentiate between the two isolates EG.SmE2 and EG.SmE1 respectively. The different restriction map of EG.SmT3 compared to other S. typhimurium isolates con rmed the variability in its 16S RNA sequence and consequently its exceptional characteristics as a strong multidrug resistant bacteria.
The phylogenetic tree con rmed the same identity ratios on the roots of clades. It concluded the taxonomic evidence of these isolates and the relative divergence of EG.SmT3 isolate that could attribute to proposed mutations caused by the vigorous use of antibiotics in Egyptian poultry farms. Hence, the continual usage of antibiotics could simulate changes in conserved genomic regions such as 16S rRNA sequence.

Conclusion
The ve Salmonella enterica serovars isolates showed multi-drug resistant characteristics. The isolate EG.SmT3 showed the most multi-drug resistant pro le with relative divergence in sequence similarity. The vigorous use of antibiotics could stimulate mutations in 16S rRNA gene sequence. In further investigation, the isolate EG.SmT3 could be used as a good and strong model for multi-drug resistant Salmonella Typhimurium to assess biocontrol agents against foodborne salmonellosis.