Isolation, Identification, and Genetic Characterization of Antibiotic Resistance of Salmonella Species Isolated from Chicken Farms

Salmonella is a major cause of foodborne outbreaks. It causes gastroenteritis in humans and animals. This micro-organism causes severe illness in chickens and has a major impact on chicken productivity and the poultry industry. This study aimed to address the prevalence of Salmonella infection in broiler chicken farms in Kafrelsheikh, Gharbia, and Menofeya provinces in Egypt during 2020–2022. This work also aimed to evaluate the genetic characterization and antibiotic resistance of the isolated Salmonella strains. Clinical signs and mortalities were observed and recorded. In total, 832 samples were collected from 52 broiler flocks, including 26 from both one-week-old and 6-week-old chicken farms from different organs (liver, intestinal content, spleen, and gallbladder). The prevalence of Salmonella infections was reported in the study region to be 36.54%. Of the 26 one-week-old farms surveyed, 11 (42.31%) and 8/26 (30.77%) of the six-week-old broiler chicken farms had Salmonella infections. Recovered isolates were serotyped as 9 (47.37%) S. enteritidis O 1,9,12, ad monophasic H: g, m: -, 6 (31.58.%) S. shangani 2, (10.53%) S. gueuletapee 1, (5.26%) S. II (salamae), and 1 (5.26%) untypable. The results showed that Salmonella infection was predominant in one-week-old chicks compared to infection in six-week-old and uninfected flocks. All Salmonella isolates were resistant to ampicillin and erythromycin, while all isolates were sensitive to ciprofloxacin, chloramphenicol, and levofloxacin. The isolates also contained 10.53% (2/19) streptomycin, 10.53% (2/21) gentamicin, 15.79% (3/19) doxycycline, and 26.32% (5/19) lincomycin and colistin. The phenotypically resistant Salmonella samples against ampicillin, erythromycin, and macrolide harbored blaTEM, blaSHV, ermB, ereA, mphA, and ermB, respectively. This baseline data on Salmonella spp. prevalence, serotyping, and antibiotic profiles are combined to define the antimicrobial resistance to this endemic disease. Elucidation of the mechanisms underlying this drug resistance should be of general importance in understanding both the treatment and prevention of Salmonella infection in this part of Egypt.


Introduction
Chicken is a signifcant source of eggs and meat. Te poultry production-related industry is one of the economically important agro-industry components [1].Salmonella spp. is one of the most causative agents of diseases in poultry and avian species [2]. It causes heavy economic losses because of its high mortality rate and reduced production rate in poultry [3]. Te economic cost of the Salmonella spp. outbreak has been estimated to be $11.6 billion in the USA [4] and more than €3 billion in the European Union [5].
Poultry is one of the most preferable reservoirs for Salmonella spp., which will allow it to transmit to humans through food [6]. Poultry meat is thought to be the most common source of Salmonella infection in humans, accounting for roughly 40% of clinically reported cases [7].
Salmonella spp. is a Gram-negative bacteria that belongs to the family Enterobacteriaceae [8]. Salmonella spp. is an opportunistic zoonotic organism that infects human and animal cells through contaminated food and the environment [9]. It infects a wide variety of cells, such as M cells, epithelial cells, dendritic cells, and macrophages [10]. It can survive both in the absence and presence of oxygen [11].
Salmonella spp. chickens are usually divided into three classes based on the diseases they cause [12]. Te frst class comprises nonmotile, chicken-adaptedSalmonellae, which include S. gallinarum, which causes fowl typhoid, and S. pullorum, which causes pullorum disease in chickens [13]. Fowl typhoid (FT) and pullorum disease (PD) are septicemic diseases that are usually most signifcant in growing and adult chickens. In mature poultry, symptoms of FT and PD include reduced egg production, reduced fertility, decreased hatchability, anorexia, and increased mortality [14].
Te second class of Salmonella that afects birds is the invasive, nonhost-specifcSalmonella, which can infect more than one host, including animals and humans, and is called paratyphoid Salmonella in birds. Tis type of bacteria causes paratyphoid in birds, and it is of zoonotic concern. Te paratyphoid Salmonella includes 10-20 serovars. S. enteritidis, S. typhimurium, S. shangani, S. gueuletapee, and S. II salamae are the most important serovars [15].
Salmonella enteritidis, S. typhimurium, S. shangani, S. gueuletapee, and S. II salamae have been reported to be the most common salmonellae isolated from Egyptian poultry farms [16]. Tey are transmitted horizontally between farms and vertically to the progeny through trans-ovarian infection [16]. Te clinical manifestations of paratyphoid infections are most predominant in young chickens, especially in the frst few weeks of life. Te most common symptoms associated with paratyphoid disease in broilers include depression, anorexia, and diarrhea, with high mortalities, especially in the frst week of life. While in adult birds, the infection is asymptomatic, and the infected birds are considered carriers, which are the most dangerous source for the shedding of bacteria in meat and eggs of zoonotic concern (Tiwari, Swamy, et al.).
Te third class of Salmonella is neither host-adapted nor invasive and may cause disease in humans and other animals [14].
Te widespread use of antibiotics on poultry farms as growth promoters or prophylactics as well as for treatment can raise concerns about antibiotic resistance, which has been reported in many Salmonella spp. serovars [17]. During the second half of the twentieth century, there were two signifcant breakthroughs in the epidemiology of nontyphoidal salmonellosis throughout the world [18]. First, multidrug-resistantSalmonella typhimurium strains, such as S. typhimurium DT104, have arisen, and second, Salmonella enteritidis has emerged as a prominent poultry and egg pathogen [19].
Amoxicillin (β-lactam antibiotic) competitively inhibits penicillin-binding protein 1. By producing an enzyme called a β-lactamase, which attacks the β -lactam ring; bacteria frequently become resistant to β-lactam antibiotics. Prophylactic β-lactam resistance develops through four main mechanisms: the production of a β-lactamase enzyme (primarily in Gram-negative bacteria), low expression of external membrane proteins, alterations in the dynamic site of penicillin-binding proteins (PBPs), and active efux [20]. Tere are genes that are associated with resistance to β-lactamasebla TEM-1 , bla TEM-2 , and bla SHV-1 . Te β-lactam ring of penicillin is hydrolyzed by bla TEM β-lactamases, which is how they work. Tere are three types of SHVs (sulfhydryl variables): 2b, 2be, and 2ber. Penicillin and frstand second-generation cephalosporins are hydrolyzed by type 2b; third-generation cephalosporins are hydrolyzed by type 2be; while clavulanic acid and tazobactam are resistant to type 2br. Every year, new β-lactamase variants are recorded, and this poses a challenge to the medical feld [21].
Erythromycin stops bacteria from producing their protein by attaching to the bacterial cell membrane and the 50S subunit of the ribosome. A small 30S subunit and a large 50S subunit make up the bacteria's ribosome. Te latter has at least 30 proteins and 23S rRNA. Erythromycin inhibits protein synthesis by attaching to the 50S subunit. Erythromycin ribosomal methylase is a ribosomal enzyme that modifes the 50S subunit's binding site for erythromycin. It is encoded by the ermB gene. Te modifcation gene markedly reduces the afnity of erythromycin for its target [22]. Macrolides, including erythromycin, inhibit bacterial protein synthesis by binding at the exit tunnel of the 50S ribosomal subunit. Tey do this by preventing peptidyl transferase from adding the growing peptide attached to tRNA to the next amino acid. It also inhibits bacterial ribosomal translation [23]. Macrolide inactivation also occurs by phosphotransferases encoded by mphA and mphB [24]. A resistance enzyme that preferentially inactivates 14-membered macrolides (such as erythromycin, telithromycin, and roxithromycin) over 16-membered macrolides is encoded by the mphA gene (e.g., tylosin and spiramycin). It phosphorylates macrolides in a GTP-dependent manner at the 2′-OH hydroxyl group of the desosamine sugar of macrolides [25]. Resistance to macrolides may also be due to the ereA gene (erythromycin resistance esterase type I) [26]. Tis encodes the erythromycin esterase enzyme, which causes enzymatic hydrolysis of the macrolactone ring [23].
Serotyping is a basic biomarker for investigating the epidemiology status of Salmonella infections, and it's frequently used to allocate the source of contamination during epidemics [27]. Tis method was established by White and Kaufmann based on the detected phase-shift fagella antigen and fagella H, somatic O antigen [2]. Te method addressed is considered the reference one for the serotyping of Salmonella spp. Serotyping of Salmonella spp. has many advantages, including details regarding the disease's severity, the source of contamination, and the pattern of resistance. Molecular characterization methods have been used to identify diferences between Salmonella strains. Tese methods include PCR, pulsed-feld gel electrophoresis (PFGE), random amplifcation of polymorphic DNA (RAPD), etc. [28].
Tis study aimed to isolate and identify Salmonella spp. from diferent provinces in Egypt. Te study also concludes the investigation of antimicrobial resistance against 11 diferent clinically relevant antimicrobials and the molecular characterization of resistance-attributed genes.

Sampling Strategy and Salmonella Isolation.
Tis study has conveniently targeted 52 broiler chicken focks (Avian 48, Abdelsalam Hegazy Company), of which 26 were oneweek-old chick focks and 26 were six-week-old birds. Tese farms were investigated for Salmonella infections. Te broiler chicken focks were surveyed in Kafrelsheikh, Gharbia, and Menofeya provinces in Egypt during 2020-2022. Te birds showed diferent clinical signs, including reluctance to move, pasty diarrhea, huddling near the source of the heat, rufing feathers, dehydration, decreased body weight gain, droopy wings, lameness, and high mortalities of 9.64% ± 1.72 in one-week-old chicks from each broiler chicken farm. Four living, diseased birds were selected randomly and sacrifced. At postmortem, sections from the liver, gallbladder, spleen, and intestinal contents were collected under aseptic conditions for Salmonella isolation. Within fve hours of collection, samples were delivered to the lab and stored on ice until then. Selenite-F broth (SFB) (Oxoid, UK) was combined with one gramme of tissue from each organ and incubated statically at 37°C for an overnight period. Te enrichments were applied to XLD agar (Oxoid, UK) using a swap, and they were then incubated at 37°C overnight. One colony from each plate that appeared to be Salmonella spp. was chosen for additional examinations based on appearance [29].

Biochemical Identifcation.
Te pure pink colonies on XLD agar with black center colouration were taken as suspected colonies of Salmonella spp. According to Lamboro et al. [30], these bacterial colonies were confrmed biochemically as Salmonella spp. [30]. Te biochemical tests used for Salmonella spp. detection were IMViC reactions that included indole, methyl red, Vogues Proskauer, oxidase, and citrate utilization tests [31]. Urease hydrolysis and hydrogen peroxide production were also tested [31].

Serological Identifcation of Salmonella Isolates.
Serotyping of suspected Salmonella strains was conducted at the Animal Health Research Institute, Dokki, Giza, Egypt, according to the manufacturer's instructions (Denka Seiken Co., Tokyo, Japan). Briefy, the isolates were examined with an omnivalent A-67. Te positive isolates were tested with anti-Salmonella A-E and anti-Salmonella F-67. Te samples were identifed by using anti-Salmonella antibodies grouped by specifc O antigens (2, 4, 7, 8, etc.). Te samples were tested for grouped anti-Salmonella H antigen phases 1 and 2.

Genomic DNA Extraction and Purifcation.
A single colony was collected from each plate and inoculated into fve ml of selenite-F broth SFB (Oxoid, UK) throughout the course of an overnight period at 37°C. One minute of 13000 rpm centrifugation was performed on one milliliter of bacterial culture broth in a microcentrifuge tube. After removing the supernatant, the bacterial pellets were heated at 95°C for 10 minutes while being homogenized with water devoid of nucleases. Finally, the boiled lysates were centrifuged, and the supernatant was removed to create DNA templates that were stored at − 80°C until use [32].

Molecular Detection of the Salmonella Genus and Antimicrobial Resistance-Associated Genes.
Te ompC gene was used as a specifc determinant for Salmonella spp. detection [33]. Te amplifcation of ompC PCR was performed using primers, as shown in Table 1, according to the method described by the authors of [33]. Salmonella isolates were screened for fve genes known to be associated with antibiotic resistance to ampicillin, erythromycin, and macrolides. Tese genes are bla TEM , bla SHV , and ermB, ereA, and mphA, respectively, as shown in Table 1 according to the methods described by [34,35].
Briefy, primers were utilized in a 25 μl of uniplex PCR mix, comprising 12.5 μl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 μl of each primer (20 pmol), 5.5 μl of water, and 5 μl of DNA template. Te reaction was performed in an Applied Biosystems 2720 thermal cycler. Te cycling condition started with primary denaturation at 94°C for 5 min, followed by 35 cycles and a fnal extension at 72°C for 10 min. Te specifc annealing of each gene is shown in Table 1. Te positive controls were represented by feld samples that were previously confrmed to be positive by PCR for the antimicrobial resistance-related genes in the reference laboratory for veterinary quality control on poultry production, an Animal Health Research Institute. Te Salmonella ATCC 9184 strain was used as a control positive for ompC gene detection, while sterile water was added to the PCR mix with each primer pair as a control negative.
2.6. Te Antimicrobial Susceptibility Test. Antimicrobial susceptibility testing (AST) was carried out using the Kirby-Bauer disc difusion method as recommended by the CLSI [36]. E. coli ACTC25922 and E. coli NCTC10418 were used as quality control strains during AST. Te AST for Salmonella isolates was conducted against 11 antimicrobial agents that are clinically used in the Egyptian poultry industry. Tis includes ciprofoxacin (CIP 5 μg), chloramphenicol (C 30 μg), streptomycin (STR 10 μg), gentamicin (CN 10 μg), erythromycin (E 15 μg), doxycycline (DO 30 μg), levofoxacin (LEV 5 μg), ampicillin (AM 10 μg), lincomycin (L 2 μg), norfoxacin (NOR 10 μg), and colistin (CT 10 μg). Te tested Salmonella inoculum was prepared by direct saline suspension of a nutrient broth culture from an isolated colony on selective XLD agar plates that had been incubated for 18 to 24 hours. Te bacterial suspension of tested Salmonella was adjusted in sterile saline by adding approximately one ml of overnight bacterial suspension to 4 ml of sterile saline to match the 0.5 McFarland standard (containing approximately 1-2 x 10 8 CFU/ml for American Type Culture Collection (ATCC) 2592 E. coli) by using a McFarland densitometer (Biomerieux Biotechnology, UK). Using a sterile swab, the saline suspension was applied to the Mueller-Hinton Agar plate (Oxoid, UK). Antibioticcontaining antimicrobial discs were strewn throughout the Mueller-Hinton agar surface after it had been inoculated. Overnight, the agar plates were incubated at 37°C. Using sliding calipers and interpretation, the diameters of the inhibited zones, including the diameter of the discs, were measured and observed according to the Clinical Laboratory Standards Institute (Table 2) [37].

Statistical Analysis.
Student's t-tests were employed using Microsoft Excel software for the percentage of mortalities related to Salmonella infection and the rate of isolation of Salmonellae from internal organs, according to the method described by [38].

Clinical Signs, Incidence, and Mortalities of Salmonella spp.
Samples were collected from 52 broiler chicken farms from the study regions, and clinical symptoms of Salmonella infection were gathered at the time of sampling. Te symptoms, which included diarrhea, dehydration, decreased body weight gain, lameness, and signifcant mortalities, were primarily seen in one-week-old broiler chicks. Hepatitis, hepatomegaly with necrotic foci, arthritis, typhlitis, omphalitis, myocarditis, and pneumonia were the predominant postmortem pathologies. However, the symptoms were less severe in older birds at the 6 th week of age.

Molecular Identifcation of Salmonella Isolates.
Te isolates were confdently identifed as Salmonella spp. by amplifcation of the ompC gene ( Figure 1). Te PCR confrmed 19 of the Salmonella isolates that were identifed phenotypically and biochemically.

Discussion
Salmonella species are members of the Enterobacteriaceae family. Tey are nonspore-forming, facultatively anaerobic, and Gram-negative rods [8]. Tey pose a signifcant challenge in our lives nowadays. Salmonella spp. can be found in all poultry products that are consumed by humans, including meat and eggs. However, it can contaminate other food products and infect humans, so it is considered a health-threatening organism [39]. It is responsible for a variety of poultry diseases, including fowl typhoid, pullorum, and paratyphoid diseases. Our results showed that the clinical signs of paratyphoid Salmonellae including, S. enteritidis, S. typhimurium, S. shangani, S. gueuletapee, and S. II salamae, were more severe in young birds than in older ones. Tis may be due to a defciency of benefcial microfora in the intestine of young chicks obtained from hatcheries, which makes them susceptible to infection with Salmonella. Tese results were consistent with [14]. In this study, 832 samples were collected from 52 poultry focks, and we found that 19 (2.28%) of them were positive for Salmonella spp., and the young age was more afected by the disease than the old age, as the clinical signs and mortalities were higher at the young age than others. Te isolation of Salmonella from the liver (3.36%) and gallbladder (2.88%) was signifcantly (P < 0.05) higher compared to that of the spleen and intestinal content (1.44%). Tese fndings could be explained by the high invasive ability of these motile Salmonellae. Te results found in this study were close to those of El-Sharkawy et al. [16]. Our results indicated that the isolates were serotyped as 9 (47.37%) S. enteritidis O 1,9,12, ad monophasic H: g, m: -, 6 (31.58.%) S. shangani 2, (10.53%) S. gueuletapee 1, (5.26%) S. II (salamae) and 1 (5.26%) untypable. Our results were in the same line as described by the authors of [40]. In this study, Salmonella spp. can be considered a major pathogen and an important hazard for the poultry industry, particularly young broilers due to the    high mortalities, which showed levels of (9.64% ± 1.72) in one-week-old chicken farms compared to the none infected focks (2.5% ± 0.99). Tis may be due to diarrhea dehydration, and severe lesions in the liver and other vital organs caused by infection with these motile and invasive Salmonellae. Our fndings also showed that there was no signifcant diference in mortality rates between infected and uninfected 6-week-old focks (P � 0.15). Our results were compatible with the study conducted by El-Sharkawy et al. [16].
In the study area, ampicillin and erythromycin are the recommended frst-line agents used for the treatment of poultry infections. However, these antibiotics are misused because they are not used in the right doses and durations, given the high burden of developing antimicrobial resistance strains of bacteria against these agents. Tis study discovered   Journal of Tropical Medicine that all detected Salmonella strains were erythromycin-and ampicillin-resistant. Indeed, these antibiotics were the most commonly prescribed without AST. Tey were also the most easily available on the market without a prescription because they were also very cheap. A similar study revealed that Salmonella spp. was more sensitive to levofoxacin, norfoxacin, ciprofoxacin, chloramphenicol, gentamycin, streptomycin, doxycycline, and colistin, while it was more resistant to ampicillin, erythromycin, and lincomycin [41,42]. Genes responsible for extended-spectrumβ-lactamases (ESBL) production arise by a point mutation at the active site of the earlier β-lactamases and are usually plasmidmediated. In addition, ESBL-positiveGram-negative bacteria often carry genes that confer high resistance levels to many other antibiotics [43]. Tis can limit the chemotherapeutic options for ESBL-producing pathogens and facilitate the interspecies and intraspecies dissemination of ESBLs. Terefore, phenotypic detection of ESBLs among Enterobacteriaceae species is important for epidemiological purposes and for limiting the spread of resistance mechanisms.
In this study, ampicillin resistance of Salmonella spp. was dependent on the presence of bla TEM 18/19 (94.7%) and bla SHV 19/19 (100%) of isolated Salmonella. Our fnding agreed with the results described in a previously reported study by the authors of [44]. Furthermore, we found that the erythromycin resistance of Salmonella isolates was attributed to ermB 6/19 (31.58%), ereA 2/19 (10.53%), and mphA 19/19 (100%), which harbored by resistant Salmonella isolates. Similar results were observed by [44]. Te presence of at least one of these resistance mechanisms in all resistant strains may have been responsible for an increasing number of mortalities in one-week-old broiler chicken farms.

Conclusion
Tis study has been focused on giving a clear pattern of the current situation of Salmonella spp. infection in broiler chickens, especially in Egypt. Salmonella spp., including prevalence, serotyping, and an antimicrobial resistance profle. As a result, it is prudent for farmers to develop and share knowledge about salmonellosis diagnosis, treatment, and prevention protocols in order to reduce economic losses and human health risks. Limiting disease burdens would not only improve the well-being of managed broilers but also provide new avenues for achieving the WHO's global development goal of eliminating poverty and famine.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
All experimental protocols were approved by the Committee on Research, Publication, and Ethics of the Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, which complies with all relevant Egyptian legislation in research and publications.