Introduction

Chicken meat is considered one of the most consumed meats worldwide, and the average world annual production of chicken meat increased from 83.3 million metric tons in 2012 to about 103.4 million metric tons in 2023 (https://www.statista.com/statistics/237637/production-of-poultry-meat-worldwide-since-1990/). Chicken meat is widespread among Egyptian consumers across all income denominations. In Egypt, poultry meat consumption is about 1.2 billion birds per year. Furthermore, the total quantity of poultry meat consumed in Egypt increased from 1.13 million metric tons in 2016 to 1.46 million metric tons in 2020 (https://www.fao.org/faostat/en/#data/FBS).

Chicken is the leading reservoir for Salmonella, which is mainly present in the intestines of live birds. Chicken carcasses are contaminated mainly with Salmonella species owing to fecal cross-contamination, so Salmonella could cling to the skin of chicken carcasses and/or become stuck inside the skin feather follicles [1]. Live bird markets constitute the primary source for chicken carcass contamination by Salmonella species. The poor hygienic practices in live bird markets may permit the spread of foodborne pathogens to humans as live birds were housed at high intensity in cages with confined spaces until they were slaughtered and sold as freshly dressed carcasses. Chicken carcasses are contaminated mainly due to unhygienic practices during the several processing steps, such as slaughtering, scalding, plucking, evisceration, washing, and chilling. Moreover, cages, chopping boards, butchers’ hands, and knives are potential sources for contamination of chicken carcasses by Salmonella serotypes [2].

Nowadays, there are over 2700 Salmonella serotypes [3]. Salmonella species isolated from chicken carcasses include a wide variety of serovars, for instance, S. Typhimurium, S. Kentucky, S. Enteritidis, S. Infantis, S. Molade, S. Bargny, and S. Inganda [4, 5]. Although most Salmonella infections are mild, some can be life-threatening according to the serotype and host factors. Salmonella causes abundant morbidity and mortality, mostly in developing countries [6]. S. Typhimurium and S. Enteritidis caused around 75% of human salmonellosis cases, represented by fever, vomiting, diarrhea, nausea, and abdominal cramps [7].

Salmonella is one of the most important foodborne pathogens in humans and animals worldwide and has been widely related to foodborne outbreaks. Approximately 1.3 billion infection cases and 155,500 deaths annually worldwide are attributed to Salmonella [8]. In the USA, about 1.35 million infections, 26,500 hospitalizations, and 420 deaths yearly were caused by non-typhoidal Salmonella, leading to a loss of $400 million in direct medical costs [9]. Salmonellosis is a leading foodborne pathogen in Egypt; nevertheless, there was no national surveillance with reliable statistics on its health and economic load [10].

The pathogenicity of Salmonella species is based on many virulence genes that could cause severe infections, such as the invasion A (invA) gene, enterotoxin gene (Stn), and Salmonella plasmid virulence C protein gene (spvC) genes [6]. Many virulence-determinant genes are grouped in specific genomic regions known as Salmonella Pathogenicity Islands (SPIs), gained via genetic transfer across bacterial pathogens [11]. Salmonella pathogenicity islands (SPI-1 to SPI-5) are located at the large chromosomal DNA region and help in the invasion of the genus to host epithelial cells. SPI-1 and SPI-2 have many genes encoding type III secretion systems (TTSSs), which is a needle-like device that helps Salmonella to inject its effectors across the intestinal epithelial cell membrane into the cytoplasm, which permits Salmonella to rearrangement of the actin cytoskeleton in host epithelial cell, leading to ruffling (outward extension) of the epithelial cell membrane to engulf the bacteria [6]. The invA gene (SPI-1 gene) is the Salmonella marker gene that allows Salmonella to invade host epithelial cells [12]. The enterotoxin stn gene has biological activity like that of the cholera toxin (CT) and encodes a protein that causes gastroenteritis with various symptoms including nausea, vomiting, abdominal pain, fever, and diarrhea [12, 13]. The spvC gene has a significant role in the survival of Salmonella in host cells and reaction with the host defense mechanism and reduction of cytokine production, additionally, it helps systemic invasion of Salmonella in the host cells and could be used as a standard for detecting the pathogenicity of Salmonella isolates [14].

The emergence of multidrug-resistant bacteria (MDR) against the commonly used antimicrobials is alarming as it could reduce the therapeutic options for treating complicated Salmonella infection cases [7]. Annually, more than 2.8 million people in the United States have been infected by antibiotic-resistant bacteria [9]. Salmonella strains isolated from chicken samples exhibit a variety of antibiotic resistance profiles. The MDR bacteria harboring antimicrobial resistance genes can be transmitted through food of animal origin to humans, especially chicken and its giblets [15]. In recent years, Salmonella isolates have demonstrated high levels of resistance to the most clinically important antimicrobials, such as cephalosporins and fluoroquinolones, leading to many morbidity and mortality cases globally [16].

The β-lactams antibiotics have a top place in the antibacterial armamentarium and the widespread emergence of multidrug-resistant bacteria carrying beta-lactamase genes among foods of animal origin is considered a worrisome threat to public health. Beta-lactamases are bacterial enzymes that hydrolyze the β-lactam ring in β-lactam antibiotics. The most common β-lactamase resistance genes are blaOXA, blaCTX, blaSHV, and blaTEM [17]. The most predominant β-lactamases in multi-drug-resistant Salmonella isolates are the CTX-M family, followed by TEM and SHV [18]. Extended-spectrum β-lactamases (ESBLs) confer resistance to third-generation cephalosporins (cefotaxime, ceftriaxone, and ceftazidime) [19].

The emergence of MDR Salmonella strains, particularly resistant ones to the most critically important antimicrobial classes such as polymyxin, fluoroquinolones, and third- and fourth-generation cephalosporins, is worrisome. The current study, therefore, intended to determine the prevalence, virulence genes (invA, stn, and spvC genes), antimicrobial resistance profiles, and β-lactamase resistance genes (blaOXA, blaCTX-M1, blaSHV, and blaTEM) of Salmonella strains isolated from native chicken carcasses in Egypt marketed in Mansoura, Egypt, as well as highlight the hazard of isolated MDR-, colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars to public health.

Materials and methods

Collection and preparation of samples

A total of 150 freshly dressed chicken samples were purchased from various poultry shops with various sanitation levels in Mansoura city, Egypt during the period from July 2022 to November 2022. Whole chicken carcasses were separately packed in sterile polyethylene bags, held at 4 °C in an insulated ice box, and transported within an hour to the Laboratory of Food Hygiene, Safety, and Technology Department, Faculty of Veterinary Medicine, Mansoura University, Egypt, wherein the bacteriological analyses for Salmonella were performed immediately.

Isolation of Salmonella

The preparation of chicken samples, isolation, and identification of Salmonella was done according to the methodology recommended by the Food Safety and Inspection Service of the United States Department of Agriculture [20]. Each chicken carcass was weighed (ranging from 1.0 kg to 1.8 kg), placed in a Whirl–Pak bag, and then 400 ml of sterile buffered peptone water (BPW; CM0509B; Oxoid Ltd., Basingstoke, UK) was added. The carcass was rinsed manually for 10 min to ensure that BPW was in contact with the external and internal surfaces of the chicken carcass, and then the chicken rinsate was mixed with buffered peptone water and incubated at 37 °C for 20–24 h.

Salmonella isolation was performed according to the International Organization for Standardization (ISO) [21]. From the incubation, an enrichment was done by inoculating 0.1 ml of cultured buffered peptone water into 10 ml of Rappaport–Vassiliadis broth (RV; CM0669; Oxoid Ltd., Basingstoke, UK) then incubated at 42 °C for 20–24 h. After incubation, a loopful from each enriched broth was streaked onto the selective solid media; xylose-lysine-desoxycholate (XLD) agar (Oxoid, CM0469; Oxoid Ltd., Basingstoke, UK) then the inoculated plates were incubated at 37 °C for 24 h. All typical presumptive Salmonella colonies (pink with or without black center) on XLD agar were picked up and cultured onto nutrient agar plates (CM0003; Oxoid Ltd., Basingstoke, UK) and incubated at 37 °C for 24 h. Presumptive Salmonella colonies were exposed to additional confirmation by biochemical, molecular, and serological identifications. Biochemical tests conducted were triple sugar iron (TSI; Oxoid Ltd., Basingstoke, UK) test, urease test (CM0053B, urease Agar Base (Christensen), Oxoid Ltd., Basingstoke, UK), indole production (SIM medium, CM0435, Oxoid Ltd., Basingstoke, UK), methyl-red (MR) test (MRVP medium, CM0043, Oxoid Ltd., Basingstoke, UK), Simmons citrate (Titan media, India) test, and Voges-Proskauer (VP) test (MRVP MEDIUM, CM0043, Oxoid Ltd., Basingstoke, UK). Isolates confirmed biochemically to be Salmonella were serologically identified.

Serological identification of Salmonella isolates

PCR-verified Salmonella isolates were classified into serovars according to the Kauffmann–White scheme by slide agglutination test depending on monovalent and polyvalent O and H antisera (Denka-Seiken, Tokyo, Japan) [22].

Molecular detection of virulence genes in Salmonella isolates

The biochemically verified Salmonella isolates were further confirmed by applying the polymerase chain reaction (PCR) targeting the invA, stn, and spvC genes. According to the manufacturer's prescript, the genomic DNA of suspected Salmonella isolates was extracted using QIAamp® genomic DNA extraction kits (QIAGEN, Germantown, MD, USA).

Detection of the invasion gene (invA) was performed using the forward (5′-ACAGTGCTCGTTTACGACCTGAAT-3′) and the reverse (5′-AGACGACTGGTACTGATCGATAAT-3′) primer sequence sets, which yield an amplified band size of 244 bp [23]. Detection of the enterotoxin gene (stn) was performed with the forward (5′-CTTAATCGCGCCGCCATGCTGTT-3′) and the reverse (5′- CATGAACTGGCGCAGGTGAT-3′) primer sequence which produces an amplified DNA size of 480 bp [24]. The detection of the Salmonella plasmid virulence gene (spvC) was performed with primer sequence sets (forward: 5′-ACCAGAGACATTGCCTTCC-3′; and reverse: 5′-TTCTGATCGCCGCTATTCG -3′) which yield an amplified DNA size of 467 bp [25].

The PCR amplification of invA, stn, and spvC genes was applied using a SimpliAmp thermal cycler (Thermo Fisher Scientific Inc, UK). The protocol of PCR cycling for the three genes detected was done as an initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 5 min. Ten microliters of each amplified PCR product were electrophoresed in 1.5% agarose gel (Puregene™, India) for 50 min at 95 V and then visualized under an ultraviolet transilluminator (acculab, Montréal, Québec, Canada). A 100-bp DNA ladder (Solarbio; Beijing Solarbio Science & Technology Co., Ltd., China) was used as a marker for PCR products.

Antibiotic susceptibility testing for Salmonella isolates

Antibiogram profiles of 129 molecularly-verified Salmonella isolates against sixteen antimicrobials related to eleven antibiotic classes were done using the disk-diffusion method on the Mueller–Hinton agar (MH; CM0337; Oxoid Ltd., Basingstoke, UK) for all the antibiotics tested except the polymyxins class (Colistin), where the minimum inhibition concentration (MIC) was applied and determined by broth microdilution technique according to Clinical and Laboratory Standards Institute guidelines [26]. Salmonella isolates with colistin MICs ≥ 4 μg/ml were interpreted as resistant.

The isolates were tested against various classes of antibiotics which included polymyxins class (Colistin), Carbapenems (Meropenem, MEM—10 μg), Sulfonamides (Trimethoprim/Sulphamethoxazole, SXT—25 μg), Quinolones (Nalidixic acid, NA—30 μg; Levofloxacin, LEV—5μg; Ciprofloxacin, CIP—5 μg), Tetracyclines (Tetracycline, TE—30 μg), Aminoglycosides (Gentamicin, CN—10 μg), Cephalosporins (Cephalothin, KF—30 μg; Cefaclor, CEC—30 μg; Cefotaxime, CTX—30 μg; Cefepime, FEP—30 μg), Macrolides (Azithromycin, AZM—15 μg), Phosphonic antibiotics (Fosfomycin, FOS—50 μg), Extended-spectrum beta-lactamases (Ceftazidime/Clavulanic acid, CAZ/CLA—10/30 μg), Glycopeptides (Vancomycin, VA—30 μg). All antibiotic discs were purchased from Oxoid (Oxoid Ltd., Basingstoke, UK).

According to the antimicrobial resistance profiles, Salmonella isolates were classified into multidrug-resistant (MDR) if they showed resistance to at least one antimicrobial agent in three or more antimicrobial classes, extensively drug-resistant (XDR) when they were resistant to all tested antimicrobial classes except one or two antimicrobial classes, while considered pan drug-resistant (PDR) when they showed resistance to all tested antimicrobials in all antimicrobial classes [27]. The MAR “multiple antibiotic resistance” index was calculated for all Salmonella isolates as the ratio of the number of antimicrobials to which an isolate was resistant to the total number of antimicrobials tested [28]. MAR index > 0.2 implies high-risk contamination and the misuse of antibiotics.

Detection of β-lactamase resistance genes

Cefotaxime-resistant Salmonella isolates (n = 82) were tested using multiplex polymerase chain reaction targeting the β-lactamase resistance genes. The adopted primer set sequences and DNA amplification protocol were previously described by Perez et al. [29] for blaOXA, blaCTX-M1, and blaTEM genes and Ogutu et al. [30] for the blaSHV gene. Primers used in this study were constructed to yield 564, 655, 713, and 800 bp for blaOXA, blaCTX-M1, blaSHV, and blaTEM, respectively. The protocol of PCR cycling for these genes was done as an initial denaturation at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 61 °C for 35 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 8 min. Ten microliters of each amplified PCR product were electrophoresed in 1.5% agarose gel for 90 min at 80 V and then visualized under an ultraviolet transilluminator. A 100-bp DNA ladder was used as a marker for PCR products.

Statistical analysis

Data analysis was performed using the SPSS program (SPSS Inc., Chicago, IL; v 21). The distribution of virulence genes and resistance rates of Salmonella serovars isolated against different antimicrobial agents tested was determined using the chi-square (χ2) test.

Results

One hundred and fifty freshly dressed native chicken carcasses were collected from different poultry shops to isolate Salmonella species using standard bacteriological techniques, comprising pre-enrichment in BPW, selective enrichment in RVS broth, and cultivating on the surface of XLD agar. All presumptive isolates were subjected to biochemical tests, serological identification, and PCR targeting the invA gene. Salmonella isolates were examined for the existence of two selected virulence genes (stn and spvC) and antimicrobial susceptibility testing by Kirby–Bauer disc diffusion method for all (n = 16) antimicrobial agents tested, except for colistin, where the MIC was determined by broth microdilution technique. Additionally, cefotaxime-resistant Salmonella isolates (n = 82) were tested using multiplex PCR targeting the β-lactamase resistance genes, including blaOXA, blaCTX-M1, blaSHV, and blaTEM genes.

Phenotypic characteristics of the recovered Salmonella isolates

Conventional cultural and morphological characteristics revealed 357 (51/150; 43%) suspected Salmonella isolates. The morphological characters of presumptive Salmonella colonies on XLD were pink colonies with or without black centers. Based on the biochemical tests, Salmonella isolates are Voges-Proskauer, urease, and indole negative, and positive TSI, methyl red, and Citrate utilization tests.

Prevalence of Salmonella spp. in freshly dressed chicken carcasses

Of the 357 presumptive Salmonella isolates determined based on conventional cultural and biochemical identification methods, only 129 isolates from 27 native chicken carcasses were confirmed as Salmonella depending on molecular identification of the invA Salmonella marker gene with an overall prevalence of 18% (27/150). Salmonella prevalence rate decreased after molecular confirmation compared to conventional cultural and biochemical identification methods, indicating the accuracy and reliability of molecular techniques.

Serotypes of Salmonella isolates recovered from chicken carcasses

The molecularly confirmed Salmonella isolates (n = 129) were serologically identified into 12 different Salmonella serotypes. Salmonella Kentucky, S. Enteritidis, S. Typhimurium, and S. Molade were the most prevalent serotypes with an incidence of 19.4% (25/129), 17.1% (22/129), 17.1% (22/129), and 10.9% (14/129), respectively. Moreover, S. Tsevie, S. Takoradi, S. Inganda, S. Muenster, and S. Labadi were detected with an incidence of 6.9% (9/129), 6.2% (8/129), 6.2% (8/129), 5.4% (7/129), and 4.6% (6/129), respectively. While the less common serotypes were S. Giza (2.3%, 3/129), S. Chester (2.3%, 3/129), and S. Apeyeme (1.6%, 2/129) (Fig. 1).

Fig. 1
figure 1

Serological identification of the 129 Salmonella enterica Serovars isolates recovered from native Egyptian chicken carcasses

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Prevalence, and distribution of virulence genes among Salmonella isolated from chicken carcasses

Only 129 of the 356 isolates detected by conventional cultural and biochemical identification methods were molecularly confirmed as Salmonella via PCR amplification of the 244-bp DNA fragment of the invA marker gene specific for Salmonella species. The other virulence genes detected were stn, and spvC, which were amplified at a molecular size of 480, and 467 bp, respectively (Fig. 2). All tested Salmonella isolates were positive for both invA and stn genes, while only 31.8% (41/129) of isolates examined were positive for the spvC gene (Fig. 2).

Fig. 2
figure 2

Representative agarose gel electrophoresis for PCR assay of the virulence genes detected in Salmonella species. A invA (244 bp); B stn (480 bp); C spvC (467 bp) in Salmonella isolates recovered from native Egyptian chicken carcasses. M; DNA marker (100-bp gene ladder). C+; Control positive, C–Control negative. Ten microliters of the PCR product were separated by electrophoresis on 1.5% agarose gel and visualized under UV light

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Antimicrobial resistance of Salmonella enterica serovars isolates (n = 129) recovered from native Egyptian chicken carcasses

The Salmonella isolates in the current study showed a high resistance rate of 92.3% (119/129), 82.9% (107/129), 82.2% (106/129), 82.2% (106/129), and 63.6% (82/129) against vancomycin, nalidixic acid, cefepime, colistin, and cefotaxime, respectively (Table 1), while they exhibited a mediate resistance rate of 51.9% (67/129), 50.4% (65/129), 50.4% (65/129), 48.8% (63/129), 47.3(61/129), and 42.6% (55/129) against ceftazidime/clavulanic acid, levofloxacin, tetracycline, ciprofloxacin, cefaclor, and cephalothin, respectively (Table 1). On the other hand, Salmonella isolates showed lower resistance rates of 32. 6% (42/129), 31% (40/129), 28.7% (37/129), 11.6% (15/129), and 10.1% (13/129) towards gentamicin, sulfamethoxazole/trimethoprim, Fosfomycin, azithromycin, and meropenem, respectively (Table 1).

Table 1 Antimicrobial susceptibility of Salmonella enterica serovars isolates (n = 129) recovered from native Egyptian chicken carcasses
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Classification of Salmonella isolates based on their antibiotic resistance profile and their multiple antibiotic resistance (MAR) index

The antimicrobial resistance profile of all Salmonella isolates (n = 129) examined toward 16 antimicrobial agents revealed 48 different patterns and 121 (93.8%) of the 129 Salmonella-verified isolates were resistant to at least 3 antibiotics (Table 2). Furthermore, 3.9% (5/129), 14.7% (19/129), 75.2% (97/129), and 6.2% (8/129) of Salmonella isolates tested were categorized according to their antibiotic resistance phenotype into pan-drug-resistant, extensively drug-resistant, multidrug-resistant, and low drug-resistant, respectively (Table 2). Amazingly, five isolates (3.9%, 5/129) comprised 4 Salmonella Enteritidis isolates and 1 Salmonella Typhimurium isolate revealed resistance to all antimicrobial agents tested with a MAR index equal to 1 (Table 3).

Table 2 Antimicrobial resistance profile and MAR indexes of Salmonella serovars isolates (n = 129) from native Egyptian chicken carcasses
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Table 3 Classification of Salmonella enterica server isolates (n = 129) according to their antimicrobial resistance profile against the 16 antimicrobial agents tested
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Distribution of β-lactamase resistance genes among MDR Salmonella isolates

In the present study, β-lactamase-resistance genes encompassing blaOXA, blaCTX-M1, blaSHV, and blaTEM were identified in cefotaxime-resistant Salmonella isolates at molecular sizes of 564, 655, 713, and 800 bp, respectively (Fig. 3). Thirty-one (37.8%) of the 82 cefotaxime-resistant Salmonella isolates tested were β- lactamase producers and had at least one of the blaOXA, blaCTX-M1, or blaTEM β-lactamase resistance genes (Fig. 4). The distribution of β-lactamase-resistance genes among the positive 31 Salmonella serovars indicated that the blaTEM was the most predominant β-lactamase resistance gene and was identified in 25.6% (21/82) of the isolates, followed by blaCTX-M1 and blaOXA genes, which were detected in 19.5% (16/82) and 17.1% (14/82) of Salmonella isolates tested, respectively (Fig. 4). Conversely, the blaSHV gene was not detected in any of the Salmonella isolates examined. Interestingly, two isolates comprised one Salmonella Kentucky and one Salmonella Typhimurium among the 31 β-lactamase producers isolates had the three identified β-lactamase resistance genes: blaOXA, blaCTX-M1, and blaTEM (Fig. 3 & Table 4).

Fig. 3
figure 3

Representative agarose gel electrophoresis for multiplex PCR screening of β-lactamase-resistant genes demonstrated by blaOXA (564 bp), blaCTX-M1 (655 bp), blaSHV (713 bp), and blaTEM (800 bp) detected in Salmonella isolates recovered from the native Egyptian chicken carcasses. M; DNA marker (100-bp gene ladder). C+ Control positive, C– Control negative. Lane 1: S. Enteritidis (blaCTX-M1- and blaTEM-positive); Lane 2: S. Labadi (blaTEM-positive); Lane 3: S. Apeyeme (blaCTX-M1-positive); Lane 4: S. Kentucky (blaOXA-, blaCTX-M1-, and blaTEM-positive); Lane 5: S. Enteritidis (blaTEM-positive); Lane 6: S. Typhimurium (blaCTX-M1- and blaTEM-positive); Lane 7: S. Typhimurium (blaCTX-M1-positive); Lane 8: S. Apeyeme (blaOXA-positive); Lane 9: S. Kentucky (blaTEM-positive); Lane 10: S. Kentucky (blaCTX-M1- and blaTEM-positive); Lane 11: S. Enteritidis (blaOXA-positive); Lane 12: S. Kentucky (blaCTX-M1-positive). Seven microliters of the PCR product were loaded and separated by electrophoresis on 1.5% agarose gel and visualized under UV light

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Fig. 4
figure 4

Distribution of the identified β-lactamase resistance genes among the cefotaxime-resistant Salmonella isolates (n = 82)

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Table 4 Correlation between the phenotypic and genotypic profile of multidrug resistance β-lactamase-producing Salmonella enterica serovars (n = 31) identified among Salmonella isolates retrieved from native Egyptian chicken carcasses
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Discussion

Prevalence of Salmonella spp. in freshly dressed chicken carcasses

Salmonella is a leading foodborne pathogen and has been widely linked to severe foodborne outbreaks cases worldwide. Chicken is the main reservoir of Salmonella, which is mainly present in the intestines of live birds [1]. Furthermore, live bird markets are the prime source of Salmonella contamination of chicken carcasses. In Egypt, consuming poultry is controlled by cultural legacies, as most Egyptian consumers prefer to go to live poultry shops to select chicken to be slaughtered and receive freshly dressed chicken carcasses. However, most of these shops lack hygienic practices during the slaughtering and processing techniques. In the current study, 357 presumptive Salmonella isolates were identified based on conventional cultural morphological characteristics (pink colonies with or without black centers on XLD agar) and biochemical identification methods. The suspected Salmonella isolates were tested by PCR targeting Salmonella marker gene, the invA gene. A total of 129 isolates from 27 native chicken carcasses were confirmed as Salmonella with an overall prevalence of 18% (27/150). A similar prevalence rate of Salmonella species in chicken carcasses was reported in Egypt by Abd-Elghany et al. [24], who found that 16% of whole chicken carcasses examined were contaminated with Salmonella spp. By comparison, a higher prevalence of Salmonella in chicken carcasses was reported by other researchers; for instance, Salmonella species were detected in 29.4% (50/170) of whole chicken carcasses examined in Egypt [4]. Moreover, 25.1% (156/622) of chicken carcasses in the abattoir environment of Taiwan [2] and 36.4% (138/ 379) of chicken carcasses in two different commercial poultry processing plants in Canada [31] were contaminated with Salmonella spp.

Leakage of crop and intestinal contents at the time of the evisceration process are considered the leading sources of poultry contamination by Salmonella during slaughtering and processing procedures [32]. Chicken carcasses can be also contaminated with Salmonella species due to improper cleaning and sanitation procedures, inadequate chilling and storage temperature, the presence of insects and rodents, and poor personal hygiene in poultry shops [2], besides the contaminated knives, wooden tables, weighing scales, scalding water, chilling tanks, processing equipment such as plucking machines, and cross-contamination from one carcass to another.

Serotypes of Salmonella isolates recovered from chicken carcasses

Salmonella serotypes isolated from chicken vary among geographic regions; S. Kentucky is the most prevalent serotype in the present study, which is consistent with a previous study conducted by Awad et al. [5], who found that S. Kentucky was the dominant serovar among Salmonella isolates from retail chicken meat in Egypt with an incidence of 22.6% followed by S. Molade with an incidence of 6.5%. Nonetheless, S. Typhimurium, S. Enteritidis, and S. Kentucky were the most prevailing serovars recovered from chicken meat [4, 5, 24, 33].

Among the identified 129 Salmonella isolates recovered from freshly dressed native chicken carcasses examined in the present study, 9 were serotyped as S. Tsevie at a percentage of 6.9%, which seemed higher than the 3.9% of S. Tsevie identified among recovered Salmonella isolates from broiler chicken flocks in Qalyubiya Governorate, Egypt [34]. On the other hand, 8 (6.2%), 8 (6.2%), 7 (5.4%), and 6 (4.6%) of the 129 isolates recovered in the current study were serotyped as S. Takoradi, S. Inganda, S. Muenster, S. Labadi, respectively; similarly, such serovars were identified among Salmonella isolates isolated from chicken carcasses collected from different shops and supermarkets distributed in Mansoura city, Egypt [5, 24]. Interestingly, the least prevalent Salmonella serovars in the current study encompass S. Giza, S. Chester, and S. Apeyeme, which were identified only among 3, 3, and 2 of the 129 Salmonella isolates, respectively. Previous studies also indicated the identification of S. Giza, S. Chester, and S. Apeyeme in low incidences among Salmonella isolates from chicken samples examined in different governorates in Egypt [12, 35, 36], which require more monitoring to protect public health.

Prevalence, and distribution of virulence genes among Salmonella isolated from chicken carcasses

In the current study, all Salmonella isolates tested were positive for both invA and stn genes, while only 31.8% (41/129) of isolates examined were positive for the spvC gene. These results are closely similar to those reported by many researchers. For instance, all Salmonella serovars isolated from chicken carcasses collected from different shops and supermarkets distributed in Mansoura city, Egypt had both invA and stn genes [5, 24]. On the other hand, 25.3% (42/166) of Salmonella isolates from chicken carcasses examined harbored the spvC gene [23], while 39.9% of Salmonella enterica serovar Typhimurium recovered from retail raw chickens in China, were positive for the spvC gene [37].

The frequency distribution of the spvC gene among the 12 different Salmonella serovars identified indicated that S. Kentucky (n = 12) harbored a high frequency of the spvC gene, followed by S. Enteritidis (n = 10), S. Typhimurium (n = 9), S. Tsevie (n = 3), S. Takoradi (n = 2), S. Muenster (n = 2), S. Giza (n = 2), and S. Chester (n = 1), while spvC gene is absent in S. Molade, S. Inganda, S. Labadi, and S. Apeyeme. The spvC gene has a prime role in the systemic invasion of the genus in the host cells and could be used as a standard for detecting virulent Salmonella strains [14]. Consequently, Salmonella isolates from chicken carcasses harboring the spvC gene constitute a tremendous public health issue and need a strict monitoring program to avoid the spread of such virulent isolates via food of poultry origin.

Antimicrobial resistance of Salmonella isolates and their classification based on the resistance profile and the multiple antibiotic resistance (MAR) index

The spread and emergence of antimicrobial resistance have been related to the overuse or abuse of antibiotics in animals and humans [38]. In the veterinary field, antibiotics are frequently used as therapeutic, growth promoters, or to enhance the efficiency of food utilization and weight. Multidrug resistance has emerged worldwide as a growing threat to public health threat. Several recent studies concerning the emergence of MDR pathogens from different origins increase the necessity for rationalizing antibiotic usage in veterinary and human medicine [11, 39,40,41,42,43]. Salmonella serovars with MDR patterns can produce a variety of multidrug resistance plasmids that harbor resistance genes that mediate resistance to many antimicrobials. Recently, Salmonella isolates have undergone several genomic changes and acquired resistance against broad-spectrum cephalosporins through mutated genes that encode for extended-spectrum β-lactamases, hydrolyzing antibiotics with β-lactam rings [6].

The high antimicrobial resistance of Salmonella isolates in the current study toward vancomycin, nalidixic acid, and cefepime suggests that these antibiotics are widely used in veterinary medicine. Likewise, Salmonella enterica isolates from chicken meat in Turkey exhibited a high resistance rate of 98.8% (83/84) and 89.3 (75/84) towards vancomycin and nalidixic acid, respectively [44].

Surprisingly, 82.2% (106/129) of Salmonella enterica isolates in the present study were resistant to colistin; however, colistin is not the drug of choice for treating Salmonella infection. A previous study from our laboratory revealed that 39.2% (62/158) of the identified Salmonella enterica serovars isolates, recovered from poultry (whole duck, pigeon, and quail carcasses) collected from Mansoura, Egypt, were resistant against colistin [10]. In this study, 51.93% of isolates were resistant to ceftazidime/clavulanic acid, which is a better screening method for the extended-spectrum beta-lactamases (ESBL) in the Enterobacteriaceae family [45]. Additionally, 50.4%, 32.6%, and 31% of isolated Salmonella strains in the current study were resistant to tetracycline, gentamicin, and sulfamethoxazole/trimethoprim, which are widely used in veterinary medicine as growth promoters, broad-spectrum antibiotic or prophylaxis. In this context, Siriken et al. [44] in Turkey found that 91.6% (77/84), 32.1% (27/84), and 4.8% (4/84) of Salmonella enterica isolates from chicken meat were resistant to tetracycline, sulfamethoxazole/trimethoprim, and gentamicin, respectively.

Salmonella enterica isolates in the current study revealed a high resistance rate toward the cephalosporin antibiotics encompasses cephalothin, cefaclor, cefotaxime, and cefepime, which constitute a leading global problem as cephalosporins, especially the third- and fourth-generation are the critically important antimicrobials for salmonellosis treatment. Amazingly, the resistance of Salmonella isolates against cephalosporins followed the order: cefepime (fourth-generation cephalosporin) > cefotaxime (third-generation cephalosporin) > cefaclor (second-generation cephalosporin) > cephalothin (first-generation cephalosporin), which indicates the improper use and overuse of the third- and fourth-generation cephalosporins in poultry industry. Likewise, most cephalosporin-resistant isolates from poultry in Korea obtained after 2016 were mainly resistant to third- and fourth-generation cephalosporins [46].

Fluoroquinolones are highly effective broad-spectrum antibiotics used mainly for treating human salmonellosis. Due to the wide use of fluoroquinolones in human and animal medicine, high resistance rates of 50.39% and 48.84% were observed against levofloxacin and ciprofloxacin, respectively. By comparison, 63.1% and 44.2% of Salmonella isolates from raw chicken meat in Colombia were resistant to ciprofloxacin and levofloxacin, respectively [47]. Moreover, 30.8% (8/26) of Salmonella enterica serovars recovered from broiler chickens and chicken carcasses in Egypt were resistant to ciprofloxacin [48]. Fluoroquinolone-resistant Salmonella serovars isolated from chicken carcasses are alarming as Fluoroquinolones are the mainstay antibiotics for complicated salmonellosis cases.

Salmonella enterica isolates in the current study revealed a low resistance rate of 28.7%, 11.6%, and 10.1% toward fosfomycin, azithromycin, and meropenem, respectively. Fosfomycin displays substantial activity against Gram-negative pathogens involving Salmonella spp. The widespread of fosfomycin-resistant Salmonella strains constitutes a crucial public health threat as fosfomycin could be an effective treatment option. Likewise, 15.4% of Salmonella isolates from broiler chickens and chicken carcasses in Egypt were resistant to azithromycin [48]. On the contrary, 100% of Salmonella isolates from broiler carcasses in Colombia were susceptible to imipenem [47]. The emergence of meropenem- and azithromycin-resistant Salmonella isolates poses a tremendous public health issue, as they obstruct treatment options for salmonellosis and could increase morbidity and mortality rates.

The average multiple antibiotic resistance (MAR) index for the 129 isolates tested was 0.505, with 82.2% (106/129) of Salmonella isolates showing a MAR index above 0.2. A MAR index greater than 0.2 indicates the abuse and excessive use of antimicrobial agents in poultry farms [27]. Therefore, establishing a strict monitoring system to rationalize antimicrobial usage in poultry farms is crucial to protect public health from transferring antimicrobial-resistant bacteria to humans via food of animal origin.

Distribution of β-lactamase resistance genes among MDR Salmonella isolates

Extended-spectrum β-lactamases (ESBLs) confer resistance to third-generation cephalosporins (cefotaxime, ceftriaxone, and ceftazidime) [19]. The most common genetic variant of ESBL is CTX-M [49]. The β-lactamase genes provide resistance to many β-lactam antibiotics, especially cephalosporins (cefotaxime) [10]. A former study revealed that the blaTEM was detected in Salmonella serovars isolated from broiler chickens and chicken carcasses in Egypt [48]. On the other hand, another study indicated that most of the ESBL-producing Salmonella strains (n = 9) isolated from diseased and apparently healthy farmed chickens carried blaTEM and blaSHV genes, whereas the minority possessed blaOXA [12]. The emergence of multidrug-resistant (MDR) Salmonella species harboring beta-lactamase genes among foods of animal origin highlights the need for surveillance strategies to diminish the usage of antibiotics in veterinary medicines and prevent the transmission of such resistant strains to humans. Consequently, the implementation of the Hazard Analysis Critical Control Point (HACCP) could reduce the hazard of transmission of such pathogenic strains to humans via chicken carcasses.

Conclusion

The emergence of colistin-, cefepime-, and levofloxacin-resistant Salmonella serovars among Salmonella isolates from native chicken is worrisome because these antibiotics are the critically important antimicrobials used for treating complicated salmonellosis cases. The current study revealed that native chicken carcasses marketed in Mansoura, Egypt, are contaminated with multidrug-resistant Salmonella enterica serovars, which constitutes a tremendous threat to public health. The most predominant Salmonella serotypes are S. Kentucky, S. Enteritidis, S. Typhimurium, and S. Molade. All Salmonella isolates examined harbored both invA and stn genes, with 31.8% of isolates carrying the spvC gene, which is detected only in highly pathogenic Salmonella strains. Furthermore, 31 (37.8%) out of 82 cefotaxime-resistant Salmonella isolates tested were β-lactamase producers and had at least one of the following β-lactamase resistance genes: blaTEM, blaCTX-M1, and blaOXA, Therefore, establishing a strict surveillance system to restrict antibiotic use in poultry farms is decisive in protecting public health from transmitting antimicrobial-resistant bacteria to humans via food of poultry origin. Besides, more studies are requested on the emergence and development of antimicrobial-resistant Salmonella, which carries many virulent and resistant genes in chicken carcasses.