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.