Phenotypic and Genotypic Characterization of Salmonella enterica Serovars Isolated from Imported Poultry

| Monitoring of imported 1-day-old poultry is mandated in Egypt to prevent the possible introduction of new Salmonella serovars into the country’s poultry industry. Such serovars are considered to be a major public health threat. We examined 391 imported poultry flocks for the presence of salmonellae (231 duckling, 84 chick, and 76 turkey poult), serotyped all isolated salmonellae, and performed antimicrobial susceptibility testing. Molecular profiles were also constructed based on results of conventional polymerase chain reaction assays to detect virulence genes ( stn , avrA , and sopB ) and antibiotic resistance genes ( blaTEM , tetA(A) , and qnrS ) in the Salmonella isolates. Thirty Salmonella strains were isolated from the 391 samples (7.7%). By poultry type, salmonellae were isolated from 21 of 231 (9.1%) duckling samples, 6 of 84 (7.1%) of chick samples, and 3 of 76 (3.9%) turkey poult samples. Serotyping of the isolates identified 16 different serovars: S . Enteritidis, S. Typhimurium, S. Sinstorf, S. Muenster, S. Vejle, S. Cuckmere, S. Indiana, S. Infantis, S. Koenigstuhl, S. Macallen, S. Nchanga, S. Neftenbach, S. Newlands, S. Nigeria, S. Nyborg, and S. Regent. The isolates showed variable degrees of antibiotic resistance across species. All tested Salmonella strains harbored the virulence and antibiotic resistance genes, with the exception of qnrS , which was found in only 50% of the isolates. In conclusion, examination of imported poultry is a critical point of control to prevent poultry from becoming a reservoir for human health hazards, including salmonellae with antimicrobial resistance phenotypes .

Uncontrolled and indiscriminate use of antibiotics for growth promotion or prophylaxis, especially in low-and middle-income countries, can lead to the emergence of antibiotic resistance to fluoroquinolones and extended-spectrum beta-lactam antibiotics in the poultry sector (Badr et al., 2015;Velhner et al., 2018). MDR salmonellae are significant for both human and animal health because they can lead to illness that is unresponsive to antibiotic treatment (Chen et al., 2013).
Antibiotic resistance is transmitted between bacteria through mobile genetic elements such as plasmids, transposons, and integrons. Such transmission results in healthy animals becoming carriers for antibiotic-resistant bacteria, which may subsequently cause human infections that are difficult to treat (Mthembu et al., 2019).
Bacterial self-defense against antibiotics involves developing modifications such as decreased cell permeability, alteration or replacement of the target, and enzyme inactivation that serve as mechanisms enabling antibiotic resistance (Frye and Jackson, 2013). For example, bacteria that are resistant to beta-lactam antibiotics produce beta-lactamase, an enzyme that can destroy the beta-lactam ring and thus inactivate the antibiotics (Mthembu et al., 2019). In salmonellae, many extended-spectrum beta-lactamases (ESBLs) exist, with tem, shv, and ctx-M genes being the most effective and frequent encoding genes ( Jin and Ling, 2006).
In gram-negative bacteria, tetA is the most frequent antibiotic-resistance gene, and it may be present on either chromosomes or plasmids (Pezella et al., 2004). This gene enables tetracycline resistance in salmonellae that infect humans and animals (Threlfall, 2002). Qnr proteins are a group of penta peptide repeat proteins that allow bacteria to evade the effects of quinolones and develop resistance to these antibiotics. Qnr genes prevent or limit ciprofloxacin from disabling bacterial DNA gyrase and topoisomerase IV, enzymes that are necessary for bacterial DNA synthesis and thus growth (Tran and Jacoby, 2002).
Various virulence genes are important for Salmonella pathogenesis, such as sopB and avrA, which encode T3SS proteins that are involved in the Salmonella-induced in-flammatory response and have anti-apoptotic roles. Together, the two proteins delay apoptosis of epithelial cells, which aids salmonellae in avoiding host adaptive immune response and in constructing a stable intracellular niche (Wu et al., 2011). Salmonella enterotoxin (stn) is an important virulence factor that causes diarrhea (Chopra et al., 1999). Some authors have reported that stn is not a virulence factor because they did not find any decrease in virulence phenotypes when the stn gene was deleted in tested mutant (Nakano et al., 2012). In contrast, earlier research (Chopra et al., 1999) found that inactivation of the stn gene reduced the ability of Salmonella Typhimurium ability to induce intestinal fluid accumulation.
The present study was conducted to determine the presence of salmonellae in chicks, ducklings, and poults being imported into Egypt. The isolates were studied with regard to their phenotypic characteristics and identified by serovar. In addition, their antimicrobial resistance profiles and genotypic profiles were defined based on important virulence genes (stn, avrA, and sopB) and antibiotic resistance genes (blaTEM, tetA(A), and qnrS).

etHics approval
Birds were handled in accordance with the regulations for collecting samples from live animals, and this study was approved by the animal care committee of the Animal Health Research Institute.

saMpling
We obtained 391 samples from imported poultry flocks (231 ducklings, 84 chicks, and 76 turkey poults) from January to December 2019, by collecting the paper-lined the boxes used to transport the birds. Each box contained 30 young birds. The collected samples represented the pooled meconium samples from these 30 birds/box.

isolation and cHaracterization of salMonellae
Salmonella spp. were isolated and identified according to ISO 6579-1: 2017. The paper -lined the box (average 15-20gm) was placed in pre-enrichment medium 1:10 dilution (buffered peptone water; Oxoid, UK), which was subsequently incubated at 37°C for 16-18 h. Next, 0.1 mL of the pre-enrichment medium was transferred to modified semisolid Rappaport-Vassiliadis medium (MSRV, LabM, UK) and incubated at 41.5°C for 24 h. In addition, 1 mL of the pre-enrichment medium was transferred to MKTTn broth (LabM, UK) and incubated aerobically 37°C for 24 h, and then streaked onto XLD (LabM, UK) and SS (Oxoid, UK) agar plates, which were subsequently incubated at 37°C for 24 h aerobically. Selected colonies were then identified by biochemical tests (urea agar, triple sugar iron,

serotyping of Salmonella isolates
Next, the isolated Salmonella species were serotyped according to ISO 6579-3: 2014, and the serotypes were classified according to the Kauffman-White scheme (Grimont and Weill, 2007) using Salmonella antiserum (Sifin Co., Japan).

Molecular identification
A conventional polymerase chain reaction (PCR) assay was used to detect virulence and antibiotic-resistance genes in Salmonella isolates. DNA extraction was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH), with modifications of the manufacturer's recommendations. Briefly, 200 µL of the sample suspension was incubated with 10 µL of proteinase K and 200 µL of lysis buffer at 56°C for 10 min. After incubation, 200 µL of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer's recommendations. Nucleic acid was eluted with 100 µL of elution buffer provided in the kit. The oligonucleotide primers used were supplied from metabion international AG (Germany) and are listed in Table 1. Each 25-µL reaction mixture contained 12.5 µL of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µL of each primer (20 pmol concentrations), 5.5 µL of water, and 5 µL of DNA template. The reaction was performed in an Applied Biosystems 2720 thermal cycler.
The PCR products were separated by electrophoresis on 1.5% agarose gel (AppliChem, GmbH, Germany) in 1× TBE buffer at room temperature using gradients of 5 V/ cm. For gel analysis, 20 µL of conventional PCR products were loaded into each gel slot. GeneRuler 100 bp DNA Ladder (Fermentas, Thermo, Germany) was used to determine the fragment sizes. The gel was photographed with a gel documentation system (Alpha Innotech, Biometra, Germany), and the data were analyzed through computer software.

prevalence of Salmonella
Among the 391 imported poultry flock samples (231 duckling, 84 chick, and 76 turkey poult) collected, 30 (7.7%) were positive for Salmonella. Based on species, 21 of 231 (9.1%) duckling samples, 6 of 84 (7.1%) chick samples, and 3 of 76 (3.9%) poult samples yielded Salmonella isolates as shown in Table (2). Serotyping of the Salmonella strains isolated from the three different bird species revealed a broad range of serovars. Poults had three different serotypes (S. Enteritidis, S. Muenster, and S. Cuckmere), and chicks had two strains for both S. Typhimurium and S. Vejle and one each for S. Enteritidis and S. Sinstorf. Ducklings had four S. Enteritidis strains, three S. Vejle strains, and two S. Nyborg strains, as well as one strain for each of the following serotypes: S. Typhimurium, S. Sinstorf, S. Muenster, S. Indiana, S. Infantis, S. Koenigstuhl, S. Macallen, S. Nchanga, S. Neftenbach, S. Newlands, S. Nigeria, S. Nyborg, and S. Regent as shown in Table (3).

antiMicrobial susceptibility
All strains showed variable degrees of antibiotic resistance as shown in Table 4. The most frequent resistance phenotypes involved clindamycin and lincomycin, with all species being resistant to these antibiotics. Nalidixic acid resistance was found in 57.2% of the Salmonella isolates from ducklings and 16.7% of those from chicks. A High level of trimethoprim-sulfamethoxazole resistance was found in isolates from chicks and poults, with 50% and 66.7% of isolates being resistant, respectively. Resistance to ampicillin was found for 38% of isolates from ducklings and 33.3% of those from both chicks and poults. Tetracycline resistance was observed for 38.1% of isolates from ducklings and 33.3% of those from chicks. Streptomycin resistance was associated with 38.1% of isolates from ducklings, 16.7% from chicks, and 66.7% from poults. Lower levels of resistance were found for cefotaxime, with 33.3% of duckling isolates showing resistance, and for ciprofloxacin, with 28.6% of duckling isolates and 33.3% of both chick and

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June 2021 | Volume 9 | Issue 6 | Page 826

Molecular identification
To determine the virulence and antibiotic resistance profiles for all of the isolated Salmonella spp. (30 isolates), PCR was performed for the related genes. We found stn, avrA, and sopB virulence genes in all tested samples. With regard to antibiotic resistance, all tested strains carried blaTEM and tetA(A) genes, while the qnrS gene was reported in 50% of the isolates (Table 5) and Fig. (1, 2, 3, 4, 5 & 6).
On association relationship between species, resistance patterns, related antibiotic resistance genes and the virulence genes individually with its serotype as shown on  (2002) reported similar results, isolating S. Enteritidis from 7 of 231 (3%) of meconium samples from poults. In addition, rates of 4.3% and 4% were reported by Abdallaha et al. (2015) and Osman et al. (2014b), respectively, for poults imported into Egypt, while Sorour et al. (2016) and Osman et al. (2010) isolated Salmonella from 11.3% (6/53) and 12.6% of imported poults to Egypt, respectively.
The prevalence of Salmonella in ducklings in our study was substantially similar to the 2.1% found by Wang et al. (2020) isolated from China and the 6.45% reported by Badr et al. (2015) for imported ducklings into Egypt; se

Advances in Animal and Veterinary Sciences
June 2021 | Volume 9 | Issue 6 | Page 829      Recently, antibiotic resistance has been increasing in Salmonella species isolated from animal origin with increasing emergence of drug-resistant strains. The common use of antibiotics to promote growth and to treat diseases in the poultry industry adds to the potential risk of the dissemination of MDR salmonellae (Abdallaha et al., 2015). In particular, antibiotic resistance of Salmonella associated with hatchlings is due to dipping eggs in an antibiotic solution or inoculation of hatching eggs with antimicrobial agents (Kabir, 2010).
With regard to virulence, some researchers (Wallis et al., 1999) have argued that the stn gene cannot be considered as a Salmonella virulence factor and it is not related to Salmonella enterotoxicity, while other research groups have reported that the stn gene is specific for all Salmonella serotypes (Lee et al., 2009). This finding highlights the stn gene as a reliable marker for Salmonella screening (Chopra et al., 1999). In the current study, the stn gene was detected in all tested samples, indicating that all isolates had the potential to produce the heat-labile exotoxin that is one of the main sharing agents in diarrhea (Van Asten and Van