Antimicrobial Resistance and Virulence Genotyping of Different Salmonella Serovars Isolated from Chickens in Egypt

| Twenty eight Salmonella strains representing 9 Salmonella serovars (S.Agama, S.Blegdam, S. Enteritidis, S.Gueuletapee, S.Infantis, S.Kentucky, S.Montevideo, S.Typhimurium and S.Virchow) were previously isolated, purified, and identified in our laboratory from freshly dead and diseased chickens suspected to infect with Salmonellosis. In the present study, antimicrobial resistance to 15 different antimicrobials and virulence genotyping to those Salmonella strains were performed. The significantly higher rate of resistance was detected against amoxicillin-clavulanic acid (AMC) and ampicillin (AMP) (85.7% and 78.5% respectively) comparing to the significantly lower rate of resistance detected against etapenem (ETP), gentamicin (GEN), ciprofloxacin (CIP), and norfloxacin (NX) (0 %, 0%, 3.5%, and 3.5% respectively). High resistance to cephalosporin antibiotics were also reported in this study. Resistance to 3 antimicrobials or more were identified in 17 out of 28 tested Salmonella strains. Interestingly, two S. Typhimurium strains were resist to 9 and 12 out of 15 antimicrobials used in this study. A multiplex Polymerase chain reaction (PCR) targeting 17 virulence genes was performed for virulence genotyping among different strains. Interestingly, all 17 virulence genes were detected in S. Infants and one strain of S. Agama. From a public health aspect, continuous resistance of Salmonella spp. to antimicrobials represent a serious public health hazard. Furthermore, Identification of virulence genes can help us to further understanding of Salmonella pathogenesis.

However, multiple antimicrobial resistant Salmonella strains developed due to haphazard use of antimicrobials at recommended doses or at sub-therapeutic doses which representing a public health hazard (Antunes et al., 2016;EFSA, 2013) . Thus, continuous monitoring of antimicrobial resistance have a high priority.
The severity of Salmonella infection in human and animals is depending on the presence of virulence genes (Ammar et al., 2016). Several virulence genes have been reported. Virulence genes encoded proteins such as: invA, orgA, prgH, spaN, tolC, sipB, pagC, msgA, spiA, sopB, lpfC, pefA, and spvB which responsible of adherence, invasiveness, entry to non-phagocytic cells, survival within macrophage, and growing within the host. Other virulence genes (sitC and iroN) were involved in iron acquisition, while cdtB was responsible of toxin biosynthesis (Skyberg et al., 2006). Thus, detection of virulence genes among different Salmonella serovars is always required. Therefore, the aim of our study was study virulence genotyping of 28 Salmonella strains representing 9 Salmonella serovars (S.Agama, S.Blegdam, S. Enteritidis, S. Gueuletapee, S. Infantis, S. Kentucky, S. Montevideo, S. Typhimurium and S. Virchow) by Multiplex PCR technique targeting 17 virulence genes. Moreover, the resistance profile of those Salmonella strains to 15 antimicrobials was performed.

bActeRiAl stRAins
Twenty eight Salmonella strains representing different Salmonella serovars (S.Agama, S.Blegdam, S.Enteritidis, S. Gueuletapee, S.Infantis, S.Kentucky, S.Montevideo, S.Typhimurium and S.Virchow) were used in this study. These Salmonella strains were previously isolated, purified, and identified in our laboratory from sick chickens suspected to infect with Salmonellosis.

Antibiotic sensitivity AssAy
Antimicrobial susceptibility testing to Salmonella strains belong to different serovars against different antimicrobials (Table 1) were determined by disk diffusion method (Bauer et al., 1966). Briefly, adjustment of bacterial inoculums to the 0.5 McFarland standard, streaking onto Mueller-Hinton agar plates, placing standard antibiotic disks (HIMEDIA®), and aerobic incubation at 37°C for 24 h were subsequently performed. The diameter of inhibition zone were measured and Salmonella strains were categorized to resistant, intermediate, or susceptible to different antimicrobials according to the CLSI guidelines (CLSI, 2017).

PolyMeRAse chAin ReAction (PcR)
Bacterial DnA extraction and multiplex Pcr amplification: Extraction of bacterial DNA was done for each Salmonella strain according to extraction kit instructions (GF-1 bacterial DNA extraction kit, vivantis, Malaysia). All bacterial DNA were kept in -20. Primers used in this study are listed in (Table 2). Three sets of multiplex PCR were accomplished for each sample to amplify different virulence genes (Skyberg et al., 2006) as follow: (set 1) amplified spvB, spiA, pagC, cdtB, and msgA. While (set 2) amplified invA, sipB, prgH, spaN, orgA, and tolC. Finally (set 3) amplified iroN, sitC, lpfC, sifA, sopB, and pefA. Amplification was performed in a 50 µl reaction mixture that included 1 µl of template DNA, 25 µl of master mix (Cosmo PCR Master Mix, UK), 2.5 µl of 50 mM MgCl2, 0.5 µl of 10 µM forward and reverse primers, and the reaction mixture was completed to 50 µl using dd H2O. Twenty five amplification cycles were run after 5 min at 95 C as follow: 30 sec at 94 C, 30 sec at 66.5 C, and 2 min at 72 C, with a final cycle of 10 min at 72 C, followed by a hold at 4 C. PCR products obtained were subjected to horizontal gel electrophoresis in 1.5% agarose, and the size of the amplicons was determined by comparison with DNA marker (VC 100bp Plus DNA Ladder, vivantis).

stAtisticAl AnAlysis
Antimicrobial resistance rates were analyzed using the chisquare test and GraphPad Prism 5.

DIScuSSIon
A total of 28 Salmonella strains which representing 9 Salmonella serovars (S.Agama, S.Blegdam, S.Enteritidis, S.Gueuletapee, S.Infantis, S.Kentucky, S.Montevideo, S.Typhimurium and S.Virchow) were used in this study to perform antimicrobial resistance profile and virulence genotyping. In our study, a high resistance rates among different Salmonella strains were detected in AMC and AMP indicating the limited therapeutic value of these antibiotics to control salmonellosis. Salmonellae resistance to beta-lactam was previously reported in Egypt (Ammar et al., 2016;Khairy, 2015), Turkey (Siriken et al., 2015), Pakistan (Shah and Korejo, 2012), Brazil (Oliveira et al., 2006). Moreover, the resistance rates of Salmonella spp. to cepha-losporin antibiotics, CF (2 nd generation), CTX (3 rd generation), and CPM (4 th generation), were detected as 32%, 28.5%, and 17.8% respectively. Development of resistance against cephalosporins was previously detected (Abo-Amer and Shobrak, 2015; Elkenany et al., 2019;Mir et al., 2015) which has a public health consequences as these antimicrobials are used to treat serious Salmonella infections in human. Unfortunately, the resistance rate of Salmonella strains tested in this study to 3 or more antimicrobials was 65%. Interestingly, two S. Typhimurium strains were resist to 9-12 out of 15 antimicrobials. Thus, increasing resistance rates and multiple antimicrobial resistance among Salmonella strains (Ammar et al., 2016;Elkenany et al., 2019;Yu et al., 2021) could be due to haphazard use of antimicrobials at recommended doses or at sub-therapeutic doses which representing a public health hazard. Antimicrobial resistance among Salmonella is a serious public health problem that needs to be monitored continuously. Furthermore, using of alternatives instead of antibiotics to control salmonellosis in poultry is required.
We performed a multiplex PCR targeting 17 virulence genes of Salmonellae related to adherence, invasiveness, entry to non-phagocytic cells and killing of macrophages, survival within macrophage, growing within the host, iron acquisition, and toxin biosynthesis. Genes required for host recognition and invasion (invA, prgH, tolC, lpfC, and sopB) and also required for survival of Salmonella within macrophages (spiA and msgA) were detected in all Salmonella strains tested in this study. Both cdtB and pefA virulence genes were rarely detected (7% and 21% respectively) which agree with results previously reported (Skyberg et al., 2006). Moreover, cdtB gene which responsible for toxin biosynthesis (Haghjoo and Galan, 2004) was only detected in both strains of S. Agama while PefA gene encoded by virulence plasmid was detected in S. Agama (2/2) and S. Typhimurium (4/7). Interestingly, all virulence genes were detected in S. Infants and 1 out of 2 S. Agama tested in this study. S. Agama was previously isolated from the poultry environment, dead birds, and apparently healthy birds in Nigeria (Ahmed et al., 2019). Furthermore, it is a zoonotic pathogen as it was a cause of diarrhea (Bélard et al., 2007;Kudaka et al., 2006), neonatal meningitis and septicemia (Heaton et al., 2015).

concluSIon
Antimicrobial resistance profile and virulence genotyping to Salmonella strains representing 9 Salmonella serovars previously isolated and identified in our laboratory were performed in this study. Multidrug resistant Salmonella strains were described and many virulence genes were detected among Salmonella strains. Finally, continuous monitoring of antimicrobial resistant Salmonella strains, using of