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Genetic Correlation of Virulent Salmonella Serovars (Extended Spectrum β-Lactamases) Isolated from Broiler Chickens and Human: A Public Health Concern

This study aimed to detect the virulent Salmonella serovars (including ESBLs producing) isolated from broiler chickens and humans. Three hundred broilers and sixty human fecal samples were bacteriologically examined. Thirty (10%) and fourteen (23.4%) Salmonella isolates were recovered from broiler and human samples, respectively. The most predominant serovar was S. enteritidis and S. typhimurium. All Salmonella isolates were confirmed by conventional PCR-based invA and ompA genes. Multidrug resistant (MDR) isolates were screened for the detection of adrA and csgD biofilm-associated genes, which were found in all isolated serovars except one S. typhimurium and 2 S. infantis of chicken isolates that were devoid of the adrA gene. Moreover, MDR isolates were screened for detection of seven resistance genes including ESBLs and other classes of resistance genes. Chicken isolates harbored blaTEM, int1, blaCTX and qnrS genes as 100, 27.8, 11.1 and 11.1%, respectively, while all human isolates harbored blaTEM, int1 and int3 genes. The genetic correlations between virulent Salmonella serovars (including antimicrobial resistance) avian and human origins were compared. In conclusion, the high prevalence of virulent ESBL producing Salmonella serovars in broilers and humans with genetic correlations between them might be zoonotic and public health hazards.


Introduction
Salmonellae are very important bacterial pathogens affecting the poultry industry and human health [1]. Their principal habitat is the intestinal tract of humans, animals and birds, causing significant morbidity and mortality worldwide [2]. The Centers for Disease Control and Prevention (CDC) divided genus Salmonella into two species-Salmonella enterica and Salmonella bongori [3]. There are approximately 2600 serovars of Salmonella have been identified depending on the differences in their lipopolysaccharide layer with regard to their somatic (O) and flagellar (H) antigens [4]. The identification of salmonellae biochemically and serologically are time consuming; therefore, molecular methods based on DNA technology are recommended [5].
Salmonellae are invasive bacteria and their ability to infect and invade a host depends on the existence of many virulence factors. At least 60 genes are located on Salmonella pathogenicity islands (SPIs), which play the main role in Salmonella pathogenicity-especially SPI-1 and SPI-2-encoding structural proteins forming needle-like complexes allowing the insertion of the bacterial proteins into the host cells and modulating the cellular functions and immune pathways [1]. The invasion-related (invA) gene is located within SPI-1 on the chromosome encoding inner membrane protein of the bacteria specific for invasion of the epithelial cells of the host [6]. The invA gene is one of the most common PCR targeting genes and its amplification is considered an international standard for the determination of Salmonella [7]. Moreover, among outer membrane proteins (OMPs), OmpA is a major Salmonella antigenic protein, which is common to the majority of the stimulatory fractions; therefore, the ompA gene is highly specific for the detection of Salmonella serovars [8].
Antimicrobial misuse in therapy, prophylaxis, or as growth promoters has been implicated as a risk factor in the development and spreading of antimicrobial resistance (AMR) [1] leading to the limitation of therapeutic options available for the human salmonellosis treatment [9]. B-lactams; penicillins and cephalosporins, are drugs of choice for the treatment of Salmonella [10]. The extended spectrum β-lactamases (ESBLs) and plasmidmediated ampC β-lactamase are essential causes of antimicrobial resistance (AMR) [11]. In Enterobacteriaceae, resistance to cephalosporins is connected with the production of large spectrum β-lactamase as ESBLs and ampC β-lactamase [11]. ESBLs are widely reported all over the world and have been associated with successful enterobacterial clones having huge epidemic potential [12,13]. ESBLs are plasmid-mediated β-lactamase enzymes that hydrolyze penicillins, 3rd and 4th generation cephalosporins and monobactams with an oxyimino side chain [14]. ESBLs coding plasmids also carry resistance genes for other antimicrobial classes as aminoglycosides [15].
As it is very important to have knowledge about the behavior of Salmonella strains against antimicrobial agents, this study was planned to record the updates of virulence and ESBL among Salmonella serovars recovered from broiler chickens as well as of human origin and seeking the detection of correlations between broiler and human Salmonellae.

Samples
A total of 360 fecal samples (300 from broiler chickens and 60 from humans) were collected in plastic screw-top tubes containing sterile 10 mL buffered peptone water (BPW) (Oxoid Ltd., Basingstoke, UK) under complete hygienic conditions and transported in an ice box to the laboratory of the Faculty of Veterinary Medicine, Beni-Suef University, for bacteriological examination. Regarding the history of chicken samples, they were collected from 30 broiler chicken flocks resembling five Egyptian governorates during 2019-2020 with a total no. of 300,000 birds aged 1 to 14 days that were suffering from diarrhea, unabsorbed yolk sac, enteritis, arthritis and conjunctivitis. For human samples, all were collected from 60 patients aged 30-40 years during 2020, who were suffering from enteritis and hepatic problems.

Isolation, Identification and Serotyping of Salmonella Isolates
Next, 0.1 mL of BPW broth was transferred into tubes containing 10 mL Rappaport Vassiliadis broth (RVB) medium and was incubated at 41.5 • C for 24 h. A loopful from RVB was streaked onto xylose-lysine-deoxycholate (XLD) agar plates (Oxoid Ltd., Basingstoke, UK) and was incubated at 37 • C for 24 h. The colonies were examined for their cultural characteristics and morphological appearance. The suspected Salmonella colonies, red colonies with black centers, were subjected to Gram's staining and biochemical identification [16]. Moreover, suspected Salmonella isolates were serotyped according to the Kauffman-White scheme using slide agglutination with standard "O" and "H" antisera (Difco, Sparks, MD, USA) [16].

Conventional Polymerase Chain Reaction (cPCR) on Salmonella Isolates
All Salmonella isolates were confirmed by cPCR using invA and ompA virulence genes. Moreover, MDR Salmonella serovars were screened for their existence in 2 adrA and csgD biofilm-associated genes as well as 7 resistance genes including ESBL genes; bla TEM, bla OXA-1 , bla CTX , bla MOX and class 1, 2 and 3 integrons (int1, int2, int3), as well as the quinolone resistance gene (qnrA and qnrS).
Genomic DNA of the selected Salmonella isolates was extracted using the QIAamp DNA mini kit (Qiagen, Germany, Gmbh) according to the manufacturer's instructions. The primers sequences and amplified products for the targeted genes for Salmonella isolates are depicted in Table 1.

PCR for Screening Virulence and Resistance Genes in Salmonella Isolates
All the chicken and human Salmonella serovars were confirmed using PCR as they all harbored both invA and ompA genes (100%). Regarding adrA and csgD biofilm-associated genes in MDR Salmonella serovars, all tested chicken isolates (n = 18) harbored the csgD gene (100%) while 15 (83.3%) harbored the adrA gene. Meanwhile, all human isolates harbored both genes (100%) ( Table 4).    Regarding resistance genes in MDR Salmonella serovars, all tested chicken isolates harbored the bla TEM gene (100%) while five (27.8%) harbored the int1 gene and two isolates harbored both bla CTX and qnrS genes (11.1% for each). On the other hand, bla OXA-1 , bla MOX , int2, int3 and qnrA genes were not found. Meanwhile, all human isolates harbored bla TEM , int1 and int3 genes (100%) while the other resistance genes were not found (Table 4).

Correlation between Virulence Genes and Antimicrobial Resistance among Salmonella Isolates from Broilers and Human
The results in Table 5 revealed that the virulence genes-invA, ompA, csgD and adrAexisted in all MDR Salmonella serovars recovered from broiler chicken and human fecal samples except one S. typhimurium and two S. infantis isolates from broiler chickens, which did not have the adrA gene. A detailed analysis displayed an association of resistance phenotypes with potential virulence genes. The present study confirmed the relation between AMR and virulence genes in Salmonella serovars of avian and human origins.

Discussion
Avian Salmonellosis causes great morbidity, mortalities and consequently great economic losses in poultry industries with public health and zoonotic hazards [25]. This study included bacteriological examination of 300 broiler chicken and 60 human fecal samples and a molecular characterization of major virulence and AMR-associated genes. The obtained results showed that 30 Salmonella serovars (10%) were recovered from broilers while 14 serovars (23.4%) were recovered from 60 human fecal samples with a total percentage of 12.2%. The total prevalence was slightly lower than that reported by Tegegne [26] who recovered Salmonella from different sources with an overall prevalence of 16.9%. Regarding broilers, the obtained results were nearly similar to those of other studies [27]; 9.2%, and [28] 12%, while it was much lower than that recorded by other authors; [29] 30.5% and [26] 42.9%. The most frequent serovars in broilers were S. enteritidis (20%), S. infantis (16.6%), S. typhimurium and S. kentucky (10% for each). The most prevalent serotypes were S. enteritidis, S. typhimurium, S. pullorum, S. vejle, S. amsterdam, S. infantis, S. petersburg, and S. atakpame [30]. These results run parallel to those recorded by [1,28] as S. enteritidis, S. typhimurium and S. kentuky and S. infantis were the most frequent broiler chicken isolated serovars. For human samples, such prevalence was double that recorded in Ethiopia (11.3%) [26]. The most frequent human serovars were S. enteritidis and S. typhimurium (35.7% for each), S. anatum (21.4%) and S. derby (7.1%). These results coincided with those recorded by other authors [26,31] who recorded that S. enteritidis, S. typhimurium and S. derby were the most dominant non-typhoidal Salmonella serotypes in humans.
The virulence of Salmonella is related to its capability to invade a host cell, replicate and resist both digestion by macrophages and destruction by complement components of plasma [1,6]. The invA gene-based PCR assay is considered an international standard procedure for the detection and confirmation of Salmonella spp. and mostly reduces the number of false-negative results commonly occurring in diagnostic laboratories [7]. Recently in Nigeria, 75% of Salmonella isolated from hospitalized febrile patients harbored the invA gene [32]. Moreover, the ompA virulence gene plays an important role in bacterial adaptation to external environment stresses, which cause adhesion, invasion and host tissue damage [33]. The ompA gene was conserved among various Salmonella serovars and can be used, with high sensitivity and specificity, for Salmonella detection in food and clinical samples [8,34]. In our study, all the isolated Salmonella serovars were confirmed by cPCR using invA and ompA virulence genes. Additionally, MDR Salmonella serovars were screened for the presence of adrA and csgD biofilm-associated genes which are responsible for cellulose production. The four tested genes were found in almost all chicken and human Salmonella isolates except for one S. typhimurium and two S. infantis chicken isolates which lack the adrA gene. The high expression of adrA and csgD genes, which are necessary for regulating cellulose production in Salmonella spp., showed a strong biofilm production capacity. These results are supported by those recorded by previous studies [35] in which all Salmonella isolates harbored the invA gene while 90% contained csgD gene.Ćwiek et al. [36] recorded the prevalence of adrA and csgD as 100.0% for S. enteritidis. The obtained results were consistent with others who detected them in S. enteritidis and other Salmonella serovars [18,37,38]. The presence of these genes and their expression might be useful in Salmonella biofilm production via involvement in the synthesis of biofilm extracellular polymeric components [33]. Another study showed the presence of the csgD gene in all Salmonella isolates [39].
Antimicrobial agents are an effective tool for treating clinical diseases and maintaining healthy animals and human health. However, there is an increasing resistance of Salmonella to conventional therapeutic antimicrobial agents such as penicillins, sulfonamides, cephalosporins, amoxicillin-clavulanic and fluoroquinolones, which have been increasingly misused [31]. Antimicrobial-resistant Salmonellae create a public hazard, diminishing the therapeutic options available in the treatment of human salmonellosis [28]. In this study, 60% of broiler chicken Salmonella serovars were MDR, exhibiting resistance to β-lactams, cephalosporins, sulfonamides and fluoroquinolones. Meanwhile, only 28.6% of human Salmonella serovars were MDR, exhibiting lower resistance against β-lactams, cephalosporins, sulfonamides, fluoroquinolones and other antimicrobials used. These results resembled those recorded by other studies [31,36].
Antimicrobial resistance of Salmonella is one of the major virulence factors that enables the bacteria to survive and to be more pathogenic and toxigenic. ESBLs and plasmidmediated ampC β-lactamase are essential causes of AMR [11]. ESBLs are one of the main causes of β-lactam resistance, including penicillin, 1st, 2nd and 3rd generations' cephalosporins and monobactams by hydrolysis of antibiotics, among Gram-negative bacteria [40], and they can be inhibited by β-lactamase inhibitors such as clavulanic acid [14]. ESBLs coding plasmids may also carry additional β-lactamase genes and other resistance genes for other classes of antimicrobial [15]. This can restrict the treatment options for ESBLproducing pathogens and enhance the intra-and inter-species spreading of ESBLs [12]. There are many groups of ESBLs but those most frequently found in clinical Gram-negative isolates are TEM-, SHV-, OXA-, CMYand CTX-Mtypes [1,41].
In the current study, all broiler chicken and human MDR Salmonella isolates were investigated using cPCR for seven resistance genes including ESBL and other AMR-associated genes (bla TEM , bla OXA-1 , bla CTX , bla MOX , int1, int2, int3, qnrA and qnrS). Regarding broiler chicken isolates, only bla TEM , int1, bla CTX and qnrS genes were represented as 100, 27.8, 11.1 and 11.1%, respectively. Meanwhile, all human isolates included bla TEM , int1 and int3 genes (100%). These results agreed with those obtained by Abdel Aziz et al. [42], who detected bla TEM and int1 in all the tested Salmonella serovars recovered from broilers and humans. Additonally, the obtained results supported the fact that TEM-1 is the most popular plasmid mediated β-Lactamase in resistant Gram-negative bacteria [43]. The high bla TEM gene frequency in our study also corresponded with Zhu et al. [44]. In other studies, the bla TEM gene existed in 51.6% of Salmonella isolates [45] whileĆwiek et al. [36] detected it in 63.6% of isolates. The existence of ESBLs in all samples in our study indicated a shortage of infection barriers between humans and broilers.
Integrons are genetic elements that can recognize and capture movable gene cassettes carrying the AMR genes leading to MDR dissemination and limiting the chemotherapeutic options available for infectious diseases [42]. Regarding integrons genes, our results supported that int1 (class 1 integrons) was the most frequent in Salmonellae [46]. Additionally, the int1 gene was found in all Salmonella serovars recovered from poultry and humans [47], while it was found in another study in 82% of isolates [48]. The presence of int1 in Salmonellae was significantly associated with their AMR and this clearly indicates their important role in the dissemination of AMR genes [42].
The results recorded in this study strongly indicate a correlation between virulence genes and resistance to the most commonly used antimicrobial agents. The relation between virulence and AMR among Salmonella serovars occurred because of genetic determinants of AMR-and virulence-associated genes [49]. AMR-and virulence-associated genes may be linked in the same replicon [50]. Finally, the detection of chickens-to-human Salmonella strains' transmission during farming was widely demonstrated and this may occur through the food chain or by direct contact with live broiler chickens during industrial production [25,51].

Conclusions
ESBLs producing virulent Salmonella serovars are a hazard to food safety and public health, creating severe therapeutic problems in the future. The relationship between human and broiler chicken strains strongly indicates that the poultry sector may be considered a very important reservoir for ESBL-producing Salmonella, which transmits to humans through the food chain and/or direct contact. However, further studies are badly needed to evaluate the expression of virulence genes using qPCR and compare them with non-virulent serotypes devoid of ESBL genes, which may indicate a variable measurable quantitative analysis of the virulence of the serovars isolated from the chicken farms in order to assess a correlation with the presence and activity of ESBL.