Resistance profiles, virulence and antimicrobial resistance genes of XDR S. Enteritidis and S. Typhimurium

Avian salmonellosis is concomitant with high financial crises in the poultry industry as well as food-borne illness in man. The present study is designed to investigate the emergence of Salmonella Enteritidis and Salmonella Typhimurium in diseased broilers, resistance profiles, and monitoring virulence and antibiotic resistance genes. Consequently, 450 samples (cloacal swabs, liver, and spleen) were collected from 150 diseased birds from different farms in Giza Governorate, Egypt. Subsequently, the bacteriological examination was done. Afterward, the obtained Salmonella isolates were tested for serogrouping, antibiogram, PCR monitoring of virulence (invA, stn, hilA, and pefA), and antimicrobial resistance genes (blaTEM, blaCTX-M, blaNDM, ermA, sul1, tetA, and aadA1). The total prevalence of Salmonella in the examined diseased broilers was 9.3%, and the highest prevalence was noticed in cloacal swabs. Among the recovered Salmonella isolates (n = 35), 20 serovars were recognized as S. Enteritidis and 15 serovars were identified as S. Typhimurium. Almost 60% of the retrieved S. Enteritidis serovars were extensively drug-resistant (XDR) to seven antimicrobial classes and inherited sul1, blaTEM, tetA, blaCTX-M, ereA, and aadA1 genes. Likewise, 25% of the recovered S. Enteritidis serovars were multidrug-resistant (MDR) to six classes and have sul1, blaTEM, tetA, blaCTX-M, and ereA resistance genes. Also, 66.7% of the retrieved S. Typhimurium serovars were XDR to seven classes and have sul1, blaTEM, tetA, blaCTX-M, ereA, and aadA1 genes. Succinctly, this report underlined the reemergence of XDR S. Typhimurium and S. Enteritidis in broiler chickens. Meropenem and norfloxacin exposed a hopeful antimicrobial activity toward the re-emerging XDR S. Typhimurium and S. Enteritidis in broilers. Moreover, the recurrence of these XDR Salmonella strains poses a potential public health threat.


Introduction
Salmonellosis is a serious zoonotic disease that has significant public health importance.Non-typhoidal Salmonellae are imperative foodborne pathogens associated with the digestive tract of animals and birds (Hunter and Watkins 2018).Salmonella is a ubiquitous pathogen that causes clinical or subclinical infection in asymptomatic birds, known as carriers.Globally, nontyphoidal Salmonella is incriminated in more than 150 million reports of gastroenteritis as well as about 56,000 mortalities every year (Mezal et al. 2014).Salmonella infection results in tremendous financial losses in the poultry industries due to treatment costs, and poor growth, and high mortalities.Moreover, it causes food poisoning in man (Shafiullah et al. 2016).
Salmonella is a Gram-negative, facultative anaerobic bacterium belonging to the family Enterobacteriaceae.Most Salmonella serovars are motile except S. Pullorum and S. Gallinarium (Shafiullah et al. 2016;Zhao et al. 2017).S. Typhimurium and S. Enteritidis are the utmost predominant non-typhoidal Salmonella species that incriminated in gastroenteritis in both humans and animals.The infection is commonly associated with diarrhea, fever, vomiting, and severe abdominal pain 12-36 h after ingestion of the contaminated food (Mezal et al. 2013;Zhao et al. 2020).Salmonellosis in poultry leads to prolonged fecal shedding and severe infection in hens and chicks.In experimentally infected birds with S. Enteritidis, disparities in mortalities, severity of infection, rate of production of contaminated eggs, and fecal shedding were noticed.The severity of infection is affected by the inoculum size, Salmonella serovar, and bird age (Kumar et al. 2019;Li et al. 2020).
Salmonella is an optional intracellular pathogen.The pathogenicity of Salmonella is governed by various determinants, which are regulated by its capability to attach to the host cells, invade different cells, intracellular survival, and multiplication in the host enterocytes (Eng et al. 2016).The main virulence determinants that exert a vital role in Salmonella pathogenicity include adhesion and invasion to the target cells, intracellular survival and growth, iron acquirement, and toxin production.Several virulence-determinant genes are assembled together in definite genomic elements called Salmonella Pathogenicity Islands (SPIs), gained by genetic transfer between bacterial pathogens (Tamang et al. 2014;Litrup et al. 2010).The invA gene is the most prominent virulence gene of Salmonella that exerts a remarkable role in host invasion.Moreover, the invA gene is conserved in different Salmonella species.Furthermore, other genes such as stn (Salmonella-enterotoxin), hilA, and pefA (the plasmidencoded fimbriae) are the key virulence determinant genes associated with salmonellosis (Gole et al. 2013;Webber et al. 2019).
The present study directed to determine the prevalence of S. Enteritidis and S. Typhimurium in diseased broilers, antimicrobial susceptibility testing, and PCR-based screening of virulence (invA, stn, hilA, and pefA) and antibiotic resistance genes (bla TEM , bla CTX-M , bla NDM , ermA, sul1, tetA, and aadA1) in the recovered Salmonella serovars.

Sampling
Approximately 450 samples (cloacal swabs, liver, and spleen; n = 150 for each) were collected from 150 diseased broilers (4-6 weeks old age) from commercial farms in Giza Governorate, Egypt (from April to May 2021).The examined diseased broilers suffered from diarrhea, depression, and reduced growth performance.Post-mortem examination of sacrificed and recently dead chickens revealed dehydration, enlarged congested liver, and enlarged spleen.Moreover, the postmortem findings were uniform in most of the examined birds.Samples were obtained aseptically, placed in an ice box, and conveyed to the laboratory immediately for bacteriological examination.

Isolation and identification of Salmonella
The obtained samples (1 g of each liver and spleen sample) were inoculated in 9 ml buffered peptone water (BD Difco, Thermo Fisher Scientific, Waltham, USA) and incubated at 37 °C for 18 h.Afterward, 0.1 ml of the incubated broth was inoculated in 10 ml of Rappaport-Vassiliadis broth (BD Difco, Thermo Fisher Scientific, Waltham, USA), a selective enrichment medium, and left incubated at 42 °C for 18 h.Then a loopful from the incubated broth was streaked on Xylose Lysine Deoxycholate agar (XLD), Hektoen Enteric Agar (HEA), and MacConkey agar (BD Difco, Thermo Fisher Scientific, Waltham, USA) plates and left incubated for 24 h.at 37 °C (ISO 6579-1 2017;Abd El-Aziz et al. 2021).The identification of Salmonella was performed consistent with Gram's staining, cultural features, and the biochemical reactions (oxidase, Voges-Proskauer, catalase, H 2 S production, methyl red, nitrate reduction, sugar fermentation tests, indole production, citrate utilization, and urease test) according to Quinn et al. (2002).Besides, the identification of Salmonella was ensured genetically by the PCR amplification of the invA gene (Oliveira et al. 2003).

Serological typing
The retrieved Salmonella isolates were subjected to serological identification consistent with (Kauffmann and Das Kauffmann 2001) using diagnostic polyvalent and monovalent Salmonella "O" and "H" antisera (Sifin Diagnostics, Gmbh, Berlin, Germany).

PCR monitoring of virulence determinant and resistance genes in the obtained Salmonella serovars
PCR was used to monitor the distribution of the virulence (invA, hilA, stn, and pefA genes) and resistance genes (bla TEM , bla CTX-M , bla NDM , ermA, sul1, aadA1, and tetA) among the recovered Salmonella serovars.The gDNA of the tested Salmonella serovars was extracted using a genomic DNA extraction Kit (Invitrogen, Carlsbad, USA).Moreover, positive (positive strains obtained from the A.H.R.I, Egypt) and negative controls (reactions with DNA-free reactions); were used.The used primers (Thermo Fisher Scientific, Karlsruhe, Germany) and PCR protocols were clarified in Table 1.The amplified PCR products were screened by the agar gel electrophoresis (1.5% agarose stained with 10 mg/ml ethidium bromide).Afterward, the gel was photographed.

Statistical analysis
The obtained data were analyzed using the Chi-square test (SAS software, 9.4 M6, SAS Institute, Cary, NC, USA), whereas a p-value < 0.05 points to a significant difference between the obtained data.The findings of the antibiogram were illustrated by a heatmap using Graph-Pad Software (version 8.0.1, GraphPad Software Inc., La Jolla, CA, USA).A heatmap with hierarchical clustering was accomplished to illustrate the occurrence of the antimicrobial resistance phenotypes and antimicrobial resistance genes in the retrieved serovars using the "Pheatmap" package in R software (version 4.0.2;https:// www.r-proje ct.org/).Also, the R-software was used to estimate the correlation coefficient between phenotypic resistance patterns and resistance genes.Moreover, the association between different variables was performed.

Phenotypic traits of the retrieved Salmonella serovars
The retrieved Salmonella colonies were transparent with a black center on Hektoen Enteric agar, red colonies with a black center on XLD, and small pale (non-lactose fermenter) smooth, transparent colonies on MacConkey agar.Moreover, the microscopical examination revealed Gram-negative, non-spore-forming rods.Furthermore, the obtained Salmonella serovars tested positive for citrate utilization, catalase, methyl red, H 2 S production, and nitrate reduction tests.In contrast, the isolated Salmonella serovars tested negative for oxidase, urease, Voges-Proskauer, and indole tests.

Prevalence of Salmonella serovars in the examined diseased birds
Herein, the total prevalence of Salmonella in the examined diseased broilers was 9.3% (14/150).In the present study, 35 (7.8%) Salmonella isolates (20 S. Enteritidis and 15 S. Typhimurium) were isolated from 450 bacteriologically examined samples collected from 150 diseased birds.The prevalence of S. Enteritidis was 5.3%, 5.3%, and 2.7% in the examined cloacal swabs, liver, and spleen samples.Moreover, the prevalence of S. Typhimurium was 4%, 3.3%, and 2.7% in the examined cloacal swabs, liver, and spleen samples.There is no significant difference in the distribution of Salmonella serovars amongst the examined samples (p > 0.05%), as clarified in Table 2 and Fig. 1.
A non-significant difference (p > 0.05) was recorded in the dissemination of virulence genes in the tested Salmonella serovars.Contrariwise, there was a marked variation (p < 0.05) in the distribution of resistance genes between the recovered Salmonella serovars.

Discussion
Avian salmonellosis is concomitant with high financial crises in poultry farms and severe food-borne illness in man globally (Alam et al. 2020).This work is designed to investigate the occurrence of S. Enteritidis and S. Typhimurium in diseased broilers, resistance profiles, and PCR detection of virulence and resistance genes.
In the present work, Salmonella was isolated from diseased birds suffering from diarrhea and reduced growth performance.The PM examination revealed dehydration, an enlarged congested liver, and an enlarged spleen.Similar results were described by Cocciolo et al. (2020), who recorded that diarrhea, ruffled feathers, anorexia, and pale combs are the predominant clinical signs of Salmonella infection in poultry.Salmonella infection has an adverse economic impact on the poultry industry due to losses in production, costs of treatment, and mortalities.Moreover, it has public health importance due to the potential transmission to humans, causing foodborne illness (Wajid et al. 2019).
The postmortem inspection of infected birds with Salmonella usually exhibited enlarged liver with necrotic foci, enlarged friable spleen, and marked intestinal necrotic foci (Kakooza et al. 2021).
Herein, the retrieved Salmonella serovars exposed the typical phenotypic, culture, and biochemical features of Salmonella in agreement with Islam et al. (2016).Likewise, the total prevalence of Salmonella in the examined diseased broilers was 9.3%, where 35 Salmonella isolates (20 S. Enteritidis and 15 S. Typhimurium) were isolated from 450 samples.Moreover, the highest dissemination of Salmonella was noticed in the cloacal swabs.A higher prevalence (35%) of Salmonella in broiler chickens was confirmed by Alam et al. (2020).Besides, the highest incidence of Salmonella in cloacal swabs was previously reported by Karim et al. (2017).The existence of Salmonella in cloacal swabs suggests that bird droppings might represent vehicles for the shedding and transmission of Salmonella among chickens (Islam et al. 2016).The emergence of non-typhoidal Salmonella serovars in diseased broiler chickens was previously highlighted by Barua et al. (2013) and Alam et al. (2020).The occurrence of S. Enteritidis and S. Typhimurium infection in broiler chickens suggests the probability of their transmission to human consumers leading to severe food-borne illness (Jajere 2019).Disproportions in the incidence of Salmonella could be due to management strategies, biosecurity, sanitary measures, the season of sampling, geographical disparity, environmental stress, species, immune status, and age of the bird (Kumar et al. 2019).Regarding the antibiogram of the retrieved Salmonella serovars, the tested serovars disclosed significant resistance to various classes, for example, tetracyclines, cephalosporins, macrolides, β-Lactams, sulfonamides, and aminoglycosides.These outcomes were nearly consistent with those confirmed by Wajid et al. (2019) and Lapierre et al. (2020).The existence of MDR Salmonella serovars is deliberated as a public health concern.The uncontrolled use of antimicrobial agents in the poultry industries, harboring or acquiring Salmonella to several resistance genes, resistant plasmids, and integron classes: are the chief causes that recommend the occurrence of these superbugs (Zwe et al. 2018).Hygienic measures and the use of alternatives to antibiotics such as probiotics, prebiotics, and organic acids could reduce the application of antibiotics in poultry farms (Tellez-Isaias et al. 2021).
Concerning the dissemination of virulence determinant genes, the tested S. Enteritidis and S. Typhimurium serovars usually carried invA and stn virulence genes, followed by hilA and pefA genes.These findings nearly agreed with those confirmed by Ramatla et al. (2020) and Mubarak et al. (2021).The invA gene, the most conserved gene in Salmonella species, encodes for a protein, which initiates the invasion of Salmonella to the host enterocytes.PCR detection of the invA gene is an accurate and reliable diagnostic tool for the identification of Salmonella species such as S. Enteritidis and S. Typhimurium (Shanmugasamy et al. 2011;Rodriguez et al. 2015).Likewise, Salmonella enterotoxin, encoded by the stn gene, is presumed the key virulence determinant that is incriminated in diarrhea.The detection of the stn gene is valuable for the diagnosis of Salmonella infection as it is unique to the Salmonella species (Lee et al. 2009).Moreover, the hilA gene codes the OmpR/ToxR family transcriptional regulator, which triggers the expression of invasion genes due to external stimulators (Thung et al. 2018).Furthermore, the pefA gene is responsible for the adhesion of the pathogen to the host enterocytes (Webber et al. 2019).
With reference to the phenotypic resistance profiles and the dissemination of resistance genes, most of the obtained S. Enteritidis and S. Typhimurium serovars were XDR to 7 classes possessing sul1, bla TEM , tetA, bla CTX-M , ereA, and aadA1 genes.Multiple-drug resistance is one of the foremost risks to public health worldwide.It was developed attributable to the inappropriate application of antibiotics in poultry farms and the health sector, and the transmission of resistance genes among bacterial pathogens, the presence of resistant plasmids and integrons classes (Soler and Forterre 2020;Rodríguez-Beltrán et al. 2021).The resistance to sulfonamides, penicillins, tetracyclines, and cephalosporins is mainly attributed to the presence of sul1, bla TEM , tetA, and bla CTX-M resistance genes, respectively (McMillan et al. 2019).Likewise, the aminoglycosides resistance occurred via the enzymatic modification pathway enhanced by adenylyltransferase (coded by the aadA1 gene) with subsequent inactivation of aminoglycosides antibiotics (Ramirez and Tolmasky 2010).Besides, the resistance of Salmonella serovars to erythromycin is commonly enhanced by erythromycin esterase (encoded by the ereA gene) (Katiyar et al. 2020).Worryingly, in the present study, one S. Enteritidis serovar is carbapenem-resistant carrying the bla NDM gene reflecting a public health threat.A previous investigation (Parvin et al. 2020) revealed the occurrence of carbapenem-resistant Salmonella strains carrying the bla NDM-1 in chicken meat in Bangladesh as a first report.
Concisely, this study underscored the re-emergence of XDR S. Enteritidis and S. Typhimurium serovars in diseased broilers.The retrieved S. Enteritidis and S. Typhimurium serovars usually carried the invA and stn virulence genes, followed by hilA and pefA genes.Most of the obtained S. Enteritidis and S. Typhimurium serovars were XDR to several classes and inherited sul1, bla TEM , tetA, bla CTX-M , ereA, and aadA1 genes.Meropenem    As a result, it inspires the predictable application of antibiotic susceptibility and the correct application of antibiotics in the poultry industry and health sector.

Fig. 1
Fig. 1 Prevalence of Salmonella serovars between various examined samples collected from diseased broilers

Fig. 2
Fig. 2 Demonstrates the antimicrobial susceptibility of the obtained a S. Enteritidis and b S. Typhimurium serovars isolated from diseased broilers

Fig. 4
Fig. 4 The dispersal of virulence and antimicrobial resistance genes in the tested a S. Enteritidis and b S. Typhimurium serovars from diseased broilers

Fig. 5
Fig. 5 A heatmap shows the occurrence of the antimicrobial resistance phenotypes and antimicrobial resistance genes between the tested a S. Enteritidis and b S. Typhimurium serovars.Blue squares indicate the presence of phenotypic and genotypic resistance; red squares indicate the absence of antimicrobial resistance

Fig. 6
Fig. 6 The heatmap reveals the correlation coefficient (r) among tested antibiotics and resistance genes detected in a S. Enteritidis and b S. Typhimurium serovars

Table 1
List of used primers used in this study

Table 2
The prevalence of Salmonella isolated from examined diseased broilers (n = 35)

Table 4
Dissemination of virulence and resistance genes in the tested Salmonella serovars

Table 5
Multi-drug resistance profiles and dissemination of resistance genes among Salmonella serovars SXT, AM, OX, and NOR sul1, bla TEM , tetA 0.36