Serotyping, Antimicrobial Resistance Prole and Virulence Genes of Salmonella Serovars Isolated from Human, Animals and Birds

The study was undertaken to investigate the prevalence, antimicrobial susceptibility, antimicrobial resistance and virulence genes of Salmonella isolates recovered from human and different species of animals and birds. Out of 88 (7.15%), 21 (23.86%) belonged to Salmonella enterica subsp. enterica serovar Weltevreden, 22 (25%) to serovar Enteritidis, 16 (18.2%) to serovar Typhi and 14 (15.9%) to serovar Newport, while 7 (7.95%) isolates were found to be untypable. Among the 88 isolates, 45.45% showed resistance to ampicillin, 61.36% to tetracycline, 61.18% to cefotaxime, 65.90% to gentamicin, 48.86% to trimethoprim, 11.36% to ceftriaxone, 10.22% to chloramphenicol, and 7.95% each to ciprooxacin and cefepime. Most of the isolates were susceptible to a low MIC ( ≤ 0.25 µg/ml) of Cefepime, Cefotaxime, Ciprooxacin, Ceftriaxone and Co-trimoxazole and a moderate MIC (0.5µg/ml − 4µg/ml) of Ampicillin, Tetracycline, Gentamicin and Chloramphenicol. The resistance genes, blaTEM, tetA and dfrA12 were most prevalent, irrespective of the host of origin of the isolates. While invA was used for molecular detection of Salmonella, other virulence genes, viz. sipA, sipB, sipC, stn and T2544 were also detected in all (100%) the Salmonella isolates. Total 69.32 % of tested samples were found to be contaminated with multi-drug resistant (MDR) Salmonella and various virulence genes were present among the isolated serovars. Another virulence-associated gene, T2544 (pagN) could also be found in all the isolates, irrespective of serovar or host of origin suggesting the possibility of using this gene as a marker for identication of pathogenic Salmonella isolates. This study highlights the importance of continuous monitoring and surveillance for pathogenic salmonellae and their potential risks to both human and animal. of their source of origin. The sefC gene is present in 45 (51.14%) isolates from human, cattle, wild bird, pig, tiger and poultry. The genes sopB and sopE were present in 76 (86.36%) and 55 (62.50%) isolates, respectively, while pefA, sefC and fepA genes were present in 70 (79.54%), 45 (51.14%) and 57 (64.77%) isolates, respectively recovered from human, cattle, wild bird, pig, tiger, Gecko gecko and poultry. Currently, T2544 and sopE are re-annotated as pagN and sopE2, respectively.


Introduction
Bacteria under the genus Salmonella are found in the intestinal tract of many animals, including cattle, pigs, horses, other mammals, reptiles, amphibians, and poultry (e.g., chickens, ducks, geese, and turkeys) are capable of causing infections in these species (Hale et al. 2012). Salmonella infection in human typically manifests as acute gastroenteritis that develops 12-72 hours after exposure. Young children, persons > 65 years of age, and immuno-compromised persons are at greater risk for serious complications, including septicemia, joint or brain infections, and death (Giannella et al. 1996). The genus Salmonella consists of two species, namely Salmonella bongori and Salmonella enterica (Issenhuth-Jeanjean et al. 2014). Salmonella bongori is classically called as the Salmonella of lizards and it is mostly found in reptiles causing diarrhoea. Salmonella enterica is predominantly found in human and animals causing severe diarrhoea and fever. The species S. enterica has six subspecies namely enterica, salamae, arizonae, houtonae, indica and diarizonae. All of these subspecies have different serotypes. So far, a total of 2659 serotypes of S. enterica have been reported. Out of that, S. enterica subsp. enterica has the highest 1586 serotypes (Issenhuth-Jeanjean et al. 2014). Although all serotypes must be considered as potential human pathogens, only a limited number of serotypes are attributed to be the cause of infection in humans and animals. Most of the enteric diseases in human and animals with severe diarrhoea and fever are caused by different serotypes of S. enetrica subsp. enterica. This subsp. is one of the leading causes of zoonotic food-borne disease worldwide (Voetsch et al. 2004). It is estimated that in 2019, there were 2,12,500 and 4,100 cases of infections due to drug resistant non-typhoidal Salmonella and drug resistant Salmonella Typhi, respectively only in USA (CDC report; 2019). Antimicrobial Resistance Surveillance and Research Network (AMRSN) of India has categorized Salmonella (typhoidal and non-typhoidal) into enteric fever pathogens and diarrhoeagenic bacterial organisms groups out of a total of six groups chosen by ICMR (Walia et al. 2019) for developing a comprehensive plan on Anti-microbial resistance (AMR) surveillance in India. Salmonella species, speci cally those which are uoroquinolone resistant are categorized as "high priority" by WHO (WHO Report, 2017). MDR Salmonella carrying several classes of virulence genes have been detected in duck meat in China (Chen et al. 2020). Outbreaks of S. Typhi recently occurred in Bangladesh (Tanmoy et al. 2018) and Pakistan (Klemm et al. 2018) with the presence of XDR strains resistant to Ceftriaxone and several other antibiotics. Salmonella Typhimurium showed a high frequency of occurrence in poultry (41.40%) and humans (43%), and S. Weltevreden was found to be of zoonotic signi cance in India and has been recorded as one of the ve most frequently isolated serovars (Kumar et al. 2009). In Assam, isolation of S. Weltevreden, S. Choleraesuis, S. Paratyphi B and S. Typhimurium from pigs (Rajkhowa et. al 2018), and S. Enteritidis, S. Gallinarum, S. Typhimurium, S. Newport and S. Indiana from poultry (Rahman et al. 1997;Rajkhowa et. al 2018) have been reported.
Emergence and spread of antimicrobial resistance among zoonotic Salmonella has become a public health threat. Importantly, Salmonella strains having "clinically important resistance" to some agents like extended spectrum cephalosporins and uoroquinolones have been isolated from livestock (Li et al. 2013). In most developing countries, misuse and overuse of antibiotics has contributed to the increasing trend of multi-resistance in Salmonella (Eddra et al. 2017). Salmonella with antibiotic resistance in contaminated products could infect humans directly or transmit their resistance genes to human pathogens through the food chain, leading to failure of antibiotic treatment and may pose a serious threat to human health. The aim of the current study was to investigate the prevalence, antimicrobial resistance and virulence gene pro les of Salmonella serovars isolated from human and different species of animals and birds.

Materials And Methods
Collection of samples A total of 1231 different samples consisting of faecal swabs from diarrhoeic (83) and apparently healthy (60) human, apparently healthy (101) and diseased (165) cattle, diarrhoeic (60) and apparently healthy (30) pigs, diarrhoeic (53) and apparently healthy (50) goats, diarrhoeic (302) and apparently healthy (103) poultry, apparently healthy (208) wild birds, apparently healthy (8) Gecko gecko, apparently healthy (6) tigers and mice (2) were collected from different parts of Assam. Type of samples was either faecal matter or part of intestine, in case of dead animals. Fresh samples were collected in sterile sample containers or in Carry-Blair medium, in case of anticipated delay from collection to processing by not more than 48 hours.

Isolation
Immediately after receiving the samples in the laboratory, they were put in pre-enrichment non-selective medium (sterile buffered peptone water broth) and incubated at 37 0 C for overnight. It was followed by inoculation of 1 ml overnight broth into 9 ml of selective enrichment (Selenite broth or Rappaport Vassiliadis soy peptone) broth and incubated at 37 0 C for overnight. Both broth cultures were kept at constant shaking at 250 rpm in a shaking incubator.
Overnight turbid broth was inoculated to Brilliant Green Agar (BGA) and suspected positive colonies were picked and streaked on MacConkey's Lactose Agar (MLA). Both plates were incubated overnight at 37°C. For preliminary identi cation of Salmonella, the suspected cultures were subjected to biochemical tests, viz. indole, urease and H 2 S production, as well as production of lysine and ornithine decarboxylase. Additionally, growth characteristics on Triple Sulphate Iron (TSI) agar slants were also studied for determination of K/A reaction.

Molecular detection of Salmonella
Reference strains of Salmonella used in this study (Table. 1) were obtained from Microbial Type Culture Centre (MTCC), Chandigarh, American Type Culture Centre, USA and National Institute of Cholera and Enteric Diseases, ICMR, (NICED), Kolkata. The isolates were grown in Luria Bertani (LB) broth for overnight at 37°C and 2 ml of the overnight cultures were centrifuged at 10,000g for 10 minutes. The supernatants were discarded and the pellets were suspended in a total volume of 100 µl of 1X TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and boiled at 100°C for 10 minutes. After boiling, the cell suspensions were immediately cooled on ice for 10 minutes and centrifuged at 15,000g for 10 minutes at 4°C. The supernatant was then collected without disturbing the sediment. Isolated DNA was quanti ed in Nanodrop 1000 and stored in -20°C for future use as template DNA for PCR (Choudhury et al. 2016).
The isolates were con rmed as Salmonella by detecting the speci c gene invA by simplex PCR. The speci c primer pairs used (Galan et al. 1992) for this purpose was 5 / -ACCACGCTCTTTCGTCTGG-3 / and 5 / GAACTGACTACGTAGACGCTC-3 / . A reaction mixture of 25 µl was prepared with 12.5 µl of Dream Taq PCR master mix (Fermentus), 0.5 µl each primer (10 pmol/µl), 1.0 µl of template DNA and 10.5 µl of nuclease-free water. PCR condition was standardized at 94°C for 5 minutes followed by 30 cycles at 94°C for 30 seconds, at 56°C for 1 minute and at 72°C for 1 minute. Final extension was given at 72°C for 5 minutes followed by in nite halt at 4°C. The ampli ed products were run on 1.5% agarose gel, and visualized and documented in Gel Doc XR + (Bio-Rad).

Serotyping
The Salmonella isolates were sent for serotyping at National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India, and National Salmonella and Escherichia Centre (NSEC), Central Research Institute, Kasauli, Himachal Pradesh, India.

Antibiotic Susceptibility Testing and MIC
Antibiotic sensitivity and MIC values were determined by disc dilution method and E-test using 6 mm antibiotic disc (HiMedia) and Ezy MIC TM (HiMedia) strips, respectively, according to Clinical and Laboratory Standards Institute guideline (CLSI Guideline, 2017) [19]. Pure colonies from agar plate were inoculated in Luria Bertani broth for 6-8 hours to get log phase growth. Following incubation, the turbidity was adjusted to match that of a 0.5 McFarland standard. The container of discs and strips were removed from the -20°C to equilibrate to room temperature for 30 minutes. Sterile 20% Muller Hinton Agar (SRL) plates were inoculated by swabbing with sterile cotton swab (HiMedia) evenly. Antibacterial discs and Ezy MIC TM strips were placed on the surface aseptically as per manufacturer's instructions and kept for incubation at 37 o C for overnight. Zone of inhibition and breakpoint values were recorded after the overnight incubation. A total of nine antimicrobial agents were used for both the tests. Ampicillin (AMP), Tetracycline (TET), Cefotaxime (CTX), Cipro oxacin (CIP), Gentamicin (GEN), Chloramphenicol (CHL), Cefepime (CPM) and Ceftriaxone (CTR) were common for both, whereas Trimethoprim (TR) used in disc diffusion method was replaced by Co-trimoxazole (COT) in E-test (Table. 2).
Resistance and virulence gene screening Salmonella isolates exhibiting resistance to different antimicrobial agents were then subjected to PCR for detecting resistance genes of concerned antibiotics.
Resistance genes blaOXA, bla TEM , blaPSE -1, dfrA1, dfrA12, tet(A), tet(B) and tet(G) were detected using reported primers and PCR conditions (Table. 3). PCR reaction mixture constituents were same as mentioned earlier for invA gene detection. All the Salmonella isolates were screened for the presence of a total of 11 virulence genes including invA universally present in all isolates. The primer sets used for PCR ampli cation of sipA, sipB, sipC, stn, sopB, sopE, pefA, sefC, fepA and pagN along with PCR conditions are listed in Table. 4. PCR reaction mixture constituents were same as mentioned earlier. The ampli ed products were run on 1.5 % agarose gel and visualized under Gel Doc XR + (Bio-Rad, USA).

Isolation and identi cation
Out of 1231 samples examined, Salmonella was recovered from 88 (7.15%). Among the 663 clinical samples collected from man and animals with a history of diarrhoea, 68 (10.26%) were positive for Salmonella, while out of 568 samples collected from apparently healthy animals and birds, 20 (3.52%) were positive for Salmonella (Table 5). All the 88 Salmonella isolates recovered from different sources fermented glucose, mannitol and dulcitol, but did not ferment lactose and sucrose. All the isolates were positive for methyl red test, citrate utilization and hydrogen sulphide production but were negative for indole production, Voges-Proskauer (VP) test and urease production. The isolates showed yellow butt, black middle and pink top on inoculation into Triple Sugar Iron (TSI) Agar slant.      (Table 6). In case of human samples, S. Typhi (62.50%) was the most frequently isolated serovar, followed by serovars Weltevreden and Newport ( Figure. 1). On the other hand, S. Newport (52.63%) was found to be the most abundant serovar in cattle followed by serovars Weltevreden and Enteritidis ( Figure. 2).   A total of 61 (69.32 %) isolates were found to be multi-drug resistant (MDR) and one among these isolates showed resistance to four antimicrobial agents (Cefotaxim, Tetracycline, Gentamicin and Ampicillin). Salmonella Weltevreden isolates showed resistance to Cefotaxime (92.31%) and Tetracycline (69.23%). Among the human isolates, 100% resistance was observed against Cefotaxime, Trimethoprim and Tetracycline. However, no resistance was shown against Ceftriaxone and Cefepime. Among the poultry isolates, 100% resistance was observed against Cefotaxime, while no resistance was shown against Cefepime. Salmonella Newport isolates showed 100% resistance against Gentamicin and 77.78% resistance against both Ampicillin and Cefotaxime. Among the cattle isolates, Gentamicin and Ampicillin resistance was found to be 100%, Cefotaxime resistance was 80% with no resistance to Ceftriaxone. Among S. Enteritidis isolates, Cefotaxime, Trimethoprim and Ampicillin resistance was found to be 100%, 92.86% and 35.71%, respectively. Among the poultry isolates, Ampicillin, Cefotaxime and Tetracycline resistance was found to be 50%, 100% and 60%, respectively. In S. Typhi, which is a human host-speci c serovar, 83.33 % resistance was found against Cefotaxime, Gentamicin and Tetracycline each, while resistance to Chloramphenicol and Ceftriaxone were found to be 8.33% and 25%, respectively. µg/ml, whereas the corresponding values were between 0.5 µg/ml and 8 µg/ml for S. Typhi isolates (Figures. 3). For S. Typhimurium, the MIC values were found to be between ≤ 0.125µg/ml and 4 µl/ml for Cefepime, Cefotaxim, Cipro oxacin, Ceftriaxone and Co-trimoxazole, while for the rest of the drugs, it varied from 4 µl/ml to 32 µl/ml (Table 9). Table 9 Antimicrobial resistance phenotypes of Salmonella isolates (n = 88).  (Table 10). Virulence Gene Detection All the 88 Salmonella isolates were subjected to simplex PCR for detection of 11 important virulence genes ( Table 11). The different serovars of Salmonella showed varability in their virulence gene pro les. While invA was used as the internal control for molecular detection of Salmonella, virulence genes sipA, sipB, sipC, stn and T2544 were also detected in all (100%) the Salmonella isolates, while fepA gene was present in 57 (64.77%) isolates belonging to serovars Enteritidis (12), Weltervreden (14), Typhi (14), Newport (8), Litch eld and Idikan (one isolate each), and Typhimurium (2) and the untypable (5) isolates. The rest four virulence genes sopB (86.36%), sopE (62.5%), pefA (79.54%) and sefC (51.14%) were found to be present in varying percentage among the Salmonella serovars. Maximum numbers (5) of the 17 isolates carrying all the eleven genes under study were recovered from wild birds and human. Out of all the 88 isolates screened, a total of 11 (12.5%) isolates belonging to serovars Weltervreden (7) and Typhi (4) were found to be positive for all eleven genes, while three other untypable isolates also carried all eleven genes. The distribution of virulence genes according to the source of recovery of the isolates revealed that invA, sipA, sipB, sipC, stn and T2544 genes were present in all the isolates irrespective of their source of origin. The sefC gene is present in 45 (51.14%) isolates from human, cattle, wild bird, pig, tiger and poultry. The genes sopB and sopE were present in 76 (86.36%) and 55 (62.50%) isolates, respectively, while pefA, sefC and fepA genes were present in 70 (79.54%), 45 (51.14%) and 57 (64.77%) isolates, respectively recovered from human, cattle, wild bird, pig, tiger, Gecko gecko and poultry. Currently, T2544 and sopE are reannotated as pagN and sopE2, respectively.

Discussion
Salmonella infections are one of the major global public health problems. During the last decade, antimicrobial resistance and multi-drug resistance of Salmonella spp. have increased to a great extent, especially in the developing countries commensurating with increased and indiscriminate use of antimicrobial agents in the treatment of humans and animal diseases. In the present study, an attempt was made to isolate Salmonella from faecal and intestinal samples of human, animals and birds, to study serotype distribution, their antimicrobial resistance patterns, and to detect important antibiotic resistance genes and Salmonella-speci c virulence genes. e, h, 1, 2, while 7 (7.95%) isolates were found to be untypable. Salmonella Enteritidis is reported as the most common serotype worldwide (65% of the isolates), followed by S. Typhimurium (12%) (Galanis et al. 2006). This was in close agreement with the present ndings. In animals, Typhimurium is the commonest serovar recovered in India followed by Weltevreden (Kumar et al. 2009). This was also in partial agreement with the present ndings.
Higher level of resistance shown by the Salmonella isolates to ampicillin, gentamicin, cefotaxime and tetracycline might be attributed to frequent and longterm use of these drugs as therapeutic agents both in man and in animals. Although chloramphenicol has long been used as one of the drugs of choice for treatment of salmonellosis in human, it was interesting to observe that lesser number of the isolates tested in the present study exhibited resistance to this drug. This might be attributed to the current trend of prescribing uoroquinolones like cipro oxacin more frequently in human medicine to treat cases of enteric fever. Comparatively higher percentage of isolates from cattle and poultry were found to exhibit resistance to most of the antimicrobial agents tested. It might be due to more frequent use of antimicrobial therapy in these two species compared to other species to control and prevent infectious diseases.
Recently, Chloramphenicol, Ampicillin and Trimethoprim resistant S. Typhi that were also Ceftriaxone resistant created havoc in Pakistan and Bangladesh (Klemm et al. 2018 However Ceftriaxone, another 3rd generation cephalosporin, along with Cefepime, a 4th generation cephalosporin, was effective against most of the isolates, irrespective of host and serovars. Interestingly, a moderate level of resistance was shown by the isolates to Ampicillin. This may be due to the current practice of very limited use of a β lactam antibiotics without a β lactamase inhibitor (like Clavulinic acid) in the treatment regime for human and livestock diseases.
Most of the isolates from human and cattle were resistant to Gentamicin but moderate resistance was observed in case of isolates from other host species. This may be due to the abundant use of the drug in these two species for treatment purpose. Cipro oxacin, a uoroquinolone, has still remained effective, as less resistance was shown by the isolates of all serovars against this drug. Isolates from Cattle, Poultry and Pig showed moderate level of resistance to Chloramphenicol, a lifesaving ICU category drug and very low resistance was exhibited by the isolates from human, particularly of serovars S. Weltevreden and S. Newport.
The increase in antimicrobial resistance is a threat to global health. Trimethoprim is commonly used in the treatment of urinary tract infections (UTI) in all parts of the world. However, soon after the introduction of the drug, trimethoprim resistance was reported in several species (Skold, 2001 It has been observed that disruption of genes in Salmonella Pathogenicity Island (SPI) I of S. Typhimurium and S. Dublin blocks the secretion of Salmonella invasive proteins. The virulence genes of Salmonella spp. encoding ve different Sips (Salmonella invasion protein) namely sipA, B, C, D and E are capable of inducing apoptosis in macrophage (Kaur et al. 2012), and hence may play a vital role in Salmonella pathogenesis. Since stn has been found to localize and transcribe in juxtaposition to the gene, which encodes the dehydrogenase regulatory protein, a common and related protein among enteric micro-organisms, it is anticipated that stn determinant might be prevalent among all salmonellae (Prager et al. 1995). This gene in Salmonella is one of the chromosomally encoded genes that codes for production of enterotoxins. Observations from the present study indicated that the stn gene is universally present among all the Salmonella isolates, irrespective of the serovars, which was in agreement with Prager et al. (1995), and Murugkar et al. (2003). In the present study, sopB gene was detected in 86.36% of the Salmonella isolates which was in agreement with the ndings of Rahman et al. (2006) who reported that all 50 isolates of S. enterica belonging to 11 serovars carried sopB gene, irrespective of their serovars and source of isolation. The sefC gene was present in 51.14% of the isolates. The sefABC genes make up part of a complex sef operon responsible for the expression and assembly of SEF14 mbriae (Clouthier et al. 1993).

Conclusion
In our study, we examined the prevalence of Salmonella and antimicrobial resistant isolates in the fecal samples of human, animals and birds. Our ndings showed a high prevalence and serotype diversity in the area under study. Serovars S. Enteritidis, S. Weltevreden and S. Typhi were the most common serovars. Comparatively higher percentage of isolates from cattle and poultry exhibited resistance to most of the antimicrobial agents tested. It might be attributed to more frequent and rather indiscriminate use of antimicrobial agents in these two species to control and prevent infectious diseases. Moreover, the recovered Salmonella isolates exhibiting multi-drug resistance and multiple virulence genes suggested a possible risk to human and animals. Therefore, it is important to rationalize the use of antimicrobial agents to prevent vertical and horizontal transfer of MDR strains across host species. Presence of multiple virulence genes in different combinations in all the eld isolates of Salmonella as revealed by the present study indicated their possible role as a pathogen in the host species. However, a molecular level phylogenetic analysis is needed to establish the inter-serovar and inter-host relationships among the Salmonella isolates.
Declarations Figure 2 Frequency of isolation of different serovars of Salmonella from cattle