Clinical Characteristics, Antimicrobial Resistance, Virulence Genes and Multi-Locus Sequence Typing of Non-Typhoidal Salmonella Serovar Typhimurium and Enteritidis Strains Isolated from Patients in Chiang Mai, Thailand

Non-typhoidal salmonellosis (NTS) caused by ingesting Salmonella enterica contaminated food or drink remains a major bacterial foodborne disease. Clinical outcomes of NTS range from self-limited gastroenteritis to life-threatening invasive NTS (iNTS). In this study, we isolated Salmonella spp. from the stool and blood of patients hospitalized at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, between 2016–2021 (a total of 395 cases). Then, serovar Typhimurium and Enteritidis were identified and further characterized by multiplex PCR, and multi-locus sequence typing. Our data show that multidrug resistance (MDR) sequence type 34 (ST34) and ST11 are the predominant sequence types for serovars Typhimurium and Enteritidis, respectively. Most S. Typhimurium ST34 lacks spvB, and most S. Enteritidis ST11 harbor sseI, sodCI, rpoS and spvB genes. NTS can be found in a wide range of ages, and anemia could be a significant factor for S. Typhimurium infection (86.3%). Both S. Typhimurium (6.7%) and S. Enteritidis (25.0%) can cause iNTS in immunocompromised patients. S. Typhimurium conferred MDR phenotype higher than S. Enteritidis with multiple antibiotic resistance indexes of 0.22 and 0.04, respectively. Here, we characterized the important S. Typhimurium, S. Enteritidis, and human clinical factors of NTS within the region.


Introduction
One of the most frequent and impactful gastrointestinal infections on both human health and economic loss is acute nontyphoidal salmonellosis (NTS) [1,2].Over 1.35 million cases per year of NTS have been reported in the United States alone [3].The cause of NTS is Salmonella enterica which comprises more than 2500 serovars [4,5].Among all serovars, two important serovars belonging to S. enterica subspecies I that are implicated in the epidemic of NTS worldwide are Typhimurium (S.Typhimurium) and Enteritidis (S.Enteritidis) [6].Various clinical manifestations of NTS infections range from self-limited diarrhea to life-threatening invasive bacteremia [7].Children and individuals with human immunodeficiency virus (HIV) infection are at a high risk of developing invasive NTS (iNTS) [8][9][10].Throughout the last few decades, the rate of antibiotic resistance and the number of newly discovered resistance determinants in Salmonella have significantly increased [11][12][13].Multidrug-resistant Salmonella strains have become a significant problem in human and animal healthcare systems, and also in the food industry [14].
Multi-locus sequence typing (MLST) has been commonly used to determine Salmonella sequence type (ST) for epidemiological studies [5].Over time, several ST of S. Typhimurium became predominant in human and animal samples, particularly ST34 and ST19, which are the most common STs in Asia [15,16].Over the past two decades, ST34 emergence has increased significantly as supporting data showed its spread through Europe, North America, Asia, and Australia [17][18][19].Importantly, ST34 is often associated with multidrug resistance (MDR) phenotype to several antibacterial agents such as ampicillin, chloramphenicol, streptomycin, sulbactam, and tetracycline and extends to other antibacterial agents [20,21].In contrast, ST19 isolates resisted fewer antibacterial classes [22].The continuous rise in MDR S. Typhimurium has significantly challenged the current therapeutic methods to control or prevent this important foodborne illness.
S. Enteritidis typically causes acute gastrointestinal symptoms (nausea, vomiting, fever with diarrhea), like other non-typhoidal Salmonella.However, invasive strains of S. Enteritidis have emerged as a leading cause of bloodstream and other invasive infections globally [23,24].S. Enteritidis ST11 is one of the most common ST among S. Enteritidis isolates in food, animals, and humans in many countries.Previous studies identified that ST11 is associated with iNTS cases reported in Asia and Africa [25,26].Besides causing serious infections like septicemia, MDR S. Enteritidis is also resistant to commonly prescribed antibacterial drugs [27].Collectively, these data indicate an urgent need to find alternative ways of treating MDR S. Enteritidis.
Host factors such as immune status, gut microbiome, and bacterial virulence factors play a significant role in acute NTS [16,28].Several virulence factors are crucial for Salmonella as they enable it to replicate and proliferate inside the host.Major virulence factors that play a critical role in clinical outcomes of S. Typhimurium and S. Enteritidis infections including spvB, sseI, sodCI, and rpoS have been previously reported [29].The plasmid-derived spvB is essential for encoding Salmonella cytotoxin that can rearrange the host cell cytoskeleton for increasing Salmonella invasion [30].The Salmonella sseI gene encodes effector protein SseI that allows Salmonella to live inside the host cells and is essential for the systemic phase of Salmonella infection [31].The sseI gene is associated with bacterial systemic spreading and chronic infection in mice by inhibiting the migration of macrophages and dendritic cells [32].The sodCI gene encodes a phagocytic superoxide dismutase to decrease exogenous oxidative damage from the host cells [33].The rpoS gene has a regulatory function important for Salmonella stress response that increases survival of bacteria inside the host cell [34].The importance of analyzing the microbiological factors of NTS and clinical features of the patients from whom they were isolated has been demonstrated elsewhere [29].However, the study of Salmonella virulence genes, serovar, sequence type, and clinical outcomes in human NTS in Chiang Mai, Thailand, is still under investigation.

Study Design and Clinical Data Collection
A retrospective cross-sectional study was conducted at Maharaj Nakorn Chiang Mai Hospital (MNCMH) in Chiang Mai, Thailand, from October 2016 to September 2021.The MNCMH is a part of the Faculty of Medicine, Chiang Mai University, and is a super-tertiary hospital that receives referred cases throughout Chiang Mai and the nearby provinces.Stool and blood cultures positive for Salmonella spp.from the Central Diagnostic Laboratory of MNCMH were received on Salmonella Shigella (SS) agar (Oxoid Limited, Hampshire, UK).In total, 395 isolates were collected in this study.Demographic and clinical data of each patient was collected from the computer-based medical record system.Age, gender, presence of sepsis, hematocrit (Hct) level, and white blood cell count (leukocyte, neutrophil, and lymphocyte) of each patient where Salmonella spp. was detected and isolated from, upon admission to the hospital, were recorded.Sepsis was defined as having at least two of the following systemic inflammatory response syndrome (SIRS) criteria: heart rate more than 90 beats per minute, respiratory rate more than 20 breaths per minute, body temperature more than 38 • C or less than 36 • C, and total white blood cells count more than 12,000 or less than 4000/mm 3 or having band neutrophils more than or equal to 10%.

Salmonella Serotyping by a Multiplex Polymerase Chain Reaction
A total of 395 clinically isolated non-typhoidal Salmonella strains were serotyped using a multiplex polymerase chain reaction (PCR) which targeted Salmonella spp., S. Typhimurium, and S. Enteritidis as previously described [29].The sequences of primer pairs used in this study are presented in Table 1.In brief, the bacterial genomic DNA was extracted using a boiling method.The bacterial suspension was boiled at 100 • C for 5 min and centrifuged at high speed for 1 min to collect the genomic DNA in the supernatant and used as a DNA template [35].The quality and quantity of the extracted DNA were assessed by the NanoDrop 2000/2000c Spectrophotometers (Thermo Fisher Scientific, Waltham, MA, USA).Then, PCR amplification was performed in a total reaction volume of 25 µL using DreamTaq Green PCR Master Mix (Thermo Fisher scientific, Waltham, MA, USA) with 40 ng of template DNA, and a final concentration of each primer was 0.2 µM.PCRs were performed using a peqSTAR thermal cycler (Peqlab Biotechnologie GmbH, Erlangen, Germany) with the following conditions: an initial denaturation of 95 • C for 5 min; followed by 35 cycles of 95 • C for 30 s, 56 • C for 45 s, and 72 • C for 45 s; and a final extension at 72 • C for 10 min.PCR products were run on a 2.5% agarose gel stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA), and both positive and negative controls were included in each run.

Determination of Multiple Antibiotic Resistance (MAR) Index
The multiple antibiotic resistance (MAR) index, the ratio of the number of antibiotics that an isolate was resistant to, and the total number of antibiotics tested for each isolate, (120 for S. Typhimurium and 28 for S. Enteritidis) was calculated as previously described [41,42].A MAR index > 0.2 indicates that isolates are from high-risk contaminated sources with frequent use of antibiotics, whereas values ≤ 0.2 indicate that isolates are from sources that have been less exposed to antibiotic use.

Detection of Salmonella Virulence Genes
The important virulence genes for Salmonella (spvB, sseI, sodCI, and rpoS) were detected by PCR with the primer pairs listed in Table 1.In summary, one fresh colony of S. Typhimurium or S. Enteritidis was picked and resuspended in 50 µL UltraPure DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, Waltham, MA, USA).The bacterial genomic DNA was extracted using the boiling method as mentioned above.PCR was performed in 50 µL of DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions and the previous study [29].

Identification of Salmonella Sequence Type by Multi-Locus Sequence Typing (MLST)
The sequence type (ST) of Salmonella enterica isolates (n = 30) was identified by a multi-locus sequence typing (MLST) as previously described [43].In brief, the seven housekeeping genes of Salmonella, including aroC, dnaN, hemD, hisD, purE, sucA, and thrA were amplified using specific primers listed in Table 1 followed by purification with a GeneJET PCR Purification Kit (Thermo Fisher scientific, Waltham, MA, USA) before being sequenced (1st Base, Axil Scientific, Singapore).Sequences were compared with existing alleles available in the PubMLST Salmonella genome database (https://pubmlst.org/salmonella/) (accessed on 2 December 2021) to identify an allelic profile and ST of each isolate.The sequences for Salmonella seven housekeeping genes (aroC, dnaN, hemD, sucA, thrA, purE, and hisD) of the isolates in this study were deposited to the GenBank database under the accession numbers OR504765 to OR504974.

Statistical Analysis
Patient, clinical, and laboratory data were recorded in Microsoft Excel 2017 and analyzed with the Statistical Package for Social Sciences, SPSS (version 25, IBM Corp., Somers, NY, USA).Mann-Whitney U test was used for comparison of age.Pearson's chi-squared and Fisher's exact test were used to test association.

Salmonella Enterica Serovar Typhimurium and Enteritidis Strains Were Identified from Hospitalized Patients with Stool or Blood Cultures Positive for Salmonella
From 2016 to 2021, a total of 395 NTS clinical isolates were obtained from the Central Diagnostic Microbiology Laboratory of MNCMH.Serotyping of the isolates by a multiplex PCR revealed that 120 isolates (30.4%) were identified as serovar Typhimurium and 28 isolates (7.1%) as serovar Enteritidis, with 247 (62.5%) isolates being part of neither S. Typhimurium nor S. Enteritidis.However, they were not identified in this study (Figure 1A).Most S. Typhimurium and S. Enteritidis isolates were obtained from stool culture, 93.3% (112/120) and 75.0%(21/28), respectively.6.7% (8/120) of S. Typhimurium and 25.0% (7/28) of S. Enteritidis isolates were obtained from blood culture, indicating the incidence of iNTS in MNCMH (Figure 1B).Data of each isolate (N = 148) is demonstrated in Supplementary Table S2.The demographic and principal diagnosis of iNTS cases in this study is shown in Table 2.

Patients with Anemia Have a High Risk for Salmonella Typhimurium Infection
Next, we compared the demographic (age and gender) along with clinical and laboratory findings (sepsis, hematocrit, leukocyte, neutrophil, and lymphocyte counts) of 148 patients infected with either S. Typhimurium (n = 120) or S. Enteritidis (n = 28) as shown in Table S3.However, the complete blood count (CBC) data of some patients was unavailable.Our data showed that the mean age and gender were not significantly different between patients infected with S. Typhimurium and S. Enteritidis.Most patients with S. Typhimurium infection were 1 to 4-year-old children (41/120, 35.0%) followed by 19-59-year-old adults (39/120, 33.3%).Most S. Enteritidis-infected patients were adults (19-59 years old) or elderly (60-93 years old), with 46.4% (13/28) and 25.0% (7/28), respectively.The available CBC data indicated no differences in the presence of sepsis, the numbers of leukocyte, neutrophil, and lymphocyte counts between S. Typhimurium and S. Enteritidis-infected groups.Interestingly, there was a significant difference in the percentage of Hct level between the STM-infected (69/80, 86.3%) and the SE-infected group (10/18, 55.6%) (p = 0.006).By using a univariate analysis, it was found that patients with anemia (Hct level less than 13%) were at a higher risk for STM than SE infection (odd ratio of 5.018 with 95% confidence interval of 1.63-15.48).

Patients with Anemia Have a High Risk for Salmonella Typhimurium Infection
Next, we compared the demographic (age and gender) along with clinical and laboratory findings (sepsis, hematocrit, leukocyte, neutrophil, and lymphocyte counts) of 148 patients infected with either S. Typhimurium (n = 120) or S. Enteritidis (n = 28) as shown in Table 3.However, the complete blood count (CBC) data of some patients was unavailable.Our data showed that the mean age and gender were not significantly different between patients infected with S. Typhimurium and S. Enteritidis.Most patients with S. Typhimurium infection were 1 to 4-year-old children (41/120, 35.0%) followed by 19-59-year-old adults (39/120, 33.3%).Most S. Enteritidis-infected patients were adults (19-59 years old) or elderly (60-93 years old), with 46.4% (13/28) and 25.0% (7/28), respectively.The available CBC data indicated no differences in the presence of sepsis, the numbers of leukocyte, neutrophil, and lymphocyte counts between S. Typhimurium and S. Enteritidis-infected groups.Interestingly, there was a significant difference in the percentage of Hct level between the STM-infected (69/80, 86.3%) and the SE-infected group (10/18, 55.6%) (p = 0.006).By using a univariate analysis, it was found that patients with anemia (Hct level less than 13%) were at a higher risk for STM than SE infection (odd ratio of 5.018 with 95% confidence interval of 1.63-15.48).

Multidrug Resistance and ESBL-Producing Salmonella Strains Found in the Clinical Isolates
The S. Typhimurium and S. Enteritidis isolates were then tested for their susceptibility to 13 antimicrobial classes with a total of 24 antibacterial agents by a disc diffusion assay.The percentage of non-susceptible isolates between S. Typhimurium and S. Enteritidis is shown in Figure 2. Our data show that the highest incidence of non-susceptible antimicrobial agents is streptomycin (S), with 84.4% in S. Typhimurium but not S. Enteritidis.Among S. Typhimurium isolates, 82.3% are non-susceptible to ampicillin (AMP), followed by tetracycline (TE), doxycycline (DO), and amoxicillin-clavulanic acid (AMC) at 80.0%, 80.0%, and 47.3% of the isolates tested, respectively.Interestingly, S. Enteritidis isolates exhibited the highest non-susceptibility to nalidixic acid (NA), with 71.4%, followed by ciprofloxacin (CIP) and ampicillin (AMP), with 28.6% and 23.8%, respectively.Moreover, 6 out of 120 (5%) S. Typhimurium isolates, but none of the S. Enteritidis isolates produced extended-spectrum beta-lactamase (ESBL) detected by the double-disc synergy test (DDST).The source, antibiotic resistance pattern, MAR index, and virulence gene profiles of these 6 ESBL-positive S. Typhimurium isolates were shown in Supplementary Table S3.All S. Typhimurium isolates (120/120, 100%) are susceptible to ertapenem (ETP).All S. Enteritidis isolates are susceptible to most antibacterial agents tested except ampicillin (AMP), tetracycline (TE), doxycycline (DO), amoxicillin-clavulanic acid (AMC), ciprofloxacin (CIP), ceftazidime (CAZ), and nalidixic acid (NA).These data indicate that S. Typhimurium isolates in this study are more resistant to commonly used antibacterial agents than S. Enteritidis.S. Typhimurium conferred MDR phenotype higher than S. Enteritidis with multiple antibiotic resistance (MAR) indexes of 0.22 and 0.04 for S. Typhimurium and S. Enteritidis, respectively (Supplementary Tables S4 and S5).The percentage of S. Typhimurium isolates with a MDR phenotype (93/120, 77.5%) is significantly higher than that of S. Enteritidis (2/28, 7.1%) (Figure 3).Two S. Typhimurium isolates showed resistance across 10 of the 13 antibacterial agents tested, whereas the maximum resistance for S. Enteritidis was 4 classes, observed in only one isolate.
(MAR) indexes of 0.22 and 0.04 for S. Typhimurium and S. Enteritidis, respectively (Supplementary Tables S4 and S5).The percentage of S. Typhimurium isolates with a MDR phenotype (93/120, 77.5%) is significantly higher than that of S. Enteritidis (2/28, 7.1%) (Figure 3).Two S. Typhimurium isolates showed resistance across 10 of the 13 antibacterial agents tested, whereas the maximum resistance for S. Enteritidis was 4 classes, observed in only one isolate.

Detection of Essential Salmonella Virulence Genes in Clinically Isolated Strains
Next, we detected the presence of four representative genes essential for Salmonella pathogenesis in a mammalian host (spvB, sseI, sodCI, and rpoS) by PCR using the primer pairs in Table S1.The distribution of these four virulence genes across all 148 clinical Salmonella isolates is shown in Figure 4.All clinical isolates of S. Typhimurium and S. Enteritidis harbor rpoS gene.The sodCI and sseI genes were detected in all S. Enteritidis isolates.However, not all strains of S. Typhimurium contain sodCI and sseI genes (80.0% and 72.5%, respectively).Interestingly, the presence of spvB gene in S. Enteritidis is significantly higher (89.3%) than that of S. Typhimurium (5.8%).The distribution pattern of these four Salmonella virulence genes for all the strains is shown in Figure 5.Most S.

Detection of Essential Salmonella Virulence Genes in Clinically Isolated Strains
Next, we detected the presence of four representative genes essential for Salmonella pathogenesis in a mammalian host (spvB, sseI, sodCI, and rpoS) by PCR using the primer pairs in Table 1.The distribution of these four virulence genes across all 148 clinical Salmonella isolates is shown in Figure 4.All clinical isolates of S. Typhimurium and S. Enteritidis harbor rpoS gene.The sodCI and sseI genes were detected in all S. Enteritidis isolates.However, not all strains of S. Typhimurium contain sodCI and sseI genes (80.0% and 72.5%, respectively).Interestingly, the presence of spvB gene in S. Enteritidis is significantly higher (89.3%) than that of S. Typhimurium (5.8%).The distribution pattern of these four Salmonella virulence genes for all the strains is shown in Figure 5.Most S. Typhimurium isolates (65.2%) harbor sseI-sodCI-rpoS genes pattern, while most SE isolates (89.3%) have spvB-sseI-sodCI-rpoS genes pattern.The same pattern of virulence genes was observed in stool and blood-isolated S. Typhimurium and S. Enteritidis strains (Figure 5A,B, respec-tively).All blood-isolated strains also have either sseI-sodCI-rpoS or spvB-sseI-sodCI-rpoS gene patterns.
isolates.However, not all strains of S. Typhimurium contain sodCI and sseI genes (80.0% and 72.5%, respectively).Interestingly, the presence of spvB gene in S. Enteritidis is significantly higher (89.3%) than that of S. Typhimurium (5.8%).The distribution pattern of these four Salmonella virulence genes for all the strains is shown in Figure 5.Most S.

Discussion
Acute NTS is an important zoonotic foodborne illness that impacts human and animal health.Several livestock, such as pigs and poultry, are major reservoirs of drug-resistant nontyphoidal Salmonella [44].Close contact between people and animals is usually found in some areas of Thailand and is considered an important epidemiological risk factor.Previous epidemiological reports confirmed the rise of MDR NTS isolates globally and regionally [3,6].In this study, we focused on serovar Typhimurium and Enteritidis, the major human foodborne pathogens, and their increased MDR phenotype are of concern [45].We collected a total of 395 isolates of Salmonella spp.from stool or blood culture positive for Salmonella spp.from hospitalized patients at MNCMH from 2016 to 2021.
From the epidemiological studies of NTS in Thailand from 2002 to 2018, S. Typhimurium and S. Enteritidis were the major serovars detected from patient specimens, including stool and blood samples [46][47][48][49].However, variation between the geographical locations was observed.For example, 19.1% of S. Typhimurium and 19.7% of S. Enteritidis were detected from NTS patients in Northern Thailand from 2002-2007 (a total of 11,656 isolates) [10,43].In the large-scale study of Hendriksen et al., the authors reported that the top 10 serovars of nontyphoidal Salmonella in Northern Thailand are Enteritidis, Stanley, Weltevreden, Rissen, I (1),4,(5),12:i-, Choleraesuis, Anatum, Typhimurium, Corvallis, and Panama.Our study (N = 395) found that 30.4% are S. Typhimurium, 7.1% are S. Enteritidis, and 62.5% are other serovars.Most of S. Typhimurium (93.3%) and 75% of S. Enteritidis were isolated from the patient's stool, while 6.7% of S. Typhimurium and 25.0% of S. Enteritidis were collected from the patient's blood.These blood-isolated strains indicated the presence of iNTS at MNCMH during 2016-2021.
The demographic and principal diagnosis of 15 iNTS patients collected in our study (10.2%, 15 out of 148) is shown in Table 2.The age of iNTS patients can vary greatly from 2 to 76 years.Moreover, the immune status and Hct levels of patients were reported.Our data revealed no significant difference in age, gender, sepsis, leukocyte count, neutrophil count, and lymphocyte count between S. Typhimurium and S. Enteritidis-infected patients admitted at MNCMH.Interestingly, S. Typhimurium strains were significantly isolated more from anemic patients (86.3%), who have Hct levels less than 13%, than non-anemic patients (13.8%).Our study found that 80.6% of S. Typhimurium or S. Enteritidis-infected patients were anemic.This is consistent with the previous study showing that anemic patients are more susceptible to NTS [10].Katz et al. found that the hemoglobin or Hct level is inversely related to NTS bacteremia in adults in a high-income country.An increase in hemoglobin level by 1 g/dL is associated with a 28% lower incidence of iNTS.Low hemoglobin levels might reflect the poor nutritional status of patients.To our knowledge, no direct evidence indicates that S. Typhimurium infection causes anemia in NTS.However, anemia could be a significant risk factor for a subsequent S. Typhimurium infection.
In our study, S. Enteritidis can be isolated from stool and blood cultures, in accordance with the previous reports [50,51].S. Enteritidis isolated in Thailand was associated with life-threatening bacteremia in an immunocompromised host.An observational study in 2009 showed that 60.8% of S. Enteritidis isolates (n = 1517) were recovered from blood specimens [51].However, our study found that both S. Typhimurium and S. Enteritidis can cause NTS bacteremia.Our study also showed that S. Typhimurium isolates have a higher percentage of non-susceptibility to several antibacterial agents than S. Enteritidis isolates.S. Typhimurium isolates exhibited the highest resistance to streptomycin, followed by ampicillin, tetracycline, and doxycycline.The most frequent MDR pattern in ST34 S. Typhimurium in our study is streptomycin (S)-ampicillin (AMP) -TE (tetracycline)-doxycycline (DO) (15/18, 83.3%).Our results are consistent with the MDR phenotype of S. Typhimurium isolated from humans, pigs, pork, and poultry in previous reports [47,52].S. Enteritidis clinical isolates exhibited the highest resistance to nalidixic acid followed by ciprofloxacin and ampicillin, which is consistent with the previous report [51].Hendriksen et al. reported that S. Enteritidis isolates from blood and stool specimens in Thailand are resistant to ampicillin, ciprofloxacin, and nalidixic acid.A spread of the MDR ST11 S. Enteritidis between chickens and humans in Thailand had also been reported [52].We found that some S. Typhimurium isolates (5%, 6/120) were ESBL-producers, but none of the S. Enteritidis.The previous study showed that ESBL-producing Salmonella (1.9%) were detected from slaughterhouse pigs and retail market pork in the border of Thailand [11].
The first to fourth-generation cephalosporins susceptibility of the Salmonella isolates were investigated in this study.A total of 21 S. Typhimurium isolates (21/120, 17.5%) resisted the second and third-generation cephalosporins, thus defined as extended-spectrumcephalosporins (ESC) resistance strains.Moreover, 15% (18/120) of S. Typhimurium isolates were resisted to a fourth-generation cephalosporin, cefepime.The previous work illustrated that 3.8% of NTS isolated from pigs and pork in Thailand are resistant to a third-generation

Figure 3 .
Figure 3. High multidrug resistance phenotype in S. Typhimurium.Percentage of the drug resistance phenotype (MDR, resistant to 1-2 classes and completely susceptible) of S. Typhimurium and S. Enteritidis clinically isolates.The MDR was defined as non-susceptible to at least one antimicrobial agent in ≥3 classes (Total N = 120 for STM, N = 28 for SE).

Figure 5 .
Figure 5. Virulence gene-harboring pattern of S. Typhimurium and S. Enteritidis clinically strains isolated from stool culture (A) and blood culture (B).(Total N = 120 for S. Typhimurium, N = 28 for S. Enteritidis).

Figure 5 .
Figure 5. Virulence gene-harboring pattern of S. Typhimurium and S. Enteritidis clinically strains isolated from stool culture (A) and blood culture (B).(Total N = 120 for S. Typhimurium, N = 28 for S. Enteritidis).

Table 1 .
Primer pairs used in this study.

Table 2 .
Demographic and principal diagnosis of iNTS patients (15 out of 148 patients).

Table 3 .
Demographic and clinical data of hospitalized patients with stool or blood culture positive for Salmonella enterica serovars.