Non-typhoidal Salmonella contributes to gastrointestinal infections in Morogoro: Evidence from patients attending Morogoro regional referral hospital in Tanzania

Introduction Salmonella is one of the most common causes of food-borne outbreaks and infection worldwide. Non-typhoidal Salmonella (NTS) infections are common and remain a significant public health problem among important bacterial foodborne diseases. The current study aimed to establish the Non typhoidal Salmonella infection and antimicrobial resistance status among selected patients at Morogoro Regional Referral Hospital (MRRH), Morogoro Region, Tanzania, to inform clinical care management and public health interventions. Methodology A cross-sectional study was conducted using medical records and samples were collected from hospitalised and outpatients between October and December 2021. A total of 153 participants were enrolled in the study and 132 consented to being sampled. The collected samples were analysed using standard microbiological techniques. The isolates were subjected to molecular genotyping, where Polymerase Chain Reaction (PCR) was performed targeting the 16S rDNA gene. PCR products were then submitted for sequencing to establish phylogenetic relatedness. Antimicrobial susceptibility testing and resistance genes screening were also conducted. Results The phylogenetic analysis identified two Salmonella serovars; Salmonella Enteritidis and Salmonella Typhimurium. The isolates were from four adults and seven children patients. The isolates were tested against six antimicrobial agents: tetracycline, trimethoprim, gentamycin, ciprofloxacin, ampicillin and cefotaxime. Further antimicrobial assays were performed by screening 10 antimicrobial resistance genes using PCR. Overall, the highest resistance was observed in ampicillin (100%), whereas the lowest resistance was recorded for ciprofloxacin and gentamicin (9.1%). In addition, four (36.4%) of the isolates were resistant to cefotaxime and three (27.3%) to tetracycline and trimethoprim. The isolates also exhibit the presence of resistance genes for sulfamethoxazole 1&2, tetracycline (tet) A&B, Beta-lactamase CTXM, Beta-lactamase TEM, Beta-lactamase SHV, Gentamycine, Acra and acc3-1 in different occurrences. The overall prevalence of Salmonella species in Morogoro region was 8.3% (11/132) with Salmonella Enteritidis and Salmonella Typhimurium being the only serovars detected from adults and children stool samples. Conclusion Our investigation showed that both children and adults had been exposed to Salmonella spp. However, the occurrence of NTS was higher in children (5.3% (7/132) compared to adults (3.0% (4/132). To stop zoonotic infections and the development of antimicrobial resistance in the community, this calls for Infection Prevention and Control (IPC) and stewardship programmes on rational use of antimicrobials in both health facilities and at the community level.


Introduction
Salmonella is one of the most common causes of food-borne outbreaks and infection worldwide.Non-typhoidal Salmonella (NTS) infections are common and remain a significant public health problem among important bacterial foodborne diseases.The current study aimed to establish the Non typhoidal Salmonella infection and antimicrobial resistance status among selected patients at Morogoro Regional Referral Hospital (MRRH), Morogoro Region, Tanzania, to inform clinical care management and public health interventions.

Methodology
A cross-sectional study was conducted using medical records and samples were collected from hospitalised and outpatients between October and December 2021.A total of 153 participants were enrolled in the study and 132 consented to being sampled.The collected samples were analysed using standard microbiological techniques.The isolates were subjected to molecular genotyping, where Polymerase Chain Reaction (PCR) was performed targeting the 16S rDNA gene.PCR products were then submitted for sequencing to establish phylogenetic relatedness.Antimicrobial susceptibility testing and resistance genes screening were also conducted.

Results
The phylogenetic analysis identified two Salmonella serovars; Salmonella Enteritidis and Salmonella Typhimurium.The isolates were from four adults and seven children patients.The isolates were tested against six antimicrobial agents: tetracycline, trimethoprim, gentamycin, ciprofloxacin, ampicillin and cefotaxime.Further antimicrobial assays were performed by screening 10 antimicrobial resistance genes using PCR.Overall, the highest resistance was observed in ampicillin (100%), whereas the lowest resistance was recorded for ciprofloxacin and gentamicin (9.1%).In addition, four (36.4%) of the isolates were

Introduction
Salmonella infection is a major public health challenge of worldwide concern in both industrialised and developing countries and has contributed to an increased economic burden on health systems [1].Non-typhoidal Salmonella is a common disease in developing countries, particularly in sub-Saharan Africa, due to problems of poor sanitation, low hygiene, and lack of access to safe water and food, contributing to around 9% of deaths in children under 5 years of age [2,3].Most of the Salmonella spp.infected individuals present diarrhoea, high fever with prostration, and stomach cramps.Symptoms typically appear 6 hours to 6 days after exposure and last 4 to 7 days [1].Antimicrobials are only used to treat people with severe illnesses with life-threatening symptoms [4].In severe cases, hospitalisation may be required.Every year, approximately 600 million people, or nearly one-tenth of the global population, become ill, and 420 000 die, resulting in the loss of 33 million disability-adjusted life years (DALYs), accounting for 22.2% of DALYs due to diarrhoea [5].
According to the White-Kauffmann-Le Minor scheme [6], Salmonella has over 2600 serotypes, with the majority (approximately 1600 serotypes) belonging to the subspecies enterica.Though many serotypes (over 200) have been identified as pathogenic agents in humans, the two most common serotypes causing human salmonellosis are Salmonella Enteritidis (S.Enteritidis) and Salmonella Typhimurium (S.Typhimurium) [7].Both Serotypes (S.Typhimurium and S. Enteritidis) produce the symptoms of gastroenteritis and bloody diarrhoea [8].Therefore, many strain types make it challenging to diagnose the condition rapidly and accurately [9].In Tanzania, there are few reports of the occurrence of Salmonella spp.infection in both humans and animals [10][11][12] and Salmonella infection in Tanzania, concretely in Morogoro, may be higher than expected.Healthcare services in low-and middle-income countries are facing significant challenges due to antimicrobial multi-drug resistance, which is increasing rapidly due to increased global human mobility and irrational use of antimicrobials in the health and livestock sectors [13].Human infections with multi-resistant bacterial strains, including Salmonella, can result in prolonged hospitalisation and increased mortality, especially in children with invasive infections [14].Diarrhoeic cases in patients admitted to Morogoro referral hospital are common, but the aetiologies were not well elucidated.The contribution of NTS was not well known; hence, it often goes unnoticed despite its disastrous effects in terms of human suffering and economic costs.Moreover, before this study, limited evidence existed on the incidence of NTS gastroenteritis in MRRH, Tanzania.Therefore, this study aimed to establish the status of NTS and its antimicrobial profile at MRRH, Tanzania, to inform its existence and treatment consideration.
Estimating the occurrence of NTS among diarrhoeic hospitalised and outpatients in MRRH is paramount for planning strategies and developing preventive and control strategies to reduce admissions caused by diarrhoeic cases.In addition, the antimicrobial susceptibility profiles of the isolated NTS assessed will guide updating treatment guidelines in public health interventions.

Ethics statement
In the present study, ethical clearance was granted by the National Institute of Medical Research, Dar es Salaam, Tanzania (Ref No.: NIMR/HP/R.8a/Vol.IX/3808-14 th October 2021).Permission to carry out the study was also sought from the regional health management team (RHMT) (DC.65/245/01 -18 th September 2021), the Morogoro region district office and the management of the MRRH.Ethical approval for the study was given by the ethical committees of Sokoine University of Agriculture, Tanzania, with reference No SUA/AMD/R.1/8/763,approved on 18th October 2021.All study participants were informed about the study objectives and the procedures involved in enrollment participation.Before collecting faecal specimens, signed informed consent was obtained from each adult individual.Additionally, the parent or legal guardian signed a written consent form for minors under 18 years of age.

Description of the study area
The study was conducted in MRRH from October 2021 to December 2021.Morogoro town is the headquarters of Morogoro region, with a population of 986,678 in 2022 [15], located in the eastern part of Tanzania, 196 kilometres (122 mi) west of Dar es Salaam.Morogoro town lies at the base of the Uluguru Mountains [16].Morogoro residents receive health services from health institutions owned by the government and private sectors.
The study area was chosen based on the more significant number of patients attending the facility in the region.Hence, patients attending the MRRH presenting with signs suggestive of Salmonella spp.infectious, such as stomach pain symptoms and gastroenteritis, were recruited.The participants were characterised by their age, gender, marital status and occupation.The 132 stool samples from patients with gastrointestinal problems without diarrhoea as the first symptom admitted at the hospital and outpatients who went to the hospital complaining about diarrhoea originating in Morogoro were analysed.In this study, age groups were classified as follows; children under five years old, children (5-8 years), teenagers (13-19 years), younger (20-21 years), adults (22-60years) and elders (above 60 years).

Sampling and patient recruitment
Purposive sampling based on clinical signs and symptoms was used to recruit participants in this study.Participants from the four hospital wards, recovery wards (male and female), surgical wards (male and female), maternity ward (females only), laboratory and outpatients were selected for recruiting patients with assistance from the clinician depending on clinical presentation and their willingness to participate in the study.Study participants were informed about the overall objective of the study, the voluntary participation, and their rights to withdraw from the study.Recruitment was subject to obtaining written informed consent.A total of 153 patients were interviewed but only 132 consented to collect samples from them.The respondents were divided into two groups of children and adults, where for children, two subgroups were under five years old and above five years up to 13. Adults were further divided into four subgroups; teenagers (13)(14)(15)(16)(17)(18)(19), youths (20)(21), adults  and elders (60+).

Inclusion and exclusion criteria
Patients with any of the main signs/symptoms suggestive of Salmonella spp.Infections, including abdominal pain, diarrhoea and blood in stool, were enrolled for sample collection.Patients who contracted gastroenteritis of any cause after admission and patients who did not have gastrointestinal alterations were excluded from the study.

Stool sample collection
Stool samples were collected from outpatients and hospitalised patients attending MRRH during the study period.The stool samples were collected from patients admitted in the adult male recovery ward, adult female recovery ward, infectious pediatric ward, general pediatric ward, maternity ward and the laboratory.Screw-caped clean plastic containers were used for the collection of stool samples.The samples were transported to Microbiology Laboratory at the College of Veterinary Medicine and Biomedical Sciences, SUA, in an icebox within 3-4 hours of collection.They were processed upon arrival within a maximum of two hours.

Conventional identification of Salmonella spp
In the laboratory, stools were enriched in peptone-buffered water and inoculated in Selenite Fecal Broth (SFB) (Becton-Dickinson-USA) overnight.Briefly, 1 g of stool sample was enriched in 9 ml of buffered peptone water (OXOID, UK) and inoculated in Selenite Fecal Broth (SFB) (Becton-Dickinson-USA), and incubated overnight for 24 h at 37˚C.The enriched samples were cultured in Salmonella-Shigella Agar (SSA) and Xylose-Lysine-Deoxycholate (XLD) agar, according to ISO 6579 and ISO 21567 [17].All suspected Salmonella spp.colonies were picked and then subcultured on MacConkey and blood agar and incubated at 37˚C for 18-24 hours.The bacteria grown were studied for their micro-morphological characteristics, while Gram stains were performed to investigate their micro-morphological characteristics as well.Conventional biochemical tests which involve several sugars, such as Triple Sugar Iron (TSI) and enzymatic tests were performed to identify the Salmonella spp presumptively.Both slide agglutination using polyvalent antisera (Poly A-E+Vi from SSI, Denmark) and Minibact-E biochemical tests (SSI, Denmark) were used to confirm isolates.The WHO National Salmonella and Shigella Center, Institute of Health in Bangkok, Thailand, used serotyping to identify specific serovars [18].Phage typing was done for S. Typhimurium (5 isolates) and S. Enteritidis (6 isolates) according to the scheme defined by the PHLS Colindale London at the OIE-National reference laboratory for Salmonella, Instituto Zooprofilattico Sperimentale delle Venezie, Italy.

Molecular confirmation of Salmonella spp
DNA extraction.According to Carriero et al. (2016) [19], the thermal extraction technique was used to isolate the genomic DNA.A few colonies were briefly emulsified in 1.0 mL of nuclease-free water (provided by Inqaba Biotech, Hatfield, South Africa), washed by vortexing, and re-suspended in nuclease-free water.These steps were completed by boiling in a water bath at 95˚C for 5 minutes, and then the colonies were immediately placed in ice for 5 minutes.After repeating this technique, the suspension was centrifuged at 10,000 g for 10 minutes.The total genomic DNA was spectrophotometrically quantified using NanoDrop TM Lite Spectrophotometer (Thermo Scientific, Waltham, and U.S.A) and stored at -20˚C until further use [19].
PCR for the 16S rRNA gene.The amplification of the 1500bp fragment of the 16S rRNA gene was conducted by PCR using the universal bacterial primers for 16S rRNA 9F5'-GAGTT TGATCCTGGCTCAG-3', 1542R5'-AGAAAGGAGGTGATCCAGCC-3' in 20μl reaction mixture: PCR master mix (Bioneer, South Korea) constituted of 18μl of the reaction mix and 2μl of a DNA sample [20].The PCR protocol was set as follows: denaturation at 94˚C for 30 seconds, annealing at 58˚C for 30 seconds, elongation at 72˚C for 2 minutes, all these temperatures undergone 35 cycles and final extension at 72˚C for 10 minutes.The post-PCR products were run in the gel electrophoresis using 1% agarose gel and 1xTE buffer for 45 minutes at 100 V.The 16S rRNA post-PCR products were sequenced using Sanger sequencing technology (ABI sequencer) at Macrogen Europe, Netherlands.Nuclease-free water was used as a negative control, and DNA extracted from a reference standard Salmonella Typhimurium (ATCC 13311) collected from Minnesota, USA, was used as a positive control.
2.8 Antimicrobial susceptibility testing.Utilising Kirby-Bauer's disc diffusion method, antimicrobial susceptibility testing was carried out using the clinical and laboratory standards institute guidelines, [21].The tops of well-defined colonies were touched with a sterile loop and then transferred to a tube of normal saline.The colony was emulsified on the inside of the tube near the side to prevent the colony's cells from clumping together.A 0.5 McFarland standard was used as the inoculum standard, which equates to roughly 106 CFU/mL.Muller Hinton agar plates (OXOID, UK) were used for the inoculation of Salmonella bacteria, which were then allowed to dry for 15 minutes.The six chosen antimicrobial disks were placed on the plates, and finally, the plates were incubated at 35˚C for 24 hours.A sterile cotton swab was dipped into the inoculum, swirled several times, and then pressed firmly against the inside wall of the tube above the fluid level to remove additional inoculum from the swab.Disks were applied to the agar surface using an Oxoid disk dispenser and sterile forceps.Plates were incubated in the air at room temperature (34˚C) for 18 hours with the plates inverted.The following standard antimicrobial disks (OXOID, UK) were used: trimethoprim (15 μg), cefotaxime (15μg), ampicillin (15 μg), tetracycline (15 μg), gentamicin (15 μg) and ciprofloxacin (15μg) [21].Using callipers, the zones of inhibition in the cultures were quantified and evaluated.Interpretation of the results were recorded as sensitive (S), intermediate (I) or resistant (R) based on the National Committee for Clinical and Laboratory Standards Institute criteria [21].To ensure the testing performance of the effectiveness of antimicrobial discs and the caliber of the media, the standard reference strain of Escherichia coli (ATCC-25922) was utilised.The resistant group also comprised intermediate resistant strains.Non-susceptibility to three or more types of antimicrobials was the definition of multidrug resistance (MDR) [22].
2.9 Amplification of antimicrobial-resistant genes.Specific PCRs targeting 9 antimicrobial-resistant genes were performed on each pooled DNA sample using specific primers, as shown in Table 1 below.Two Duplex PCRs were conducted to detect the Sul1 and Sul2 genes and bla CTX-M and bla SHV genes.Singleplex PCR was performed on the rest of the antimicrobial genes.All amplifications were performed using the same regimen except for annealing temperatures, which were adjusted based on the manufacturer's recommendations (Table 1).The amplification regimen included an initial denaturation step at 95˚C for 15 minutes, 30 cycles of denaturation at 94˚C for 30 seconds, annealing at respective temperatures for 30 seconds, extension at 72˚C for 2 minutes, followed by a final extension step at 72˚C for 10 minutes.E. coli ATCC 25922 and Universal bla CTX-M were used as negative and positive controls, respectively.The PCR amplifications were electrophoresed in 1.5% agarose gel at 110V for 60 minutes and viewed in gel Tran's illuminator machine.

Data analysis
Data were analysed using STATA version 16 of 2018, where descriptive and inferential statistical analyses to descpribe the association of symptoms to Salmonella infection and demographic data were performed using Chi-squire.Descriptive statistics were used to present the social demographic characteristics of respondents.The data analysis was performed separately for children under 05 years old and individuals aged >05 years old.Descriptive statistical analysis was conducted using GraphPad Prism 9 software.The sequence comparison was performed by BLASTING the sequences in www.ncbi.nlm.nih.gov/BLAST to obtain the reference sequences from the gene bank.The sequence assembly from this study was conducted using Bioedit software version 7.2.All sequences (from this study and references) were aligned using MEGA XI software.The neighbor-joining phylogenetic tree was constructed using the same software.

Demographic characteristics of the participants
One hundred and fifty-three participants involved in this study comprised two broad groups of children (n = 65) and adults (n = 88).While all the participants consented to an interview, only 132 patients consented to collect stool samples of these patients with gastrointestinal problems admitted and outpatients at the hospital originating in Morogoro for analysis.Most of the patients (74 patients) came from rural areas, but some were from the Morogoro Municipality (58 patients).The sampled patients were in the following groups: children (n = 54) and adults (n = 78), further classified as teenagers (n = 6), younger (n = 4), adults (n = 59) and elders (n = 9).Of those patients, 58/132 (44%) were male and 74/132 (56%) were female.In this study, age groups were classified as follows: children (5-8 years), teenagers (9-16 years), younger (17-21 years), adults (22-60 years) and elders (above 60 years).

Isolation and molecular confirmation of Salmonella spp
Of 132 samples subjected to conventional identification, 11(8.3%) were presumptively identified as Salmonella spp.The bacteria were non-lactose fermenters on MacConkey agar, while their colonies appeared colourless with black centres on SSA and pink with a black centre on XLD.The microscopic findings revealed them as short rods in single, gram-negative bacteria.Molecular amplification of the 16S rDNA gene detected a PCR product band of 1500bp in gel electrophoresis (Fig 1).Sample number 11 contains multiple bands, which makes it appear different from the rest of the bands.These multiple bands are caused by low annealing temperatures and short annealing times.But sometimes, the nature of the sample itself can cause this condition to happen.Further optimization of PCR is supposed to be done to give good results.
The results for phylogenetic analysis displayed two important non-typhoid Salmonella serotypes; Salmonella Enteritidis (6 isolates) and Salmonella Typhimurium (5 isolates) from several sequences in the gene banks (Fig 2).The sequences from this study showed a percentage identity of 97-99% during blasting with those used in phylogenetic analysis.

The occurrence of Salmonella spp between children and adults
A comparison of the isolation rate between adults and children showed a higher infection rate in children 5.3% (7/132) than in adults 3.0% (4/132); however, the difference (P>0.05) was not statistically significant.

Phenotypic and molecular antimicrobial susceptibility assessment of isolated Salmonella spp
The results of antimicrobial susceptibility profiles of the Salmonella spp.isolates showed that all of the total isolates, 100% (n = 11), were resistant to at least one antimicrobial.It further showed that all of the Salmonella spp.isolates were resistant to ampicilin (100%, n = 11).The other antimicrobial resistance occurrences were cefotaxime (36.4%, n = 4), tetracycline and trimethoprim (27.3%, n = 3 each).Resistance to gentamicin and ciprofloxacin was 9.1% each, as observed in Table 2.The results of antimicrobial susceptibility profiles of the Salmonella spp.
isolates showed resistance to one or more classes of antimicrobials, taking into account the Salmonella isolates tested as multiple drug resistance isolates (MDR), as observed in Table 3.
There was a marked difference in resistance between the two antimicrobials (gentamicin and ciprofloxacin) and the rest of the tested antimicrobials (p<-0.05).Nine different resistant genes were detected using specific primers, as shown in Table 1.The genes include the cephalosporin-resistant gene (bla TEM gene, bla SHV gene and universal CTX-M gene), ciprofloxacin- resistant gene (arc gene), gentamicin-resistant gene (arc(3)-I gene), tetracycline-resistant gene (tetA gene), doxycycline resistant gene (tetB gene) and sulfamethoxazole-trimethoprim resistant gene (Sul 1 and Sul 2 gene).Sulfonamide-resistant genes were highly detected (Sal1 = 100%) compared with ciprofloxacin (0) and gentamicin (0) Table 3.The agarose gel pictures showing the corresponding band size have not been shown.

Association between Salmonella symptoms and patient age category
The symptoms diagnosed included diarrhoea, blood in stool, nausea, vomiting, fever, and general pains.Results showed that nausea, vomiting and general aches and pains were significantly associated with age category (p<0.01).Other symptoms like diarrhoea, blood in stool and fever had no significant association with age of the patient (Table 4).

Discussion
Non-typhoid gastroenteritis caused by Salmonella spp.continues to be an important public health issue that often requires hospitalisation in Morogoro.This is one of the few studies exploring the genetic diversity of salmonellosis in Morogoro for elders, adults, youth, and young children.Salmonella spp. is developing resistance to many used antimicrobials, which makes it relevant to this study.The overall prevalence of Salmonella spp in Morogoro region was 8.3% (11/132) (95% C.I; 5%-10.0%),with Salmonella Enteritidis and Salmonella Typhimurium being the only serovars detected from adults and children stool.A comparison of the isolation rate showed that children had a higher proportion (11.6%) compared to adults (10.5%).However, the difference was not statistically significant (P>0.05).This is concomitant to studies from Mozambique, where a high burden of NTS was reported among young children (S.Typhimurium and S. Enteritidis) [29].In most parts of the world, surveys have reported S. Enteritidis and S. Typhimurium as the major serovars found in humans [30,31].Feasey et al. [32] also reported 32% compared with 54% of invasive NTS in children <15 years of age in South Africa and Malawi, respectively.In Co ˆte d'Ivoire, the mortality rate was estimated to reach 5% from Salmonella infections.From studies carried out between 2005 and 2009 among isolated serotypes, NTS was prevalent [33].It was noted from this study that S. Enteritidis is most dominant among hospital isolates, while S. Typhimurium serovar was the least dominant.The previous studies conducted in Mukuru slum, an urban informal settlement in Kenya, reported S. Typhi was the dominant serovar followed by S. Typhimurium and S. Enteritidis [34].Previous studies in other sub-Saharan African countries also showed the dominance of S. Typhimurium [35].Different serovars of Salmonella spp.are known to be commonly detected in a given area at different times depending on the type of serovars of Salmonella circulating in food sources in a study area [36].In most other African countries, these serovars are the most frequently isolated from diarrheal diseases, which is confirmed by the present study's findings.In Tanzania there are scacy data published on contribution of non typhoidal Salmonella spp in causing gastroentelitis and their antimicrobial status in human, howover, a considerable literature is available in food animals and food products.NTS resistance to the commonly used antimicrobials calls for the implementation of national surveillance systems for antimicrobial resistance and implementation of prudent antimicrobial usage, including revision of current national guidelines for antimicrobial use.S. Typhimurium has often been associated with multiple antimicrobial resistances [37], partly due to the emergence of S. Typhimurium definitive phage type (DT) 104 worldwide.Strains of this phage type are resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline [38].Phage typing has proven helpful for classifying S. Enteritidis and S. Typhimurium strains from various sources.However, the technique has shown to be inadequately selective when just one phage type has predominated, necessitating further characterisation.
The findings in this study reported a low cephalosporin resistance but high resistance to other antimicrobials among Salmonella strains.It was indicated that the resistance was higher to ampicillin (100%) than all other antimicrobials.The antimicrobial resistance in Salmonella spp. is one of the main concerns when it comes to the issue of human infection treatment [39].Moreover, the high resistance of Salmonella spp. to ampicillin was also recorded by a study from Harare, where Salmonella spp. was resistant to ampicillin (100%), as reported by Dinkineh et al. [40].A similar finding was reported by Bilal et al. [41] in Pakistan.It is also important to note that, a study from Addis Ababa reported that 81.2%, 94.5%, and 75.7% of the isolates were resistant to ampicillin, tetracycline, and sulfamethoxazole-trimethoprim, respectively [42].
In contrast to the present study, 72.7%, 63.6% and 54.5% of Salmonella spp.isolates were shown to be susceptible to sulfamethoxazole-trimethoprim, tetracycline and cefotaxime respectively.It is also worth noting that, generally the recovered Salmonella spp.serovars in this study showed low resistance to cephalosporins and high resistance to other antimicrobials routinely used for the treatment of humans in Tanzania.The increased resistance towards these commonly used antimicrobials may have been contributed by misuse, incorrect diagnosis and failure to abide by treatment guidelines.This investigation identified a significant trend in the emergence of MDR in Salmonella Typhimurium and Salmonella Enteritidis isolates.The spectrum of resistance in NTS isolates used in this study extended further to include cefotaxime, trimethoprim-sulphamethoxazole and tetracycline, with the resistance levels agreeing with rates found in a study carried out in Thailand by Toni et al., (2018) [43].In this study, the antimicrobial resistance gene results found that 8 isolates in adults and children contain sulfamethoxazole (Sul 1 and 2) resistance gene, tetracycline (tetA) resistance gene, Bla CTXM resistance gene, Bla SHV resistance gene and Bla TEM resistance gene.In contrast, no gene was detected for tet(B), Acc(I-3) and AcrA.These results agree with those of Hedayatianfardi et al., [44].who found no genetic evidence for tet(B) presence in their experiments using PCR.However, phenotypic results showed resistance to tetracycline and sulfamethoxazole, but only sulfamethoxazole carried the resistant gene.This is because the phenotype of most isolates is influenced by specific and non-specific resistance mechanisms such as lower membrane permeability and high active efflux pumps [45].In the current study, the isolates carrying the tetA gene were phenotypically susceptible to tetracycline.Tetracycline resistance in bacteria is mediated by a group of tet genes through different mechanisms, including ribosomal protection and efflux pumps, [46].On the other hand, tet genes have different modes of action, which, in some cases, necessitate the presence of more than one type of tet gene for the bacteria to fully express phenotypic antimicrobial resistance [47].The lack of consistency in the relationship between the presence of resistance genes and phenotypic antimicrobial resistance of the isolates observed in the current study indicates the importance of accompanying the resistance gene analysis with phenotypic antimicrobial resistance testing.
Based on Salmonella spp.symptoms and patient category, adults and children suffering from Salmonella had more episodes of fever and few of them complained about visible blood in the stool.The difference, however, was statistically insignificant.The symptoms like diarrhoea, blood in stool and fever had no significant association with the age of the patient in Morogoro region.Such observations are concomitant with the findings of other studies [48] Diarrhoea was significantly related to NTS infection in the Morogoro region.However, it is important to note that these clinical symptoms might be caused by other factors, including changes in diet, which can even affect faecal consistency.

Conclusion
In comparison to clinical and microbiological characteristics of patients with gastrointestinal problems at the hospital in Morogoro, the infection rate of 8.3% NTS was established.These results, combined with a different antimicrobial susceptibility profile, warrant further investigation into the epidemiology of NTS infections.NTS is one of the more important causes of illness in children than adults in Morogoro region, especially among children less than 5 years of age, implicated to their immunity.To target the control actions to the critical points in the transmission of the pathogens to consumers, systematic surveillance of the infection sources in humans and animals, as well as the routes of the bacterial pathogens, is therefore mandatory.

Study limitation
This study had some limitations.Firstly, it was limited by information bias.Questions for children were designed to enable parents or guardians to report on their behalf (recall bias), thus underestimating the true ORs or association of causal effect.Another limitation of this study was the use of stool samples only; multiple sampling sources, such as blood, could have created a nice ground for the broad prevalence establishment.

Fig 2 .
Fig 2. Phylogenetic tree for Salmonella serotypes from this study (blue circle) and closely related taxa from the gene bank to assess the genetic similarity of the Salmonella isolates from human faeces from Morogoro.The tree was generated using the Neighbor-Joining method (p-distance model), with bootstrap values expressed as percentages of 1,000 replications.Fowlpox virus (AY453172) served as an out-group.https://doi.org/10.1371/journal.pntd.0012249.g002