Antimicrobial Susceptibility of Salmonella Isolated from Chickens and Humans in Wau, South Sudan

Background Salmonella infections are a public health problem across the globe. In South Sudan, there is little information regarding the prevalence and antibiotic resistance patterns of Salmonella. Therefore, this study assessed the prevalence and antimicrobial susceptibility of Salmonella isolates from chickens and humans in South Sudan. Fecal samples were collected and cultured on Xylose Lysine Deoxycholate Agar for the isolation of Salmonella and confirmed using biochemical tests and PCR through the amplification of the invA gene. A total of 417 fecal samples were examined, of which 270 (64.7%) were chicken cloacal swabs while 147 (35.3%) were humans' stool specimens. Results Eleven (11) Salmonella isolates were isolated from humans while nine were from chickens. All 11 isolates from humans were susceptible to sulfamethoxazole-trimethoprim, chloramphenicol, streptomycin, cefotaxime, nalidixic acid, and gentamicin. However, 4 (36.7%) isolates showed resistance to ciprofloxacin, 2 (18.9%) to ampicillin, and 1 (9.1%) to tetracycline. All chicken isolates were susceptible to chloramphenicol, streptomycin, sulfamethoxazole-trimethoprim, ciprofloxacin, cefotaxime, nalidixic acid, and gentamicin but showed resistance to tetracycline 2 (22.2%) and ampicillin 1 (11.1%). Conclusion Antimicrobial resistant isolates were isolated in both chickens and humans. Further, MDR isolates were found in both chicken and human samples, and this is a public health concern. This, therefore, calls for concerted efforts to educate producers and consumers on public health, food safety, food hygiene in food production, and enhancement of surveillance programmes on zoonotic bacteria and antimicrobial susceptibility.


Introduction
Worldwide, Salmonella has been listed among the most important food-borne pathogen that is transmitted through the consumption of contaminated food [1]. It causes approximately 1.4 million cases of disease and about 20,000 hospital cases and over 500 deaths every year [2]. A growing number of human Salmonellosis cases have been associated with the consumption of contaminated food of poultry origin, such as chicken and chicken products [3]. Besides, chicken products have also been reported to play a major role in the spreading of antimicrobial-resistant zoonotic bacterial pathogens [4] even though the hygienic standards for chicken production are quite high and usually vary from place to place [5,6]. Te problem of AMR is still rising.
Antimicrobial resistance (AMR) and particularly multidrug resistance (MDR) is becoming very common among various Salmonella serotypes that have been isolated from humans and chickens world over [7]. Te extent of AMR varies from region to region and is usually infuenced by the abuse of antibiotics in humans and animals [8]. Reports of cases of Salmonella isolates being resistant to important antibiotics have been reported dating back to the 1960s during which resistance was reported to have been limited to one antibiotic [9]. However, from the 1970s onwards, there has been an increase in the number of Salmonella isolates that have shown resistance to various clinically signifcant antibiotics, and this has been exacerbated by the recovery of such isolates in foods of animal origin [10]. Tis is a growing public health concern as human Salmonellosis caused by resistant strains of Salmonella may be difcult to be treated [10]. Since the mid-1970s, there has been an increasing trend of Salmonella isolates exhibiting MDR phenotypes worldwide [11]. Te MDR exhibited by some Salmonella isolates and other pathogens is obtained from extrachromosomal genes that may impart resistance to an entire class of antimicrobials [12]. More recently, most of the resistance genes have been associated with large transferable plasmids and other DNA mobile elements, such as transposons and integrons [9,13]. Moreover, MDR seems to be more serious in some serotypes compared to other serotypes [14,15]. Terefore, there is a need for continuous monitoring of human and animal Salmonella isolates that exhibit resistance to most antimicrobials on a global scale [16].
In South Sudan, a young country where everything is still in its infancy, there is limited information regarding Salmonella species which was confrmed by PCR amplifcation of the invA gene. Terefore, this study assessed the prevalence and antimicrobial susceptibility of Salmonella isolated from humans and chickens in Wau, Western Bahr el Ghazal state, South Sudan, to inform control strategies.

Isolation of Salmonella from Fecal Samples.
Te overall prevalence of Salmonella in the study was 4.8% (20/417), as shown in Table 1. Te prevalence was relatively higher in humans, about 7.5% (11/147) compared to 3.3% (9/270) in chickens (Table 1). Te Salmonella was further confrmed by PCR using the invA gene.
Te prevalence of Salmonella in humans and chickens based on areas where samples were collected is shown in Table 2. Te highest prevalence among human samples was from Baggari (9.4%) followed by Busuri (4.8%) ( Table 2). For chicken samples, Baggari showed the highest prevalence of 4.5.% while Busuri payam showed a relatively lower prevalence of 1.7% (Table 2).

Detection of Extended Spectrum Beta-Lactamases (ESBLs)
in Salmonella Strain. All the 20 Salmonella isolated from chickens and chicken keepers (humans) did not show any sign of growth on MacConkey agar supplemented with 2 mg/l of cefotaxime implying that all the 20 isolates were susceptible to cefotaxime.

Salmonella Serotypes Isolated from Human and Chickens.
Te Salmonella isolated from humans and chicken belonged to fve serotypes, namely, Salmonella Aberdeen, Enteritidis, Uganda, Montevideo, and Typhimurium (Table 3). Salmonella Typhimurium was the most detected serotype (Table 3). Some nontypeable isolates were detected in humans while Salmonella Uganda and Salmonella serovar Montevideo were found in chickens only (Table 3).

Antimicrobial Resistance Profles of Salmonella Isolates.
Antimicrobial resistance profles of the 20 Salmonella isolates were divided into three diferent MDR resistotype profles (Table 4). Tis resistance was based on resistance to at least three diferent antimicrobials [17]. Resistotype group 1, resistance to ampicillin, tetracycline, ciprofoxacin, and gentamicin, was represented by 3 (15%) isolates. Resistotype group 2, resistance to ampicillin, gentamicin, and tetracycline, was detected in only 1 (5%) isolates, specifcally Salmonella Typhimurium isolated from humans and chickens. However, resistotype group 3, resistance to ampicillin and ciprofoxacin, was found in three (15%) isolates. Salmonella strains isolated from the chicken source were represented in two groups of the three MDR resistotypes 1 and 2. Te S. Enteritidis strain from the humans and chickens was resistant to two antimicrobials, namely, ampicillin and tetracycline.

Discussion
Te present study investigated Salmonella in chickens and humans in Wau town of South Sudan. A previous study focused on Salmonella from chickens with prevalence of 1.1% [18]. Recently in 2017, Shereen Saad and collegues (unpublished work) investigated the prevalence of Salmonella and antimicrobial resistance in humans and chickens. However, the previous study did not involve molecular confrmation; as such, the study could not identify the serotypes circulating in the study area. Te present study thus utilized molecular techniques to identify the diferent Salmonella is an important cause of morbidity and mortality in human and animals and has thus emerged as a signifcant and growing public health and economic problem worldwide [19]. Te overall prevalence of Salmonella in this study was 7.5% and 3.3% for humans and chicken, respectively. Human isolates showed its higher prevalence compared to chicken isolates. Tis diference may have resulted from the bigger volume of human sample (stool) in comparison to the chicken droopings. Tis was lower when compared to a similar study conducted in Khartoum, Sudan, where the prevalence was 70.1% in chicken handlers and 18.1% in chickens [20]. Te diference in the prevalence was found in the current study and the previous study might be due to a larger sample size (996) that involved pooling of samples in the previous study. However, the higher prevalence of 68.2%, 72%, 25.9%, and 51.2% have been reported in Ethiopia, Tailand, Korea, and Argentina, respectively [21].
Salmonella isolated from chickens and chickens keepers were screened to determine AMR patterns. Tree (3,27.2%) isolates from chicken keepers were resistant to more than one antibiotic. Tis was not in agreement with the fndings obtained by Fadlalla et al., where the resistance of Salmonella   International Journal of Microbiology was observed in 81 human samples (93.1%) [22]. Te present study further indicated that 1 (9.1%) of the human Salmonella isolates was highly resistant (>0.3) to tetracycline and ciprofoxacin while 2 (18.9%) were resistant to ampicillin. However, another study conducted in Sudan reported higher resistance patterns, including resistance to ampicillin 29 (33.3%), nalidixic acid 28 (32.2%), and tetracycline 52 (59.8%) compared to the ones reported in the present study [22]. Furthermore, another study conducted in Sudan also reported higher resistance profles of Salmonella isolates to tetracycline 11.8% and sulfamethoxazole/trimethoprim 88.2% [20].
Te current study has also indicated that 7 (63.6%) isolates were 100% susceptible to sulfamethoxazole/trimethoprim, chloramphenicol, cefotaxime, streptomycin, nalidixic acid, and gentamicin. Tis was in agreement with the results of Fadalall et al.'s study [22] except for some isolates that exhibited moderate resistance (>0.22) to ciprofoxacin 1 (9.1%). Furthermore, the results of the current study are also congruent with a study conducted in Brazil, where 88.2% of Salmonella isolates showed resistance to sulphonamides [23]. Tis resistance of Salmonella isolates to most of the antimicrobials could be due to the presence of resistance genes that are carried on the bacterial plasmids which can be acquired by the consumption of contaminated animal products by humans [24]. Continuous use of antibiotics such as ampicillin and nalidixic acid as treatment remedies may also be the reason for development of resistance [5]. Chloramphenicol combined with ampicillin has been widely used for the treatment of human salmonellosis despite this combination being known for causing aplastic anaemia. For example, it has been a drug of choice in Brazil since 1970s [23].
Salmonellosis in humans has been a health problem in both developed and undeveloped countries with nontyphoidal Salmonella (NTS) caused by other species of Salmonella diferent from S. typhi being a major cause of secondary bacteremia associated with gastritis [19]. In the present study, S. Enteritidis and S. Typhimurium were commonly isolated in chicken keepers while nontypeable isolates of Salmonella were the most dominant serovars. Generally, these serovars showed low resistance to tetracycline and high resistance to ciprofoxacin and ampicillin which are the antibiotics commonly used in South Sudan.

Conclusions
Antimicrobial resistant isolates were isolated in both chickens and humans. Further, MDR isolates were found in both chicken and human samples which is a public health concern. Tis, therefore, calls for concerted eforts to educate consumers on public health, food safety, food hygiene in food production, and enhancement of surveillance programmes on zoonotic bacteria and antimicrobial susceptibility.

Study Site. Te study was conducted in the Western
Bahr El Ghazal State, which shares boundaries with Sudan to the North and Central African Republic to the West with coordinates of 8.6452°N, 25.2838°E, and 626.9 meters above the sea level (Figure 3, study area map). Te climate is tropical with an annual rainfall ranging between 400 and 1600 mm and temperature of 23.8°C-40°C. Of the fve payams of Wau County, two payams, namely, Baggari and Bussuri were randomly selected for the study.

Study Design.
Te study design employed was a crosssectional survey design targeting local live chickens and humans at the household level in Wau County, Western Bahr el Ghazal State, South Sudan. Sample collection was carried out between September and December 2019.

Sample Size and Sampling
Technique. Using simple random sampling, a total of 270 cloacal swabs and 147 fecal samples were collected from local live chickens and humans, respectively. Te samples were collected with sterile wooden swabs from the chicken cloacal, transferred into 10 ml sterile universal containers containing the 5 ml Cary Blair Transport Medium (Himedia), and immediately placed into a cool box containing ice packs. Te samples were then transported and stored in a refrigerator at 4°C till analysis. Before collecting human stool samples, consent was sought from community gatekeepers such as chiefs, opinion leaders, and elders. Additional consent was collected from human participants as a requirement for ideal sample collection. Stool samples (147) were carefully collected into sterile 10 ml plastic universal containers with spoons to which 5 ml Cary Blair Transport Medium has been added. Te samples were then transported and stored in a refrigerator at 4°C until analysis.

Isolations and Identifcations of Salmonella Species from the Fecal Sample.
Te samples were inoculated and incubated at 37°C in preenriched, nonselective bufered peptone water for 24 hours. An aliquot from peptone water (1 ml) was cultured in Rappaport-Vassiliadis broth in the ration of 1 to 10 for each. From the broth, a loopful was innoculated on Xylose Dextrose Agar (XLD, Oxoid, UK), Te temperature and the period of incubation was done at 37°C for 24 hours for both chicken droppings and human stool samples [25,26]. Te suspected colonies of Salmonella from each plate were collected for presumptive identifcation by biochemical tests that included oxidase, hydrogen sulphide (H 2 S), urease, indole, and fermentation of glucose, sucrose, mannitol, and lactose [27]. Furthermore, Salmonella was confrmed using PCR targeting the invA, as previously described [28].

Serological Typing of Salmonella Isolates.
Te Salmonella isolates were characterized into sero groups based on the presence of distinctive "O" antigenic factor, using O polyvalent antiserum and specifc monovalent antiserum for A-S group antigen and GROUP BC1, C2, D1, E, and G, respectively, in accordance to the manufacture's protocol for the identifcation of surface antigens and their diferentiation into serogroups. Briefy, a drop of the appropriate antiserum was placed onto a clean microscopic slide. A single colony of overnight culture on nutrient agar was picked and emulsifed in the antiserum drop to obtain a thoroughly mixed suspension. Te slide was gently rocked forward and backwards/side wards for 1 minute. Agglutination or clumping between 1 and 10 seconds was considered as a positive reaction. 5.6. Serotyping of Salmonella Strains. Salmonella strains were diferentiated into serotypes by serotyping analysis according to the method described by [28,29]. All isolates of Salmonella was referred to Deltamune (Pty) Laboratory, a South African SANAS Accredited Veterinary Laboratory, for confrmation and serotyping. Characterization was done using the method described in the Microbiological Manual, and serotyping (10.2 : 1995 CCFRA) was done based on White-Kaufman-Le Minor Scheme (WHO Collaborating Center) [30]. Te scheme discriminated serotypes on the basis of their somatic (O), fagella (H), and capsular (Vi) antigens present on the surface of Salmonella [31].

DNA Extraction for Salmonella Confrmation.
Te bacteria were cultured on nutrient agar for 24 hrs at 37°C and DNA extraction was performed by the boiling method for 10 min and centrifugation at 5000 ×g for 5 min. Te supernatant was then used for the DNA amplifcation using Salmonella-invA gene specifc primers, namely, S139 (5′GTG AAA TTA TCG CCA CGT TCG GGC AA -3′ and S141 (5′ TCA TCG CAC CGT CAAAGG AAC C -3′), as described previously [32]. Te reaction volume was 25 μl with 1 μl of the DNA template. Te following PCR conditions were used: 94°C for 60 sec of the initial denaturation followed by 30 cycles of 60 sec at 94°C, 30 sec at 56°C, 30 sec at 72°C, 2 min and a fnal extension step of 10 min at 72°C. Te amplifed PCR products were then visualised on 1.5% agarose gel stained with ethidium bromide and visualised by UV illumination alongside a 100 bp DNA ladder.

Detection of Extended Spectrum Beta-Lactamases (ESBLs)
in the Salmonella Strain. Detection of extended spectrum cephalosprinase production isolate was accomplished using freshly prepared MacConkey Agar (HIMEDIA) containing 2 mg/l of cefotaxime (Sigma-Aldrich, Munich, Germany) according to the method described in [28]. Laboratory Standard Institute (CLSI) guidelines [33]. Te antibiotic discs (Oxoid, UK) included ampicillin, sulfamethoxazole/trimethoprim, streptomycin, ciprofoxacin, cefotaxime, tetracycline, gentamicin, nalidixic acid, and chloramphenicol. Using CLSI guidelines that provide ranges for zone of inhibition, the AST on all isolates were read and grouped into Susceptible (S), Intermediate (I), and Resistance (R), and for quality control purpose, E. coli ATCC 25922 was used.

Determination of Multiple Antimicrobial Resistances
Indexing MARI. Te multiple antibiotic resistance index was calculated as follows: a/b, where "a" represents the number of antibiotics to which the particular isolate was resistant and "b" the number of antibiotics to which the isolate was exposed. MARI values >0.2 are considered signifcant indicating that the strains could have originated from sources where antibiotics are often used [34]. While MARI value <0.2 suggests the strains originate from animal sources which are less frequent exposed to antibiotics or never at all [35].

Data Analysis. Data analysis was done using Statistical
Package for Social Sciences (SPSS) version 22.

Data Availability
All data used for the study are available upon request from the corresponding author.

Ethical Approval
Te

Consent
Consent was obtained from all study participants in the study.

Conflicts of Interest
Te authors declare there are no conficts of interest in the study.