Whole Genome and Phenotypic Characterization of Novel QnrB19-positive Salmonella Nigeria serovars from Food Animals in Ilorin, North-central, Nigeria.

Background: Non-typhoidal Salmonella are major foodborne pathogens, posing serious challenges to public health and food safety worldwide. Salmonellosis in humans is commonly associated with the consumption of contaminated food, water, and direct contact with infected animals. This study aimed to characterize the distribution, diversity, virulence genotypes and antibiotic resistance of Salmonella enterica subsp. enterica serovar Nigeria, isolated from farm animals in north central Nigeria. Results: We recovered 9 different S. enterica ser. Nigeria isolates from our sampling, eight from pig and one from chicken. The antimicrobial susceptibility testing against 15 antimicrobial agents showed variable resistance proles. Whole genome sequence (WGS) analysis revealed that all 9 isolates contained a single mutation parC (T57S) substitution in addition to qnrB19, expected to confer decreased susceptibility to ciprooxacin and tet(A) expected to confer resistance to tetracycline. Furthermore, two plasmid targets were also detected in all the strains, Col(pHAD28) and IncQ1. MLST analysis showed that all 9 isolates exhibited only one sequence type, (ST-4911) irrespective of the source of isolation. A SNP-based phylogeny indicates that the 9 isolates are highly related and lack other close relatives in the pathogen detection database. Forty core (housekeeping) and accessory virulence genes were identied from different virulence loci including Salmonella Pathogenicity Islands, virulence associated plasmids (pSV), chromosomes and mbriae. Conclusion: This study provided valuable information on the resistance determinants, virulence genes, phenotypic resistance proles, plasmids and multilocus sequence typing (MLST) of Salmonella Nigeria from food animals by WGS. Highlighting the signicance of poultry and pig to the spread and emergence of Salmonella Nigeria in this region of Nigeria, therefore, there is the need for consumer's education and enlightenments on the importance of proper handling and preparation of food, this will reduce the potential risk of transmission of this pathogen. potential of the pathogen, occasionally plasmids are found in Salmonella serotypes associated with infections of humans and animals, including the Salmonella virulence plasmid. To provide a deeper understanding of S. Nigeria in this region we report the genome sequence of S. Nigeria, obtained using MiSeq Illumina instrument with the 500-cycle MiSeq reagent V2 kit (2 × 250 bp). This study aimed to provide baseline information on the distribution, molecular characterization, virulence the present study. In tandem with our study, Campioni et al. [43] detected invA, sipA, sopB, ssaR, and sifA, genes in all of the strains (100%). Similar to our work, Suez et al., [36] and [37], detected cdtB genes in their studies in Israel and US respectively. In our study, sseJ and cdtB, genes were identied this is similar to the work of Pornsukarom et al. [37] and Suez et al. [36]. The cdtB toxin (typhoid toxin) may contribute to the pathogenicity in human and animal. Multilocus sequence types result revealed that a single sequence type, ST-4911 was assigned to all the isolates investigated. characterization for further understanding of the mechanism of Salmonella serovar Nigeria in induction of pathogenesis of infection in a susceptible host.


Background
Non-typhoidal Salmonella is among the most important foodborne pathogens and it continues to pose a serious challenge to public health and food safety globally [1]. Salmonellosis in human are commonly associated with the consumption of contaminated foods, water, and direct contact with infected animals have also been implicated [2]. Gastroenteritis due to non-typhoidal Salmonella is usually a self-limiting illness and is characterized by diarrhea, fever, vomiting and abdominal cramps. Usually, children, immuno-compromised and elderly individuals are more likely to develop severe disease with a higher risk of secondary complications.
Over 2,600 Salmonella serovars have been reported, but only a few are incriminated in most cases of human salmonellosis worldwide these includes Salmonella serovars S. Enteritidis, S. Typhimurium, S. Infantis and S. Heidelberg [3], these serovars are commonly reported in poultry and swine farms. Eggs and poultry products have been incriminated as the main vehicles for the transmission of human salmonellosis that is responsible for the majority of foodborne outbreaks [4]. Because Salmonella enterica is widely distributed in the environment and global food chain production, in addition to having a large public health impact it also has a huge economic implication estimated at $11·6 billion [5].
Worldwide, Salmonella is a major cause of hospitalizations and mortality among those attributed to foodborne diseases [6] causing an estimated 93.8 million cases and 155,000 deaths per year [2]. Moreover, in Sub-Saharan Africa, invasive nontyphoidal Salmonella (iNTS) has emerged as a major cause of bloodstream infection in adults and children, with an estimated annual incidence of 175-388 cases per 100,000 children and 2000-7500 cases per 100,000 HIV-infected adults [7]. Over the last decade, the emergence of some serotypes in poultry production has been observed. Salmonella enterica serotype Nigeria has been associated with poultry and pigs [8,9], but recently it has been more frequently observed in pig farms. It was also recently encountered in a human [10] in the south west of Nigeria.
Antimicrobial resistance (AMR) is an increasing problem worldwide, and Salmonella spp. resistance to quinolone was classi ed by World Health Organization (WHO) in the high priority list. AMR in foodborne pathogens is a signi cant threat to public health, this is particularly true with nontyphoidal Salmonella, acclaimed to be the most common bacterial foodborne pathogen in the United States [11]. The Centers for Disease Control and Prevention (CDC) regard Salmonella resistance to uoroquinolones to be a serious threat to public health [12], and the Food and Drug Administration (FDA) identi ed uoroquinolones as critically important drugs for human health [13]. Therefore, ndings of decreased susceptibility to uoroquinolones or identi cation of new genetic determinants conferring uoroquinolone resistance in Salmonella is a public health concern. The emergence of antimicrobial resistance in microorganisms naturally occurs; nevertheless, the increase in the utilization of antimicrobials promotes the natural selection of resistant bacteria [14]. Generally, bacterial virulence factors have a crucial role for systemic infections, the pathogenicity of Salmonella serotypes is dependent upon the virulence potential of the microorganism and the host susceptibility to the pathogen. Bacterial virulence factors are necessary for adhesion, invasion and replication inside host cells. Genes such as invA and hilA, found in SPI, allow Salmonella to invade epithelial cells [15,16]. Besides, Salmonella outer proteins (sops) (SPI effector protein) encoded by sop gene have relevance to Salmonella virulence [17]. Meanwhile, the plasmid encoded mbriae (pefA) gene contributes to the adhesion of Salmonella to epithelial cells. Other chromosomal gene like stn, that code for enterotoxin production has been shown to be a causative agent of diarrhea. In addition, virulence plasmids carrying virulence genes such as the spv operon (Salmonella plasmid virulence) contribute to the colonization of deeper tissues among other functions. These characteristics are encoded by genes present on a wide range of genetic elements, including the bacterial chromosome, plasmids, prophages and Salmonella Pathogenicity Islands (SPIs). SPI-2, which encodes T3SS-2 that facilitates intracellular survival and replication [18]. Other SPIs are serotype speci c and increase the virulence potential of the pathogen, occasionally plasmids are found in Salmonella serotypes associated with infections of humans and animals, including the Salmonella virulence plasmid.
To provide a deeper understanding of S. Nigeria in this region we report the genome sequence of S. Nigeria, obtained using MiSeq Illumina instrument with the 500-cycle MiSeq reagent V2 kit (2 × 250 bp). This study aimed to provide baseline information on the distribution, molecular characterization, virulence genotypes, SNP-based phylogenetics, genotypic and phenotypic antibiotic resistance pro les of Salmonella serovar Nigeria isolated from food and food animals.

Study area
Samples were collected from 14 farms (pig, n = 9; poultry, n = 5) located at Egbejila, Eyenkorin, and Lasoju communities located near Ilorin metropolis, Kwara State, Nigeria The geographic position of the farms lies on latitude as shown in Fig. 1. The State is located at an elevation of 305 meters above sea level with population of 2,591,555. Its coordinates are latitudes (8° 30′N) and longitudes (5° 00′E). The state shares a common internal boundary with Niger state in the North, Kogi state in the East, Oyo, Ekiti and Osun states in the South and an international boundary with the Republic of Benin in the West. The state has an annual rainfall range of 1,000 mm to 1,500 mm. The rainy season begins at the end of March and lasts until early September, while the dry season begins in early October and ends in early March. Temperature is uniformly high and ranges between 25 °C and 30 °C in the wet season throughout the season except in July -August when the clouding of the sky prevents direct insolation while in the dry season it ranges between 33 °C to 34 °C.

Isolation and Identi cation of Salmonella
The sample in buffered peptone water (10 gram of sample to 90 milliliter of broth) was incubated at 37 ºC for 24 hours, thereafter, one milliliter was inoculated Sequencing was carried out by using the MiSeq Illumina instrument with the 500-cycle MiSeq reagent V2 kit (2 × 250 bp) in accordance with the manufacturer's guidelines. The fastq les were uploaded to NCBI's SRA database for inclusion in the GenomeTrakr's [20] open surveillance of foodborne pathogens. The GenBank accession number and other genomic statistic of the study are as shown in Table 1. Whole genome sequence analysis Raw data were downloaded locally, assembled by using SPAdes v3.8 [21], and annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [22]. In silico serotyping was performed with SeqSero v.

Results
We recovered nine Salmonella Nigeria isolates, eight from pig fecal samples and one from chicken liver ( Table 2). The genotypic resistance pro les extracted from the WGS analysis showed all the nine Salmonella Nigeria isolates possessing three similar resistance determinants ( Table 2). A previously described parC (T57S) substitution was identi ed in all the isolates. Some parC mutations are associated with reduced susceptibility to uoroquinolone antibiotics such as cipro oxacin, however the parC T57S mutation is not known to confer high-level resistance alone [24,25]. In addition to the parC mutation a plasmid mediated quinolone resistance (PMQR) gene -qnrB19, and a tetracycline resistance gene (tet(A) were identi ed. Two plasmid replicon targets, Col(pHAD28) and IncQ1, were also detected in all isolates ( Table 2).

Discussion
To the best of our knowledge, this is the rst report of whole genomic or detailed characterization of Salmonella Nigeria serovars from food animals. In this study, we reported Salmonella Nigeria serovars with a prevalence rate of 0.7%, from poultry and pig farms, this is lower than a previous reports on Salmonella in pig and poultry [26] with 2.2% in Shaoyang, China and Fashae and Hendriksen (2013) with 2.6% prevalence rate in Ibadan, Nigeria. The study highlights the prevalence of this emerging serotype on farms in the South-west and North-central region of Nigeria.
Detection of Salmonella Nigeria in this study indicates Salmonella contamination of poultry and pig farms investigated, this is of veterinary and public health signi cance because this predisposes the community to public health challenges. Contamination of poultry and pig farms with Salmonella may be attributed to low level implementation of hygiene and biosecurity measures as previously reported by Fagbamila et al. [27]. The isolation of the same serovars from pigs and chickens corroborated the important roles played by environment in the transmission of Salmonella because some of the pig farms are extensively managed, pigs roam about to scavenge in poultry farm/environment refuse dumps, furthermore rodents and wildlife animals can move from one farm to the other in search of food resulting in cross contamination as earlier reported [28,29].
This study is similar and of signi cance when compared to the earlier work by Fashae et al. [9], with report of 3.5% prevalence of Salmonella Nigeria serovars and Fagbamila et al. [8] with a prevalence of 0.3% from pooled pig feces from selected pig farms in Ibadan, southwest Nigeria, and commercial poultry farms respectively. Interestingly, Fashae et al. [10] reported the same serovar from diarrhoeic human in Ibadan, South-west, Nigeria. These reports highlight the ubiquitous nature of Salmonella in food animals, and the possibility of cross transmission to humans in these regions.
The resistance observed might be related to the indiscriminate application of antimicrobials for therapeutic or prophylactic purposes (tetracycline), it could also be due to their inclusion in feed as a growth promoter or additives especially in poultry and animal husbandry [30], this is a common practice in developing country like Nigeria, this nding is highly disturbing from a public health perspective as many of these traditional (old generation) antibiotics are still widely prescribed in human medicine due to their low cost and widespread availability [31] while the observation of resistance to some of the newer generations of antibiotics are a cause for concerns.
Resistance to sulfonamides (sulfa/trimethoprim), streptomycin, and tetracycline corroborated similar results by Fashae and Hendriksen [9] on Salmonella Nigeria serovars in Ibadan, Nigeria, these antimicrobials are widely used in the treatment and prevention of bacteria diseases by farmers without prescription by veterinarian, hence, the observed in some of the isolates. Other pertinent observations on the outcome of this study is the absence of resistance to chloramphenicol and neomycin, this may be due to the o cial ban on the use of chloramphenicol in animal, this invariably in uenced the use of a "triple" antibiotic formulation/combination (neomycin, chloramphenicol and oxytetracycline) by the farmers, this eventually may be the reason for the drastic reduction in resistance to chloramphenicol and neomycin. It is noteworthy to observe that none of the isolates showed multidrug resistant (MDR) pro les.
In this study the plasmid mediated quinolone resistance gene qnrB19, associated with decreased susceptibility to uoroquinolones, was detected in all the strains. In recent times there have been several reports of foodborne Salmonella enterica harboring quinolone resistance genes in Nigeria [9,32]. These reports showed that public health risk of plasmid-borne resistant foodborne pathogens has emerged globally, this equally con rmed that PMQR genes were located in conjugative plasmids, suggesting that PMQR genes in foodborne isolates may play a role in the spread of uoroquinolone resistance through the food chain. Therefore, continuous monitoring is necessary for all resistance determinants in relation to the severity of the risk in foods.
Similarly, we identi ed a single mutation in parC (T57S), similar to report by Kim et al. [33]. This parC(T57S) mutation usually cannot independently confer quinolone resistance, this is corroborated by previous study [33,34]. Interestingly, we detected two plasmid targets, IncQ1, a plasmid associated with resistance to tetracycline encoded by tetA, and Col(pHAD28), which has been observed to harbor quinolone resistance genes. IncQ1 plasmid is a group of nonconjugative but mobile plasmids that are stably maintained and found in a wide range of bacteria. They have been incriminated in the spread of antimicrobial resistance genes and emergence of multidrug resistant bacteria. They have been involved not only in conferring resistance to tetracyclines but to other antimicrobials; sulphametoxazole, streptomycin and tetracycline (pNUC) while the IncQ1 plasmid found in the Escherchia coli strain harbored an additional dfrA14 gene that confers resistance to trimethoprim inserted within the strA gene [35].
Generally, our results demonstrated that the PMQR gene qnrB19 is common in Salmonella enterica Nigeria isolated from food animals in Nigeria. The occurrence of these antimicrobial resistance elements in Salmonella Nigeria is of public health and food safety concern, and it indicates the need for increased surveillance for the presence of these plasmids in Salmonella strains and to assess their actual impact in the rise and spread of quinolone resistance.
Genes encoded by SPI-1 region are essential for the invasion of the intestinal epithelium, these genes includes, avrA, invA, sipA, sipB, sipC, sopA, orgA, prgH and sptP, all are involved in host cell invasion and enteropathy, they are also responsible for the ability of the pathogen to invade the intestinal epithelial cells, all of these are harbored by Salmonella enterica serovar Nigeria, only two genes, spaN, and hilA were not identi ed in all the isolates. SPI-2 encoded genes detected includes ssaB/spiC, which assists in the survival of the pathogen within the Salmonella-containing vacuoles, others includes ssaR, sseB, sseC, sseF, and sseG genes which are responsible for the intracellular survival and replication of Salmonella in the host cell, thereby enhancing the pathogenicity of Salmonella, the ttrC and spiA genes are not detected in all the isolates. The three genes investigated from SPI-3 region are misL, which enhances the long term persistence of the pathogen in host, marT, mgtB, and mgtC both enhances the survival of the pathogen within the macrophages, and also responsible for Magnesium transport system, this ultimately favors the intracellular survival of the pathogen in the host, only marT gene was not identi ed among the isolates. Encoded in SPI-5 are pipB, sopB, and csgD genes, these genes are utilized by the pathogen for intestinal epithelial invasion and colonization leading to enteric salmonellosis. sopB was identi ed, this prevents apoptosis of intestinal epithelial cells, and also play an important role in the induction of uid secretion by enterocytes, as well as in polymorphonuclear leucocytes induction in the intestine. In addition, sopB also presents inositol phosphate phosphatase activity, which is directly related to the induction of diarrhea, while csgD plays an important role in bio lm formation leading to enhanced capability of the pathogen to respond to starvation, pipA was not detected in all the isolates. From SPI-11 cdtB was the only gene identi ed in all the isolates corroborating the work of Suez et al., [36] and Pornsukarom et al. [37]. The gene cdtB is responsible for the release of the cytolethal-distending toxin, thereby assisting intra-macrophage survival of the pathogen. The gene equally causes DNA destruction in intoxicated cells, this induces cell cycle arrest, chromatin fragmentation, cell distention and nucleus enlargement. The cdtB toxin may contribute to the pathogenicity in human and animal. The presence of cdtB has been reported to be associated with higher rates of invasive disease [38], pagN genes was not detected in all the isolates under study. All of the pSV associated genes, rck, spvB, spvC, and pefA were not harbored by all the isolates investigated (Table 4). Some effector proteins are encoded on the outside the SPIs and translocated to the host cell by T3SS, these protein/factors are present in other parts of the chromosomes, these includes sifA, sifB, sopE2, and sseJ, all of these were identi ed in this study. SifA effector proteins function to alter host cell physiology and promote bacterial survival in host tissues. This protein is required for endosomal tubulation and formation of Salmonella-induced laments (Sifs), Sif formation is associated with intracellular bacterial replication, sopE2 this is involved in cytoskeleton rearrangements and stimulates membrane ru ing thereby promoting bacterial entry into non-phagocytic cells. SseJ effector proteins function to alter host cell physiology and promote bacterial survival in host tissues. This protein is required for endosomal tubulation and negatively regulates the formation of Salmonella-induced laments (Sifs) in epithelial cells. SteA is a mbriae associated genes, was detected in all the isolates it function as an effector proteins which function to alter host cell physiology and promote bacterial survival in host tissues.
The outcome of this study with regards to virulence genes is in agreement with studies of [39] that reported 100% detection of invA genes in Egypt and Rahman [40] reported 100% detection of sopB in India, but a lower rate of 41.18% was reported by (Ammar et al. [39] also in India. Han et al. [41] reported 100% of the isolates to be positive for sopB, sopE, and invA genes, similar to our study, but in contrast to our work he detected 98% of hilA, prgH, avrA, and reported spvC (78.6%), pefA (57.1%), which were absent in our study. However, in Italy, Capuano et al. [42] reported rates of lower sopE (85.7%) as compared to results of the present study. In tandem with our study, Campioni et al. [43] detected invA, sipA, sopB, ssaR, and sifA, genes in all of the strains (100%). Similar to our work, Suez et al., [36] and [37], detected cdtB genes in their studies in Israel and US respectively. In our study, sseJ and cdtB, genes were identi ed this is similar to the work of Pornsukarom et al. [37] and Suez et al. [36]. The cdtB toxin (typhoid toxin) may contribute to the pathogenicity in human and animal.
Multilocus sequence types result revealed that a single sequence type, ST-4911 was assigned to all the isolates investigated.

Conclusion
In conclusion, the data presented in this study provided valuable information on the antimicrobial and virulence genes content, the multi-locus sequence types (MLST's), and phylogenetic relationship of a newly emerging Salmonella Nigeria serovar from food animals by WGS. This study showed that poultry and pig farms contributed to the spread and emergence of nontyphoidal Salmonella Nigeria serovar in Ilorin, North-central Nigeria. We also characterized some important antimicrobial resistance genes by WGS. The absence of multi-drug resistance to majority of the commonly available antimicrobial agents that are commonly used for clinical chemotherapy in human and veterinary practices is a positive development and requires continuous monitoring and public enlightenment to sustain this status to protect public health safety since the strain was initially reported in pig, and it is now reported in poultry and human. Furthermore, identi cation of variable virulence genes that has the capacity to elicit high pathogenicity on the host is a cause for public health concern.
Therefore, systematic surveillance programs of antimicrobials, and legislation on the imported food of animal origin should be considered by the policy makers in Nigeria to curtail the spread of antimicrobial resistance infection to human. Furthermore, the isolation of Salmonella from poultry and pig meant for human consumption highlights the need for consumer's education and enlightenments on the importance of proper handling and preparation of food in order to reduce the potential risk of transmission of this pathogen. Our ndings provide useful baseline information that will bene t future researchers to use this genomic characterization for further understanding of the mechanism of Salmonella serovar Nigeria in induction of pathogenesis of infection in a susceptible host.