Antibiotics Resistance and Molecular Detection of Virulence Genes in Salmonella Spp Isolated from Ready to Eat Fruits Salad in Nasarawa and Benue State in North Central, Nigeria

1. INTRODUCTION

Foodborne illnesses, particularly those caused by Salmonella, present a major public health challenge in Nigeria, where the growing popularity of ready-to-eat fruit salads increases the risk of contamination [1]. This chapter explores the global and regional burden of Salmonella-associated diseases, the emerging role of fruit salads as a vehicle for non-typhoidal Salmonella, and the rationale for studying Salmonella sp in North Central Nigeria. The global burden of Salmonella-associated diseases remains difficult to quantify due to limited epidemiological surveillance, particularly in developing countries, yet salmonellosis—largely driven by diverse non-typhoidal serovars—accounts for millions of infections and significant morbidity and mortality worldwide, with endemic persistence across Africa, Asia, and other regions and notable incidence in developed countries primarily linked to travel-related exposures [2–5].

In North Central Nigeria, high consumption of ready-to-eat foods such as fruit salads in urban markets like Abuja and Jos, combined with poor sanitation, inadequate waste management, limited refrigeration, indiscriminate antibiotic use, and weak epidemiological surveillance systems, has contributed to the persistence and underreporting of non-typhoidal salmonellosis, resulting in increasing morbidity, sporadic outbreaks, and insufficient data for effective public health planning and intervention [5–8]. Fruit salads, although widely consumed for their nutritional value and richness in essential minerals, have been identified as potential vehicles for contamination with non-typhoidal Salmonella, often introduced during cultivation through contaminated irrigation water, as well as during harvesting, handling, and processing under poor hygienic conditions in open-air markets, while these pathogens—classified under Salmonella enterica subspecies enterica and comprising over 2,610 serovars with varying host specificity and pathogenicity—are among the leading causes of foodborne gastroenteritis, presenting symptoms such as diarrhea, fever, abdominal pain, vomiting, nausea, and loss of appetite, and in severe cases progressing to life-threatening systemic infections, particularly in immunocompromised individuals, thereby highlighting the relevance of investigating antibiotic resistance and the molecular detection of virulence genes in Salmonella spp. isolated from ready-to-eat fruit salads in Nasarawa and Benue States, North Central Nigeria [9–11].

2. Materials and Methods

2.1   Study Area

The study was carried out in two selected states within North Central Nigeria, one of the country’s six geopolitical zones. This study area comprised Nasarawa and Benue. Based on projections from the 2006 National Population Census, North Central Nigeria is home to an estimated 29.8 million people National Population Commission, 2006. The area is characterized by a blend of urban centers, peri-urban settlements, and rural communities, with vibrant markets and street vending systems that supply fresh fruits and ready-to-eat (RTE) products to a large population. [12]

2.2  Sample Collection

Total of two hundred (200) fruit salad samples were randomly collected from hawkers on the streets and markets across the two selected states in North Central Nigeria (Nasarawa and Benue), with one hundred (100) samples from each state. The samples were aseptically transferred into sterile containers, appropriately labeled based on their sampling locations (markets and streets), and transported in a sealed ice-packed container to the Department of Microbiology laboratory at Nasarawa State University, Keffi, for immediate analysis.

2.3   Preparation of Media and Inoculation for the Isolation of Salmonella Species

Preparation of Culture media

2.3.1 Nutrient Broth

Thirteen grams of dehydrated powder were dissolved in one litre of distilled water by heating to boiling, followed by sterilization through autoclaving at 121 °C and 15 lb pressure for 15 minutes [13].

2.3.2 Salmonella Shigella Agar: Sixty grams of medium was weighed and dispensed in one liter of deionized water, mixed and heated with frequent agitation and boiled for one minute without autoclaving. It was poured into sterile petri dishes and allowed to solidified [13].

2.3.3 Xylose lysine Deoxycholate: Fifty-five grams of dehydrated medium was suspended in one liter of distilled water and heated with agitation until it boiled, then transferred to a 50 ^oC water bath. It was dispensed into sterile petri dish [13].

2.3.4 Bismuth Sulphite Agar
A total of 52.33 g of the dehydrated medium was dissolved in distilled water and heated to boiling until complete dissolution was achieved. The prepared medium was then aseptically poured into sterile Petri plates and allowed to solidify under appropriate laboratory conditions [13].

2.4 Isolation of Salmonella Species
The isolation of Salmonella spp. was carried out using a modified procedure based on previously reported methods [14]. Approximately 25 g of each fruit salad sample was aseptically homogenized using a sterile blender. The homogenate was transferred into 25 mL of Nutrient Broth (Oxoid, UK) for pre-enrichment and incubated aerobically at 37 °C for 24 hours.

Following incubation, 1 mL of the enriched culture was subjected to serial dilution (10⁻³ and 10⁻⁵). Aliquots of 0.1 mL from each dilution were spread onto Xylose Lysine Deoxycholate (XLD) agar and Salmonella–Shigella Agar (SSA) in duplicate. The inoculated plates were incubated at 37 °C for 24 hours.

Presumptive Salmonella colonies were identified based on characteristic morphology, including red colonies with black centers on XLD agar and colorless colonies with black centers on SSA. Selected colonies were subcultured onto Bismuth Sulphite Agar for purification and incubated at 37 °C for an additional 24 hours. Colonies exhibiting a dark metallic sheen, associated with acid production, were considered indicative of Salmonella species.

2.5 Identification of Salmonella Species
The purified isolates were further identified using standard microbiological methods, including evaluation of cultural and morphological characteristics, along with biochemical tests such as the Indole test, Methyl Red and Voges–Proskauer (MR–VP) tests, and Citrate utilization test.

 

2.5.1 Analytical Profile Index 20E (API 20E) Kit

The API 20E kit was employed for the definitive identification of Salmonella serovars within the family Enterobacteriaceae, whereby a bacterial suspension prepared from a pure colony in sterile saline was inoculated into API 20E strips containing 20 microtubes of biochemical substrates, incubated at 37 °C for 24 hours, and subsequently interpreted based on observed color changes following the addition of appropriate reagents, with the resulting numerical profiles compared against the API 20E database to confirm the identity of Salmonella isolates.

 

2.6 Antibiotic Susceptibility Testing
The antibiotic susceptibility profile of the isolates was determined following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [15]. Briefly, three discrete colonies were selected and inoculated into 5 mL of sterile 0.85% (w/v) sodium chloride solution. The turbidity of the suspension was adjusted to match the 0.5 McFarland standard, prepared by combining 0.5 mL of 1.172% (w/v) barium chloride dihydrate (BaCl₂·2H₂O) with 99.5 mL of 1% (w/v) sulfuric acid (H₂SO₄). A sterile cotton swab was then used to uniformly spread the standardized inoculum onto Mueller–Hinton agar plates. Antibiotic discs were carefully placed on the agar surface under aseptic conditions, followed by a pre-diffusion period of approximately 1 hour. The plates were subsequently incubated at 37 °C for 24 hours. After incubation, the diameters of the zones of inhibition were measured in millimetres and interpreted in accordance with CLSI breakpoint standards.

2.7 Classification of Antimicrobial Resistance
The antimicrobial resistance profiles of the isolates were categorized based on established criteria. Isolates exhibiting resistance to at least one antibiotic in three or more antimicrobial classes were classified as multidrug-resistant (MDR). Those resistant to all but one or two classes were considered extensively drug-resistant (XDR), while isolates showing resistance to all tested antimicrobial agents were defined as pan drug-resistant (PDR).

2.8 Determination of Multiple Antibiotic Resistance (MAR) Index
The Multiple Antibiotic Resistance Index (MARI) for each isolate was calculated using the method originally proposed by Krumperman (1983) and later referenced by Mohammed et al. [16]. The index was determined using the formula:

where a represents the number of antibiotics to which a particular isolate showed resistance, and b denotes the total number of antibiotics tested against that isolate.

An isolate with MARI ≥ 0.2 is regarded to have originated from an environment where antibiotics are frequently used (Krumperman, 1983).

2.9    Molecular Detection of Salmonellasp Virulence Genes

Molecular detection of virulence genes in Salmonella sp is crucial for evaluating the pathogenic potential of isolates obtained from ready-to-eat (RTE) fruit salads. In this study, four virulence-associated genes were targeted: lpfC, linked to fimbrial adhesion and intestinal colonization; spvB, a plasmid-mediated factor involved in systemic infection; sitC, associated with iron uptake and bacterial survival; and spiA, a component of the type III secretion system (T3SS) essential for intracellular survival and host manipulation Haslida et al. [17].

Multiplex polymerase chain reaction (PCR) was employed for simultaneous amplification of these genes, using primer sets previously described by Haslinda et al. [17]. Reaction conditions were optimized to enhance sensitivity and specificity, while safer visualization methods were applied to reduce laboratory hazards. To assess the genetic diversity among isolates, ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus-PCR) and BOX-PCR were performed, both of which demonstrated high discriminatory power in differentiating Salmonella strains. In addition, statistical analyses were used to correlate the prevalence of virulence genes with antimicrobial resistance (AMR) profiles. This integrated approach provided valuable insights into the interplay between virulence and resistance in Salmonella serovars, contributing to risk assessment and public health surveillance of foodborne pathogens in North Central Nigeria.

2.9.1 DNA Extraction
Genomic DNA was isolated using a modified boiling method as described by Bilung et al. [18], selected for its rapidity and simplicity. Pure colonies obtained from MacConkey agar were inoculated into Luria–Bertani broth and incubated for 24 hours. The culture was then centrifuged at 3200 rpm for 2 minutes to pellet the cells. The pellet was washed with sterile normal saline, vortexed, and centrifuged again under the same conditions. Subsequently, the cells were resuspended in 0.5 mL of sterile normal saline and subjected to heat treatment at 90 °C for 10 minutes, followed by rapid cooling at −4 °C for 10 minutes. The lysate was centrifuged at 3200 rpm for 1 minute to remove cellular debris. Approximately 300 µL of the supernatant containing genomic DNA was transferred into sterile 2 mL microcentrifuge tubes and stored at −10 °C until further analysis. DNA concentration and purity were evaluated using a NanoDrop 1000 spectrophotometer by measuring absorbance at 260 nm, and purity was determined based on the A260/A280 ratio, with acceptable values ranging from 1.7 to 1.9.

2.9.2 Amplification of Salmonella Virulence Genes
Amplification of virulence-associated genes was performed according to Bilung et al. [18], with minor adjustments to reagent composition and thermal cycling parameters. For ERIC-PCR, primer pairs ERIC 1R (5′-ATGTAAGCTCCTGGGGATTCAC-3′) and ERIC 2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) were used in a 25 μL reaction mixture containing 1.0 μM of each primer, 2.0 μL DNA template, 1× PCR buffer, 0.2 mM dNTPs, 2.5 mM MgCl₂, and 2.0 U of Taq DNA polymerase. For BOX-PCR, primer BOX A1R (5′-CTACGGCAAGGCGACGCTGACG-3′) was employed in a similar reaction volume comprising 0.4 μM primer, 2.0 μL DNA template, 1× PCR buffer, 0.2 mM dNTPs, 2.0 mM MgCl₂, and 2.0 U Taq polymerase. Amplification was carried out in a thermal cycler under conditions outlined in Table 1. The PCR products were resolved on 1% agarose gel using a 1 kb DNA ladder and visualized with a luminescent image analyzer (LAS-4000, FUJIFILM, Japan).

2.9.3 Agarose Gel Electrophoresis
Amplified products were analyzed by agarose gel electrophoresis following the method of Bilung et al. [18]. Briefly, 8 μL of PCR product mixed with ethidium bromide was loaded into wells of a 1.0% (w/v) agarose gel alongside a molecular weight marker. Electrophoresis was conducted at 120 V for approximately 30 minutes. DNA bands were visualized under ultraviolet illumination and documented. Banding profiles were further analyzed using PyElph 1.4 software, and phylogenetic relationships were inferred by constructing dendrograms based on the unweighted pair group method with arithmetic mean (UPGMA).

2.9.4 DNA Quantification
The concentration of extracted DNA was determined using a NanoDrop 1000 spectrophotometer. The instrument was initially calibrated with 2 μL of sterile distilled water and blanked using normal saline. Thereafter, 2 μL of each DNA sample was applied to the lower pedestal, and measurements were recorded using the integrated software after lowering the upper pedestal to ensure proper contact.

2.9.5 16S rRNA Gene Amplification
The 16S rRNA gene was amplified using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′). PCR reactions were carried out in a total volume of 25 μL using a 2× DreamTaq Master Mix (Inqaba, South Africa), with primers at a final concentration of 0.4 μM and genomic DNA as the template. Amplification was performed in an ABI 9700 thermal cycler under the following conditions: initial denaturation at 95 °C for 5 minutes; 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 52 °C for 30 seconds, and extension at 72 °C for 30 seconds; followed by a final extension at 72 °C for 5 minutes.

2.9.6 DNA Sequencing of Bacterial Isolates
Sequencing of amplified DNA fragments was conducted using the BigDye Terminator v1.1/v3.1 Cycle Sequencing Kit on an ABI 3510 Genetic Analyzer (Applied Biosystems) at Inqaba Biotechnological, Pretoria, South Africa. The sequencing reaction was prepared in a total volume of 10 μL, containing 0.25 μL BigDye Terminator reagent, 2.25 μL of 5× sequencing buffer, 10 μM primer, and 2–10 ng of PCR product per 100 bp. Thermal cycling conditions included an initial denaturation at 96 °C for 10 seconds, followed by 32 cycles of denaturation at 96 °C for 10 seconds, annealing at 55 °C for 5 seconds, and extension at 60 °C for 4 minutes.

2.9.7 Phylogenetic Analysis
The obtained nucleotide sequences were initially edited and trimmed using the TraceEdit bioinformatics software to ensure sequence quality. Homologous sequences were subsequently identified through BLASTN searches against the National Center for Biotechnology Information (NCBI) database. Multiple sequence alignment was performed using ClustalX to establish sequence homology. Phylogenetic relationships among the isolates were inferred using the Neighbor-Joining method implemented in MEGA version 6.0 [18]. The robustness of the phylogenetic tree was evaluated through bootstrap analysis based on 500 replicates, following the approach described by Jonit et al. (2016). Evolutionary distances were computed using the Jukes–Cantor substitution model.

2.10 Statistical Analysis
All statistical analyses were conducted using SPSS software (version 21.0). The chi-square (χ²) test was applied to assess differences between variables. Statistical significance was determined at a probability level of P < 0.05, with results interpreted within a 95% confidence interval.

3. RESULTS

3.1 Morphological Identification of Salmonella species Isolated from Ready to Eat Fruit Salads

The morphological characteristics of Salmonella species isolates from selected ready to eat fruit salads sold in some selected states in north central Nigeria namely; sliced mixed fruits, such as watermelon, pineapple, oranges, apple and pawpaw growth with colourless colonies on Xylose-lysine deoxycholate (XLD) agar and Salmonella-Shigella agar (SSA)  with back spot and black metallic sheen on Bismute sulphite (BSA) were Salmonella species grow in unequal shape, not smooth in surface, moderate in size, convex in edge and translucent. The morphological characteristics of Salmonella species are Gram-negative rod-shaped as shown in Table 1.

3.1.2    Occurrence of Salmonella species from ready to eat fruit salad

Out of two hundred (200) samples of sliced mixed fruit salads obtained, 12 (6.0%) of Salmonella specieswere isolated as shown in Table 2, the order of percentage occurrence of Salmonella species from ready to eat mixed fruit salads from different locations were the total percentage occurrence was  6.0%. The occurrence from Nasarawa was 7.0% and Benue was 5.0% respectively.

The antibiotics resistance pattern of Salmonella species isolated fromready to eat fruit salads sold in some states in north central, Nigeria is as shown in Table 3. The order of percentage resistance of Salmonella species isolated from ready to eat fruit salads sold in Nasarawa state agnist antibiotics tested were, cefexime (42.8%), cefuroxime, nalidixic acid, cefotaxime and amoxicillin/clavulanic acid (28.5 %) and ampicillin (14.2 %). From Benue the order of percentage resistance of Salmonella species to the antibiotics tested were nalidixic acid and cefexime (60.0 %), Cefuroxime, ampicillin, cefotaxime, amoxicillin/clavulanic acid (40.0 %) and nitrofurantoin (20.0 %).

3.1.5    Multiple antibiotics resistance (MAR) index

The MAR index of Salmonella species isolated from ready to eat fruits sold in selected states in north central, Nigeria is as shown in Table 4. The Salmonella species isolated with MAR index of greater than or less than 0.2 are MAR isolates, and the commonest MAR in Salmonella speciers isolated from Nasarawa was 0.3 with percentage of occurrence of 42.8 %. The commonest MAR in Salmonella species isolated from Benue was 0.4 and 0.5 with percentage of occurrence of 40.0 %.

3.1.6    Genotypic detection of virulence genes

The result of detection of virulence genesin Salmonella speciesisolated from ready to eat fruit salads sold in some north central states in Nigeria is as shown in Table 6. Out of fifteen isolates evaluated for virulence genes, 33.3 % of the isolates harbour virulence genes. The order of virulence genes detected was, spiA and spvB genes (33.3 %) from Salmonella speciesisolated from Nasarawa, spvB genes (66.6 %). From Salmonella speciesisolated from Plateau harbors spvB (33.3 %). From Salmonella speciesisolated from Kogi harbors spvB (33.3 %). The percentage of spiA detected was 20.0 % and spvB was 26.6 %. Plates 1 and 2 show the molecular bands and weight of the virulence genes detected from Salmonella species isolated.

Plate 1. Agarose gel electrophoresis of the amplicons of virulence genes expression in the Salmonella serovarsisolates. Lane M represents the 1000 bp DNA molecular ladder. Lane 1represent the expression of the spvB (717bp)

Plate 2. Agarose gel electrophoresis of the Amplified 16SrRNA bands of Salmonella serovar isolates gene region, represents 250bp of Salmonella serovars isolates gene region. Lane M represents the 1000 bp DNA molecular ladder. Lane 1 and 2  representing Salmonella enterica subsp. enterica serovar Typhimurium,

4. DISCUSSION

The consumption of fruit salads over the decades has increased because they are important source of vitamins, nutrients and fiber [19], but food born disease outbreaks have been linked to pathogen contamination of sliced fruit or mixed fruit salads. From this study the percentage of Salmonella serovars isolated were high 6.0 % but lower than 12.1 % that was reported by Eni et al. [20] in Sango Ota, Nigeria. Also, it was observed that Salmonella sp were isolated from Nasarawa state with percentage of 7.0 % which was the highest in the two states in this study area. Ready-to-eat fruit salads are susceptible to contamination from multiple sources, including cutting utensils, improper handling during processing, contaminated surfaces such as tables and trays, rinsing water, washing containers, and packaging materials, particularly as fruits are peeled, sliced, washed, and packaged in polyethylene bags prior to sale to consumers (Eni et al. [20]). The low occurrence of Salmonella sp in some part of the study area was not supervised due to nature of processing the ready to eat mixed fruits, the water used in washing and personal hygiene of the people that prepare the mixed fruit salads these may lead to less contamination of the mixed fruit salad with Salmonella sp the contamination could have occurred as faecal contamination, and environmental factors such as contaminated air [21]. The occurrence of Salmonella sp as observed in this study was in agreement with a study earlier reported by Haslinda et al. [17] the isolation of Non-typhoidal Salmonella (NTS) isolated from fruit Salads in Terengganu, Malaysia.

The occurrence of Salmonella sp as observed from different locations of this study showed that Salmonella sp is a threat to public health because it has been reported by Ngoi et al. [22] that some species of Salmonella sp are major cause of typhoid fever and some are known to cause diarrheal that can be life threatening in children and in immune compromised individuals as reported by Ngoi et al. [22]. Isolation of Salmonella sp from stool of diarrheal patients in Malaysia; Haslinda et al. [17] reported the isolation of non-typhoidal Salmonella (NTS) from fruit vegetables Orji et al. [21] reported isolation of Salmonella spp. and Salmonella Enteritidis from healthy and none healthy individuals in Ebonyi State, Nigeria.

The low antibiotic resistance of Salmonella sp s to ciprofloxacin, nitrofurantoin, ampicillin and gentamicin as observed in this study was not expected because of the high resistance of Salmonella spp that have been reported by different authors namely Adeoti et al. [1] reported high resistance to bacteria isolated from ready-to-eat vended fruits in Sango open-market, Saki, Oyo State, Nigeria. The findings of the antibiotic susceptibility justified the need to use of broad-spectrum antibiotics from treatment of Gram-negative bacteria infection such as Salmonella sp. The low antibiotic resistance may be due to the high cost of these antibiotics and the form like the gentamicin which is in an injectable form, and the discomfort of gentamicin injection when administered, it is likely that these antibiotics may not have been abused [23]. The amazing observation here is that the Salmonella sp were highly resistance to streptomycin and sulphamethoxazole/trimethoprim and this is in agreement to a study earlier reported by Mohammed et al. [16]. The low resistance of Salmonella serovars to ciprofloxacin and nitrofurantoin justifies the use of both antibiotics as drug of choice of treatment of infection cause by Salmonella sp.

The detection of virulence genes spiA was lower than spvB genes in Salmonella sp isolates and this seems to disagree with other studies that show detection of more virulence genes such as invA and sipB as reported by Haslinda et al. [17]. The low detection of this virulence genes mentioned above as recorded in this study may be responsible for the low or high susceptibility observed in this study, as the enzymes that inactivate the drugs were not available, although the mechanism of resistance of the antibiotics to Salmonella serovars were not evaluated or studied in this study. The absence of most of the virulence genes may be why people that consume contaminated ready-to-eat fruit salads in the study area hardly come down with infection normally caused by virulence Salmonella species.

The pathogenicity of Salmonella is largely governed by genetic determinants that enable invasion, survival, and replication within host cells, and although most isolates in this study exhibited limited virulence profiles, at least two virulence genes were detected—with one isolate harboring multiple genes—highlighting their pathogenic potential, particularly through genes such as sipB and spvB, located within Salmonella pathogenicity islands and associated with the outer membrane, which function synergistically to enhance intracellular survival and expression of virulence (Eni et al. [20]; [17], [24]. Molecular characterization of the Salmonella isolates in this study revealed pairwise allelic differences among strains originating from diverse sources, including humans, animals, and the environment, with the isolates showing close relatedness to Salmonella enterica subsp. enterica serovar Typhimurium; however, sequence homology was generally low, with only one isolate exhibiting less than 95% similarity, and none showing 100% identity with sequences in the NCBI database, suggesting possible genetic variation or novel sequence types, findings that are consistent with Reeves et al. [25], who reported genomic diversity among Salmonella subspecies, and Yasin et al. [26], who demonstrated variable similarity between 16S rRNA gene sequences from ready-to-eat produce and clinical isolates, while phylogenetic analysis in the present study produced multiple clusters, with one cluster containing all representative isolates and another comprising reference sequences from NCBI, indicating a shared common ancestor among the study isolates.

Conclusion

The isolation rate of Salmonella sp from mixed fruit salads was higher in Nasarawa state than Benue stat states. The isolation rate shows that most of these mixed fruit salads may have public health implications. Also, the Salmonella sp isolated from some selected markets and cities in some North central states in Nigeria were susceptible to ciprofloxacin, nitrofurantoin, ampicillin and gentamicin and this however. The virulence genes detected include spiA and spvB. The Salmonella serovars isolated were highly related to Salmonella enterica subsp. enterica serovar Typhimurium

Compliance with ethical standards

Acknowledgments

With immensely thank all the staff in the Department of Microbiology Laboratory Nasarawa State University, Keffi for the help during the bench work and analysis.

Disclosure of conflict of interest

No conflict of interest among of the authors

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