*1Attah, C. J., 2Egah, D. Z., and 3Lar, P. M.
1Infectious Diseases Unit, Department of Paediatrics, Federal Medical Centre, Keffi, Nasarawa State, Nigeria
2Department of Medical Microbiology, Jos University Teaching Hospital, Jos, Plateau State, Nigeria
3Department of Microbiology, University of Jos, Jos, Plateau State, Nigeria
*Correspondence to: marysolspecialist2@gmail.com; attah.caleb@yahoo.com; +234 8069231690;
ORCID: 0000-0002-3261-1127
Abstract:
Background: Accurate laboratory identification of Salmonella species is essential for effective diagnosis, anti- microbial stewardship, and surveillance of enteric fever. Conventional culture and biochemical methods remain the primary diagnostic approach in many laboratories in resource-limited countries but are prone to misidentification because several members of the family Enterobacteriaceae exhibit similar phenotypic characteristics. This study evaluated the role of 16S rRNA gene sequencing in validating phenotypic identification of Salmonella isolates obtained from patients with suspected enteric fever in Nigeria.
Methodology: Blood samples collected from a total of 425 patients with suspected enteric fever were cultured using standard bacteriological procedures. Presumptive Salmonella isolates were identified by conventional bio- chemical tests, including triple sugar iron (TSI) agar reactions, urease, citrate, motility, and indole tests. Genomic DNA was extracted from all presumptive isolates, and bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) and sequenced by Sanger method. Sequence identities were determined using the National Center for Biotechnology Information (NCBI) BLAST database, while phylogenetic relationships were inferred using the
Maximum Likelihood method with bootstrap analysis.
Results: Conventional phenotypic methods identified 32 (7.5%) isolates as Salmonella from the patients. However, sequencing confirmed only 25 (78.1% of presumptive isolates and 5.9% of blood cultures as Salmonella, while 7 (21.9%) were reclassified as non-Salmonella Enterobacteriaceae, including Enterobacter oligotrophi- cus (n=3), Enterobacter hormaechei (n=1), Citrobacter freundii (n=1), Citrobacter youngae (n=1), and Klebsiella oxytoca (n=1). Using sequencing as the reference standard, phenotypic identification demonstrated 100% sensiti- vity (95% CI: 84.6–100.0), 81.6% specificity (95% CI: 66.6–91.6), positive predictive value of 78.1% (95% CI: 60.0–90.7), and negative predictive value of 100% (95% CI: 88.8–100.0), with substantial agreement (Cohen’s κ =0.78, p<0.001). Among the confirmed isolates, S. Typhimurium (n=11) was predominant, followed by untyped S. enterica (n=6), and single isolates of S. Enteritidis, S. Heidelberg, S. Infantis, S. Typhi, S. Livingstone, S. Paratyphi B, S. diarizonae, and S. bongori. Phylogenetic reconstruction grouped these isolates into four major clades with strong bootstrap support, confirming distinct evolutionary lineages within the circulating population.
Conclusion: Conventional phenotypic methods substantially overestimated the prevalence of Salmonella infection because of bacterial misidentification. Integration of 16S rRNA gene sequencing into diagnostic workflows significantly improved species confirmation, refined prevalence estimates, and provided valuable phylogenetic information that can strengthen antimicrobial resistance surveillance and public health decision-making in endemic settings.
Keywords: Salmonella, 16S rRNA, Sanger sequencing, Enteric fever, Blood culture, Phylogenetic analysis
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