Phenotypic and molecular detection of Salmonella species with antimicrobial susceptibility patterns in clinical isolates

INTRODUCTION

Salmonella is a major worldwide pathogen that causes foodborne diseases, gastroenteritis, septicemia, and invasive infections in people and animals.^[1] Contaminated meat, dairy products, and fresh vegetables act as reservoirs for Salmonella transmission, providing a significant public health risk.^[2] Salmonella isolation and identification are critical for disease monitoring and outbreak investigation, and many culture medium have been standardized to do this.^[3] Conventional culture techniques remain effective; however, quick molecular detection approaches are increasingly being employed for emergency response and food safety.^[4] Antimicrobial resistance has emerged in Salmonella strains recovered from poultry and other food sources, complicating treatment efforts.^[5] Salmonella infection in chicken is well recognized as a major source of illness, affecting both animal health and zoonotic transmission.^[6] Egyptian studies have found Salmonella enterica, Escherichia coli O157:H7, and Shigella spp. in food items, underlining the potential of several enteric infections circulating concurrently.^[7] Molecular tests have also shown the presence of S. enterica serovar Enteritidis in clinical and poultry house samples.^[8] Invasive salmonellosis is still a significant clinical concern, with unique epidemiological, diagnostic, and antibiotic resistance difficulties globally.^[9] Clinical and environmental Salmonella isolates from Saudi Arabia showed a variety of serotypes and resistance mechanisms.^[10]

Genotypic and phenotypic variety among Salmonella isolates has been extensively reported in areas such as Kenya, where Salmonella typhimurium strains vary significantly.^[11] In Egypt, locally isolated strains were described for antigen preparation, revealing both phenotypic and genotypic variation.^[12] Similar studies in Turkey have indicated antibiotic resistance patterns in chicken isolates,^[13] whereas research in Korea has shown diversity in Salmonella strains isolated from pigs and farm settings.^[14] Brazilian studies revealed phenotypic and genotypic diversity in Salmonella Typhimurium strains isolated from patients and food items.^[15] Ethiopian research found temporal links between nontyphoidal Salmonella strains in people and food animals, indicating a zoonotic cycle.^[16] Research on imported chicken has also revealed resistance to S. enterica serovars.^[17]

Bangladeshi research has discovered Salmonella in broilers and performed antibiogram tests, revealing local resistance trends.^[18] Foodborne Salmonella infection has been widely documented from a variety of sources, demonstrating its widespread prevalence.^[19] Furthermore, in Nepal, isolates from enteric fever patients showed varied susceptibility to conventional antibiotics, highlighting treatment problems.^[20] Regional investigations are critical given Salmonella’s expanding clinical and epidemiological relevance, as well as the rise of antibiotic resistance. Tripura, located in Northeast India, has minimal information on the phenotypic and genotypic characteristics of Salmonella isolates.

MATERIAL AND METHODS

RESULTS

In this study 200 clinical samples were included. Out of this total samples 7 cases (3%), bacterial growth was obtained, and 193 cases (97%), no growth was recorded. The study’s participants were 61% (n - 123) men and 39% (n - 77) women.

Molecular analysis

The amplified PCR products were electrophoresed on a 1.5% agarose gel with 10 μL of ethidium bromide solution, inspected under an ultraviolet transilluminator, and photographed. A 1 kb plus DNA ladder was used to calculate the molecular weight of the obtained bands. Following agarose gel electrophoresis, PCR products with molecular sizes of around 796 bp and DNase-RNase-free water were employed as negative controls in both the DNA extraction and PCR analysis [Figure 1].

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Figure 1: Gel electroplates.

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DISCUSSION

In the current investigation, 200 clinical samples were evaluated, with 7 (3%) demonstrating bacterial growth and 193 (97%) revealing no growth. This study’s Salmonella species isolation rate was lower than that of Nair et al.,^[1] who found a higher prevalence in diarrheagenic samples from infants and young animals who found a significant frequency of Salmonella and E. coli contamination in raw meat. Differences in isolation rate can be attributable to sample type, geographic circumstances, hygiene procedures, and infection control strategies.

The Salmonella isolates in this study showed characteristic morphological features on selective media, pale, non-lactose fermenting colonies on MacConkey agar; red colonies with black centers on XLD agar; and colorless colonies with black centers on SS agar, findings consistent with those of Neyaz et al. and Lee et al.,^[3,4] who emphasized these classical colony morphologies as reliable indicators of Salmonella isolation. Biochemically, the isolates were motile, citrate positive, methyl red positive, and generated hydrogen sulfide on TSI agar, which is consistent with normal Salmonella biochemical profiles described by Ahmed and Shimamoto and Ibrahim et al.^[7,12]

Antibiotic susceptibility tests indicated a worrying trend of MDR among the isolates. Imipenem had the maximum sensitivity 100%, followed by ciprofloxacin 80% and trimethoprim–sulfamethoxazole 60%, with intermediate sensitivity for ceftriaxone 60% and cefixime 40%. Chloramphenicol and azithromycin showed the highest levels of resistance 80% and 80%, respectively, followed by ampicillin 60%. These findings are consistent with previous publications by Castro-Vargas et al. and Maharjan et al.,^[5,20] who found widespread antimicrobial resistance among Salmonella isolates, notably to routinely used first-line medicines such as ampicillin and chloramphenicol. El-Tayeb et al. and Ince and Akan have also documented similar resistance patterns, highlighting the global spread of MDR Salmonella strains.^[10,13] According to publications by Eguale et al. and Arkali and Çetinkaya, the high susceptibility to imipenem seen here supports the idea that carbapenems are still a viable treatment choice for Salmonella species that are resistant to them.^[16,21] To stop the emergence of resistance, their application should be limited to serious illnesses.

The identity of Salmonella isolates was further confirmed by molecular validation using PCR targeting the invA gene, as all culture-positive samples generated the anticipated 796 bp amplification band. The invA gene is a commonly used genetic marker for genus-level identification because it encodes invasion-associated proteins and is highly conserved among Salmonella serovars. Mezal et al. (2014) and Seribelli et al., (2020) who used gene-targeted amplification to confirm S. enterica serovars,^[8,15] have described similar PCR-based molecular identification techniques. The successful amplification of the anticipated fragment in every isolate demonstrates the dependability and diagnostic value of PCR, especially in circumstances where culture-based techniques can be laborious, ambiguous, or linked to low bacterial load. Therefore, integrating molecular confirmation with conventional phenotypic methods enhances the accuracy of diagnosis and facilitates prompt and efficient infection control.

Overall, the current work highlights the ongoing clinical and public health relevance of Salmonella infections by showing that isolates of the bacteria show MDR to a number of widely used medicines. The comparatively low isolation rate and the observed resistance patterns, especially against conventional antimicrobial agents, highlight the significance of ongoing surveillance, prudent antibiotic use, and molecular confirmation techniques for precise diagnosis and efficient infection control. In line with local and international publications, our results provide more evidence that Salmonella is a significant nosocomial and zoonotic pathogen. However, the study’s single-center methodology and rather small sample size may limit how far the results can be applied. In order to offer more thorough knowledge of the epidemiology and antibiotic resistance patterns of Salmonella infections, additional multicentric research encompassing bigger populations and incorporating specific demographic and clinical characteristics is advised (Gast and Porter, 2020; Crump et al., 2015).^[6,9]

CONCLUSION

Using distinctive cultural, biochemical, and molecular characteristics, this investigation validates the existence of Salmonella species in clinical samples. While imipenem continued to be very successful, the isolates showed substantial resistance to ampicillin, chloramphenicol, and azithromycin, indicating MDR. The results demonstrate the concerning increase in Salmonella’s resistance to antibiotics and emphasize the need for ongoing monitoring, prudent antibiotic usage, and molecular confirmation for precise diagnosis and efficient infection management.