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Ceftriaxone and Azithromycin Minimum Inhibitory Concentration of Salmonella Typhi and Paratyphi A Blood Isolates of Children with Enteric Fever
*Corresponding author: Sulochana Putli Bai Perumal, Department of Clinical Microbiology, Kanchi Kamakoti CHILDS Trust Hospital, Chennai, Tamil Nadu, India. sulochanaputlibai@hotmail.com
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Received: ,
Accepted: ,
How to cite this article: Perumal S, Shahul Hameed M. Ceftriaxone and Azithromycin Minimum Inhibitory Concentration of Salmonella Typhi and Paratyphi A Blood Isolates of Children with Enteric Fever. Ann Child Health. doi: 10.25259/ACH_2_2025
Abstract
Objectives:
Enteric fever caused by Salmonella Typhi and Paratyphi is endemic in India. Ceftriaxone and azithromycin are commonly used to treat enteric fever, but resistance to these antibiotics is emerging. The study was done to determine the ceftriaxone and azithromycin minimum inhibitory concentration (MIC) of S. Typhi and Salmonella Paratyphi A isolated from the blood of pediatric enteric fever patients.
Material and Methods:
Isolate of S. Typhi and Paratyphi A from the blood of pediatric patients with enteric fever at Kanchi Kamakoti CHILDS Trust Hospital from September 2023 to June 2024 were tested for ceftriaxone and azithromycin MIC through the Epsilometer test.
Results:
150 Salmonella (124 S. Typhi and 26 Salmonella Paratyphi A) isolates were included in this study. All Salmonella isolates were sensitive to ceftriaxone. The mean ceftriaxone MIC of S. Typhi was 0.108 µg/mL [95% confidence interval (CI): 0.101–0.114], and that of Salmonella Paratyphi A was 0.140 µg/mL (95% CI: 0.123–0.157). All the S. Typhi isolates were sensitive to azithromycin, and the mean azithromycin MIC for S.Typhi was 6.62 µg/mL (95% CI: 5.99–7.25). Salmonella Paratyphi A showed a statistically significantly higher mean MIC for both ceftriaxone (difference of 0.032 µg/mL, P = 0.001) and azithromycin (difference of 4.61 µg/mL, P = 0.000).
Conclusion:
All S. Typhi and S Paratyphi A, in our study, were sensitive to ceftriaxone, and their mean MIC was well below the sensitive breakpoint (≤1 µg/mL). All the S. Typhi isolates were sensitive to azithromycin with a mean MIC much below the sensitive breakpoint of ≤16 µg/mL. S. Paratyphi A showed a statistically significant higher mean MIC for both ceftriaxone and azithromycin than S. Typhi.
Keywords
Azithromycin
Ceftriaxone
Minimum inhibitory concentration
Salmonella Typhi
Salmonella Paratyphi A
INTRODUCTION
Enteric fever caused by Salmonella Typhi and Paratyphi is endemic in many developing countries, including India. Appropriate antimicrobial therapy has reduced the mortality rate in enteric fever to 0.5% from 30% (without antimicrobial therapy).[1]
Ampicillin, chloramphenicol, and cotrimoxazole (ACCo) were used as standard treatment for enteric fever till the 1980s. In the past two decades, multidrug-resistant strains (resistant to ACCo) of Salmonella have emerged worldwide, especially in the Indian subcontinent.[2] Ceftriaxone and azithromycin are the antibiotics commonly used currently to treat S. Typhi and
Paratyphi infections. Increased resistance of Salmonella to third-generation cephalosporins is reported in certain parts of the world.[3]
There is a paucity of data on the current minimum inhibitory concentration (MIC) of Salmonella enterica serovar Typhi and Paratyphi to ceftriaxone and azithromycin in South India, and hence this study.
The primary objective of this study was to determine the current ceftriaxone and azithromycin MIC of 150 blood isolates of S. enterica serovar Typhi and Paratyphi from pediatric patients with enteric fever and to understand the distance between their MICs and their respective susceptible breakpoint MIC as per the Clinical and Laboratory Standards Institute (CLSI) 2023 guidelines which may have a bearing on treatment success and risk of development of resistance.
MATERIAL AND METHODS
One hundred and fifty isolates of S. Typhi/Paratyphi A obtained from blood culture (performed using BacT/Alert 3D fully automated blood culture system) of clinically suspected pediatric enteric fever patients from September 2023 to June 2024, at Kamakoti CHILDS Trust Hospital, Chennai, India were tested for the MIC to ceftriaxone and azithromycin through the Epsilometer test (E-test).
All Salmonella isolates were identified by biochemical tests and confirmed serologically with antisera. Salmonella isolates were considered sensitive to ceftriaxone when their MIC ≤1 µg/mL (based on CLSI M100, 33rd edition, 2023 guidelines). S. Typhi isolates were considered sensitive to azithromycin when their MIC ≤16 µg/mL (based on CLSI M100, 33rd edition, 2023 guidelines).
Epsilometer test
S. Typhi / Paratyphi A colonies from an 18 to 24-h-old non-selective media agar plate were suspended in sterile saline to obtain the turbidity of a standard 0.5 McFarland. A sterile cotton swab was dipped into the bacterial suspension, and while pulling out, the swab was rotated firmly against the upper inside wall of the tube to express excess fluid. The entire agar surface of the Cation-adjusted Mueller–Hinton Agar (MHA) plate was streaked with the swab 3 times, turning the plate at a 60° angle between each streaking. The lid of the plate was left ajar for 5 min to allow any excess moisture to be absorbed before applying E-strips.
Ceftriaxone and azithromycin E-strips (Himedia, Mumbai) with a concentration gradient of 0.002–32 mcg/mL (ceftriaxone) and 0.016–256 mcg/mL (azithromycin) were used. Each lot of ceftriaxone E-strip was quality checked using a quality control strain (Escherichia coli ATCC 25922) before use. Each lot of azithromycin E-strip was quality checked using a quality control strain (Staph aureus ATCC 29213) before use. Ceftriaxone and azithromycin E-strips were placed on the inoculated surface of MHA using an applicator, and plates were incubated at 37°C for 18–24 h.
After incubation, the value on the E-strip where the edge of the inhibition ellipse intersects the side of the strip was noted as the MIC of the isolate. The MIC results of S. enterica serovar Typhi and Paratyphi A for ceftriaxone and azithromycin were statistically analyzed using two-sample t-test.
RESULTS
Out of the 150 Salmonella isolates included in the study, 124 (82.7%) were S. Typhi and 26 (17.3%) were Salmonella Paratyphi A.
All S. Typhi isolates were sensitive to ceftriaxone, and the mean ceftriaxone MIC was 0.108 µg/mL [95% confidence interval (CI): 0.101–0.114]. Ceftriaxone MIC for S. Typhi ranged from 0.047 to 0.25 µg/mL (MIC50 was 0.094 µg/mL and MIC90 was 0.125 µg/mL).
All Salmonella Paratyphi A isolates were sensitive to ceftriaxone, and the mean ceftriaxone MIC was 0.140 µg/mL (95% CI: 0.123–0.157). Ceftriaxone MIC for Salmonella Paratyphi A ranged from 0.094 to 0.25 µg/mL (MIC50 was 0.125 µg/mL and MIC90 was 0.19 µg/mL) [Tables 1 and 2].
| Isolate | Mean | 95% CI |
|---|---|---|
| Salmonella Typhi | 0.108 | 0.101–0.114 |
| Salmonella Paratyphi A | 0.140 | 0.123–0.157 |
CI: Confidence interval, MIC: Minimum inhibitory concentration
| Isolate | MIC range | MIC50 | MIC90 |
|---|---|---|---|
| Salmonella Typhi | 0.047–0.25 | 0.094 | 0.125 |
| Salmonella Paratyphi A | 0.094–0.25 | 0.125 | 0.19 |
MIC: Minimum inhibitory concentration
The mean ceftriaxone MIC for Salmonella Paratyphi A was higher than for S. Typhi by 0.032 µg/mL (95% CI: 0.016–0.048), which was statistically significant (P = 0.001).
All the S. Typhi isolates were sensitive to azithromycin, and the mean azithromycin MIC for S. Typhi was 6.62 µg/mL (95% CI: 5.99–7.25). Azithromycin MIC for S. Typhi ranged from 2 to 16 µg/mL (MIC50 was 6 µg/mL and MIC90 was 12 µg/mL).
All the S. Paratyphi isolates also were sensitive to azithromycin (since CLSI mentions azithromycin breakpoints for S. Typhi only, these were used for interpreting S. Paratyphi A) and the mean azithromycin MIC for Salmonella Paratyphi A isolates was 11.23 µg/mL (95% CI: 9.66–12.80). Azithromycin MIC for Salmonella Paratyphi A ranged from 4 to 16 µg/mL (MIC50 was 12 µg/mL and MIC90 was 16 µg/mL).
Mean Azithromycin MIC for Salmonella Paratyphi A was significantly higher than for S. Typhi, with a mean difference of 4.61 µg/mL (95% CI: 3.07–6.15, P = 0.000) [Tables 3 and 4].
| Isolate | Mean | 95% CI |
|---|---|---|
| Salmonella Typhi | 6.62 | 5.99–7.25 |
| Salmonella Paratyphi A | 11.23 | 9.66–12.80 |
CI: Confidence interval, MIC: Minimum inhibitory concentration
| Isolate | MIC range | MIC50 | MIC90 |
|---|---|---|---|
| Salmonella Typhi | 2–16 | 6 | 12 |
| Salmonella Paratyphi A | 4–16 | 12 | 16 |
MIC: Minimum inhibitory concentration
DISCUSSION
After the emergence of ciprofloxacin resistance, ceftriaxone and azithromycin have become the drugs of choice for enteric fever in India. Knowledge of the current ceftriaxone and azithromycin MICs for S. Typhi and Salmonella Paratyphi A helps to monitor the emergence of antimicrobial resistance to these drugs and helps clinicians make an informed decision in selecting the antibiotic for enteric fever.
Among the 150 isolates of Salmonella included in our study, 124 isolates were S. Typhi and 26 isolates were Salmonella Paratyphi A. All the S. Typhi isolates were sensitive to ceftriaxone and azithromycin, and all the Salmonella Paratyphi A isolates were sensitive to ceftriaxone.
In a study by Dutta et al.[4] Ceftriaxone sensitivity was 100% for S. Typhi and Salmonella Paratyphi A, whereas the azithromycin susceptibility was 100% for S. typhi and 72% for Salmonella Paratyphi A. In the study by Patil and Mule[5], all the S. Typhi and Salmonella Paratyphi A isolates showed 100% sensitivity to both ceftriaxone and azithromycin.
In a multicenter study on the Surveillance of Enteric Fever in India,[6] ceftriaxone susceptibility was 100% for both S. Typhi and Salmonella Paratyphi A, whereas the azithromycin susceptibility was 99.9% and 100% for S. Typhi and Salmonella Paratyphi A, respectively.
In our study, the mean Ceftriaxone MIC was 0.108 µg/mL for S. Typhi and 0.140 µg/mL for Salmonella Paratyphi A, respectively, while the mean MIC of azithromycin was 6.62 µg/mL for S. Typhi and 11.23 µg/mL for Salmonella Paratyphi A, respectively.
Our results were comparable to Veeraraghavan et al.[6] which reported the mean ceftriaxone MIC of 0.22 µg/mL for S. Typhi and 0.20 µg/mL for Salmonella Paratyphi A, respectively, and the mean azithromycin MIC of 4.98 µg/mL for S. Typhi and 7.39 µg/mL for Salmonella Paratyphi A, respectively.
Ceftriaxone MIC50 and MIC90 of S. Typhi in our study were comparable to those of Dutta et al.[4] (MIC50 was 0.064 µg/mL and MIC90 was 0.125 µg/mL), which were lower compared to Veeraraghavan et al.[6] (MIC50 was 0.12 µg/mL and MIC90 was 0.5 µg/mL).
Azithromycin MIC50 and MIC90 of S. Typhi in our study were also comparable to those of Dutta et al.[4] (MIC50 was 8 µg/mL and MIC90 was 12 µg/mL), which was higher compared to Veeraraghavan et al.[6] (MIC50 was 4 µg/mL and MIC90 was 8 µg/mL).
Ceftriaxone MIC50 and MIC90 of Salmonella Paratyphi A in our study were comparable to Dutta et al.[4] (MIC50 was 0.125 µg/mL and MIC90 was 0.19 µg/mL), but were lower compared to Veeraraghavan et al.[6] (MIC50 was 0.25 µg/mL and MIC90 was 0.25 µg/mL).
Azithromycin MIC50 and MIC90 of Salmonella Paratyphi A in our study were lower compared to Dutta et al.[4] (MIC50 was 16 µg/mL and MIC90 was 32 µg/mL), whereas MIC90 was similar to Veeraraghavan et al.[6] (MIC50 was 4 µg/mL and MIC90 was 16 µg/mL).
In our study, the mean MIC, MIC50, and MIC90 for both ceftriaxone and azithromycin among Salmonella Paratyphi A were higher than those for S. Typhi, which was statistically significant.
The ceftriaxone and azithromycin MIC profiles for S. Typhi and Salmonella Paratyphi A in our study are comparable to other similar studies in India, with a few variations. The difference may be attributed to the antimicrobial sensitivity variation within different regions of India caused by the differences in the physicians’ antibiotic treatment choices.[6]
CONCLUSION
All the Salmonella isolates in our study were sensitive to ceftriaxone with an MIC well below the sensitive breakpoint. All the S. Typhi isolates were sensitive to azithromycin.
Both ceftriaxone and azithromycin showed a statistically significantly higher MIC for Salmonella Paratyphi A than for S. Typhi.
Even though our study did not find resistance in S. Typhi and Salmonella Paratyphi A to azithromycin and ceftriaxone, the current empirical therapy for enteric fever management in India requires regular monitoring of the antimicrobial sensitivity of S. Typhi and Salmonella Paratyphi A to ceftriaxone and azithromycin, by determining their MICs, for early detection of the emergence of antibiotic resistance.
Acknowledgment:
This research was supported by the CHILDS Trust Medical Research Foundation and Kanchi Kamakoti CHILDS Trust Hospital (KKCTH), Chennai, India.
Author contributions:
SP: Conceptualization, Methodology, Investigation, Data Curation, Formal analysis, Writing - Original Draft, MB: Writing - Review and Editing.
Ethical approval:
The research/study was approved by the Institutional Review Board at the Ethics Committee of Kanchi Kanchi Kamakoti CHILDS Trust Hospital (KKCTH) and the study was reviwed during the EC conducted on 12th August 2023 and approval was given on 1st September 2023.
Declaration of patient consent:
The authors certify that they have obtained all appropriate patient consent.
Conflicts of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript, and no images were manipulated using AI.
Financial support and sponsorship: The CHILDS Trust Medical Research Foundation (CTMRF).
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