Year : 2009 | Volume
: 52 | Issue : 2 | Page : 189--190
A simple broth-disk method to determine the minimum inhibitory concentration of ceftriaxone on Salmonella enterica serovar typhi and paratyphi
K Gopalkrishna Bhat, Amruta Tripathy, Rajasree Rajagopal, Sreeja Ramachandran
Department of Microbiology, Kasturba Medical College, Mangalore - 575 001, India
K Gopalkrishna Bhat
Department of Microbiology, Kasturba Medical College, Mangalore - 575 001
Background and Purpose: Resistance to fluoroquinolones and cephalosporins is a major problem with Salmonella enterica serovar Typhi and Paratyphi. An accurate determination of antibiotic susceptibility requires tests for minimum inhibitory concentration (MIC) of antibiotics. We describe a simple broth-disk method to determine the MIC of ceftriaxone on S. typhi and S. paratyphi .
Materials and Methods: Sixteen strains of S. typhi and two strains each of
S. paratyphi A and S. paratyphi B were used in the study. The MIC of ceftriaxone was determined using the simple broth-disk method and the conventional broth macrodilution method and the results were compared.
Results: All salmonella strains were susceptible to ceftriaxone. The results of the broth-disk and the conventional broth macrodilution method were similar.
Conclusion: The broth-disk method is a simple, reliable and cost-effective method to determine the MIC of ceftriaxone on S. typhi and S. paratyphi A.
|How to cite this article:|
Bhat K G, Tripathy A, Rajagopal R, Ramachandran S. A simple broth-disk method to determine the minimum inhibitory concentration of ceftriaxone on Salmonella enterica serovar typhi and paratyphi.Indian J Pathol Microbiol 2009;52:189-190
|How to cite this URL:|
Bhat K G, Tripathy A, Rajagopal R, Ramachandran S. A simple broth-disk method to determine the minimum inhibitory concentration of ceftriaxone on Salmonella enterica serovar typhi and paratyphi. Indian J Pathol Microbiol [serial online] 2009 [cited 2020 Nov 24 ];52:189-190
Available from: https://www.ijpmonline.org/text.asp?2009/52/2/189/48913
Typhoid fever caused by Salmonella enterica serovar Typhi is a global public health problem, responsible for 30 million cases and 600,000 deaths annually.  In 1948, chloramphenicol became the standard antibiotic for the treatment of typhoid fever. However, in 1972, chloramphenicol-resistant typhoid fever became a major problem.  Toward the end of 1980s and 1990s, S. typhi developed resistance to first-line antibiotics. Decreased susceptibility and full resistance to ciprofloxacin has been reported in S. typhi . ,,, Ciprofloxacin-resistant typhoid fever is treated with third-generation cephalosporins such as ceftriaxone. S. typhi with a high level of resistance to ceftriaxone has been reported.  Antibiotic susceptibility testing by the routine disk diffusion method may fail to detect resistance in all strains. Determination of minimum inhibitory concentration (MIC) by dilution techniques is labor intensive and takes time. With these points in mind, we developed a simple broth-disk method to determine the MIC of ceftriaxone on S. typhi .
Materials and Methods
Sixteen strains of S. typhi and two strains each of S. paratyphi A and S. paratyphi B isolated from the blood were used in the study. Isolation and identification of the bacteria were performed using standard methods. The bacteria were preserved on a nutrient agar slope at 4°C. The bacteria were subcultured on MacConkey's agar and incubated at 37°C for 18h before preparing the inoculum. Two to three identical colonies were touched with a straight wire and inoculated into a tube of nutrient broth and incubated at 37°C for 4-6h. The turbidity of the inoculum was adjusted to McFarland 0.5 standard.
Disks of diameter 6mm were punched from Whatman No. 1 filter paper and sterilized in a hot air oven at 140°C for 2h. Ceftriaxone was diluted two-folds in sterile distilled water to get antibiotic solutions of concentration ranging between 6400 and 200 µg/mL. The antibiotic solution (0.01mL per disk) was added to sterile disks to prepare disks of potency 64, 32, 16, 8, 4 and 2µg. The disks were stored at 4°C until use.
For each test, six tubes containing 1mL Mueller-Hinton Broth (Hi Media, Mumbai, India) were taken. One disk of potency 64, 32, 16, 8, 4 and 2 µg each was added separately to each test tube and shaken well to release the drug into the broth. To each tube, 0.01mL of the bacterial inoculum was added. An inoculated broth containing no antibiotic was included as growth control and a tube of uninoculated broth was used as sterility control. Quality control was performed using E. coli ATCC 25922. The tubes were incubated at 37°C for 18 h. The lowest concentration of ceftriaxone that inhibited the bacterial growth was considered as the MIC. Salmonella serovars with an MIC of ceftriaxone ≤8 µg/mL were considered susceptible, 16-32 µg/mL as intermediate and ≥64 µg/mL as resistant as per the NCCLS criteria.  The results of the broth-disk method were compared with MIC results obtained by the standard broth macrodilution test. 
Statistical analysis of the results was carried out using the Mann-Whitney U test and the χ2 test and P values S. typhi , S. paratyphi A and S. paratyphi B determined by the broth-disk and the broth macrodilution methods yielded similar results ( P = 1; [Table 1]). None of the strains of S. typhi tested were resistant to ceftriaxone [Table 2]. One strain of S. typhi was intermediate to ceftriaxone (MIC 16 µg/mL).
Multidrug-resistant typhoid fever is a global health problem. Typhoid fever being a systemic infection needs prompt and correct treatment. S. typhi is a facultative intracellular pathogen. The antibiotics that are bactericidal to S. typhi and can penetrate into tissues are useful in the treatment. Early diagnosis and prompt treatment can reduce morbidity and mortality due to typhoid fever. Therefore, correct and early detection of the susceptibility pattern of S. typhi to antibiotics is important. We observed that the broth-disk method is a useful technique in this regard. This method was first introduced by us. It provided results comparable with the broth macrodilution test. Further, the broth-disk method is a simple, reproducible and easy method that could be used even in routine diagnostic microbiology laboratories. The antibiotic disks can be stored at 4°C for at least 30 days without losing potency. In conclusion, we feel that the broth-disk method is a simple method that could be used as an alternative to the broth macrodilution test for the determination of the MIC of ceftriaxone to S. typhi .
We thank the Dean, Kasturba Medical College, Mangalore and the Professor and Head of Microbiology Department, Kasturba Medical College for their support.
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