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ORIGINAL ARTICLE Table of Contents   
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


Department of Microbiology, Kasturba Medical College, Mangalore - 575 001, India

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   Abstract 

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.

Keywords: Broth-disk method, ceftriaxone, minimum inhibitory concentration, salmonella

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;52:189-90

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 Feb 16];52:189-90. Available from: http://www.ijpmonline.org/text.asp?2009/52/2/189/48913



   Introduction Top


Typhoid fever caused by  Salmonella More Details enterica serovar Typhi is a global public health problem, responsible for 30 million cases and 600,000 deaths annually. [1] In 1948, chloramphenicol became the standard antibiotic for the treatment of typhoid fever. However, in 1972, chloramphenicol-resistant typhoid fever became a major problem. [2] 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 . [3],[4],[5],[6] 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. [7] 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 Top


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 4C. The bacteria were subcultured on MacConkey's agar and incubated at 37C 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 37C 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 140C for 2h. Ceftriaxone was diluted two-folds in sterile distilled water to get antibiotic solutions of concentration ranging between 6400 and 200 g/mL. The antibiotic solution (0.01mL per disk) was added to sterile disks to prepare disks of potency 64, 32, 16, 8, 4 and 2g. The disks were stored at 4C 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 g 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 37C 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 g/mL were considered susceptible, 16-32 g/mL as intermediate and ≥64 g/mL as resistant as per the NCCLS criteria. [8] The results of the broth-disk method were compared with MIC results obtained by the standard broth macrodilution test. [9]

Statistical analysis of the results was carried out using the Mann-Whitney U test and the χ2 test and P values <0.05 were considered significant. The power of the test was 85%.


   Results and Discussion Top


The MIC of ceftriaxone to 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 g/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 4C 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 .


   Acknowledgement Top


We thank the Dean, Kasturba Medical College, Mangalore and the Professor and Head of Microbiology Department, Kasturba Medical College for their support.

 
   References Top

1.Hiruse K, Hashimoto A, Tamura K, Kawanura Y, Ezaki J, Sagara H, et al . DNA sequence analysis of DNA gyrase and DNA topoisomerase IV quinolone resistance determining regions of Salmonella enterica serovar Typhi and serovar Paratyphi A. Antimicrob Agents Chemother 2002;46:3249-52.  Back to cited text no. 1    
2.Parry CM, Hen TT, Dougan G, White NJ, Farrar JJ. Typhoid fever. N Engl J Med 2002;347:1770-81.  Back to cited text no. 2    
3.Ahmed D, D'Costa LT, Alam K, Nair GB, Hossain MA. Multidrug resistant Salmonella enterica serovar Typhi isolates with high level resistance to ciprofloxacin in Dhaka, Bangladesh. Antimicrob Agents Chemother 2006;50:3516-17.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Kownhar H, Shankar EM, Rajan R, Rao UA. Emergence of nalidixic acid resistant Salmonella enterica serovar Typhi resistant to ciprofloxacine in India. J Med Microbiol 2007;56:136-7.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Bhat GK, Suresh K. Ciprofloxacin resistant Salmonella Typhi. Natl Med J India 1999;12:88.  Back to cited text no. 5    
6.Asna SM, Haq JA, Rahman M. Nalidixic acid-resistant Salmonella enterica serovar Typhi with decreased susceptibility to ciprofloxacin caused treatment failure: A report from Bangladesh. Jpn J Infect Dis 2003;56:32-3.  Back to cited text no. 6    
7.Saha SK, Talukdar SY, Islam H, Saha S. A highly ceftriaxone resistant Salmonella Typhi in Bangladesh. Pediatr Infect Dis 1999;18:387.  Back to cited text no. 7    
8.National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 5 th ed. Approved Standard M7-A5 NCCLS, Wayne (PA) 2000.  Back to cited text no. 8    
9.Hindler JF, Jorgensen JH. Antimicrobial susceptibility testing. In: Textbook of Diagnostic Microbiology 3 rd ed. In: Mahon CR, Lehman DC, Manuselis G, editors. Saunders, 2007. p. 319-54.  Back to cited text no. 9    

Top
Correspondence Address:
K Gopalkrishna Bhat
Department of Microbiology, Kasturba Medical College, Mangalore - 575 001
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.48913

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    Tables

  [Table 1], [Table 2]

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