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 ~  Abstract
 ~  Introduction
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  References
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ORIGINAL ARTICLE
Year : 2009  |  Volume : 27  |  Issue : 2  |  Page : 107-110
 

Role of azithromycin against clinical isolates of family enterobacteriaceae: A comparison of its minimum inhibitory concentration by three different methods


Department of Microbiology, S.C.B. Medical College, Cuttack -753 007, India

Date of Submission31-Dec-2007
Date of Acceptance26-Sep-2008

Correspondence Address:
N Chayani
Department of Microbiology, S.C.B. Medical College, Cuttack -753 007
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.45361

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 ~ Abstract 

Purpose: To determine the effect of azithromycin, a new azalide antibiotic, on clinical isolates of the family Enterobacteriaceae and to determine and compare its minimum inhibitory concentration (MIC) by disk diffusion, agar dilution and E-test methods. Materials and Methods: One hundred fifty-nine bacterial strains belonging to the family Enterobacteriaceae, isolated from different clinical samples, were tested for their susceptibility to azithromycin by disk diffusion, agar dilution and E-test methods. The MIC values were analysed and the percentages of agreement between the different methods were mentioned. Results: Of the 159 isolates of the family Enterobacteriaceae, 60.37% were E. coli followed by Klebsiella species 28.3%, Salmonella and Shigella species 3.77% and Enterobacter and Citrobacter species 1.88% each. Maximum isolates were obtained from urine 117/159 (73.58%). Azithromycin was found to be more active against Salmonella and Shigella species, showing 100% sensitivity the by E-test and 83.33% by the disk diffusion methods. In the agar dilution method, 83.33% of Salmonella and 66.66% of Shigella species were sensitive to azithromycin. The overall agreement between disk diffusion and agar dilution method was 96.8%, between agar dilution and E-test was 88% and between disk diffusion and E-test was 91.2%. Conclusion: Azithromycin may become an important addition to our antimicrobial strategies, especially for the treatment of bacterial diarrhoea and infections caused by Salmonella typhi.


Keywords: Azithromycin, enterobacteriaceae, minimum inhibitory concentration determination


How to cite this article:
Chayani N, Tiwari S, Sarangi G, Mallick B, Mohapatra A, Paty B P, Das P. Role of azithromycin against clinical isolates of family enterobacteriaceae: A comparison of its minimum inhibitory concentration by three different methods. Indian J Med Microbiol 2009;27:107-10

How to cite this URL:
Chayani N, Tiwari S, Sarangi G, Mallick B, Mohapatra A, Paty B P, Das P. Role of azithromycin against clinical isolates of family enterobacteriaceae: A comparison of its minimum inhibitory concentration by three different methods. Indian J Med Microbiol [serial online] 2009 [cited 2019 Sep 16];27:107-10. Available from: http://www.ijmm.org/text.asp?2009/27/2/107/45361



 ~ Introduction Top


One of the major limitations to successful antimicrobial therapy of enteric bacterial pathogens has been the progressive emergence of resistance to these drugs, particularly in the developing countries. [1] With a marked increase in antibiotic resistance among enteric bacterial pathogens, it has become imperative to find alternative effective antimicrobial agents. Among the oral antimicrobial agents, the fluoroquinolones and the broad-spectrum cephalosporins are the only groups whose efficacy against enteric pathogens of the family Enterobacteriaceae has not yet been compromised by acquired resistance. However, the fluoroquinolones are not yet recommended for use in paediatric patients because of articular damage caused by these drugs and the broad-spectrum cephalosporins because they are quite expensive and likely to induce TEM-like β lactamases, which can hydrolyze the broad-spectrum cephalosporins.[2]

Erythromycin is an old antibiotic that has been used to prevent infections caused by gram -ve enteric pathogens. [3] Use of erythromycin is limited by frequent G.I side effects and because of high minimum inhibitory concentrations (MICs), which would presumably not be useful for treating infections caused by those members of the family Enterobacteriaceae that invade beyond the intestinal lumen (e.g.,  Salmonella More Details and Shigella species). Azithromycin, a new azalide antibiotic, is active in vitro against a variety of gram -ve enteric bacterial pathogens. [4] In murine typhoid models, azithromycin given once daily was highly effective in clearing the infection and this activity was attributable to the remarkable property of the intracellular concentration of azithromycin in the macrophage (>100 times than in serum). [5]

In the present study, different strains of the family Enterobacteriaceae were isolated from clinical samples. The antibiotic susceptibility pattern of different isolates with special reference to azithromycin was studied by the disc diffusion method. The MIC of azithromycin was evaluated by agar dilution and E-test methods.


 ~ Materials and Methods Top


The study was carried out on 159 isolated bacterial strains belonging to the family Enterobacteriaceae. All the clinical samples were inoculated into routine culture media and identification of the isolated organism was performed by standard procedures. [6],[7]

Routine antimicrobial susceptibility testing of the bacterial strain belonging to the family Enterobacteriaceae was carried out by the Kirby-Bauer disk diffusion method. The MIC for azithromycin was determined by agar dilution and E-test methods. The vial containing azithromycin powder was obtained from Alembic Pharma Ltd, India. and dissolved in distilled water as stock solution according to the manufacturer's instruction, which was used for the agar dilution method. The E-test strip for azithromycin was obtained from Hi-Media, India. The control organism  Escherichia More Details coli ATCC 25922 was included with each set of isolates tested, which was obtained from Dr. (Mrs.) Savitri Sharma (Editor IJMM), L.V. Prasad Eye Institute, Bhubaneshwar.


 ~ Results Top


One hundred fifty-nine bacterial strains belonging to the family Enterobacteriaceae were isolated from the clinical samples. Of the 159 isolates of the family Enterobacteriaceae, 96 (60.37%) were E. coli followed by Klebsiella species 45 (28.31%). Salmonella and Shigella species were 6 each (3.77%) whereas Enterobacter and Citrobacter species were 3 each (1.88%). Maximum isolates were obtained from urine 117 (73.58%), followed by sputum and stool (7.54%) each .

On routine antimicrobial susceptibility testing by the disk diffusion method, E. coli and Klebsiella species were found to be most sensitive for amikacin (78.1% and 73.3%), followed by gatifloxacin (50% and 57.7%), respectively. Salmonella and Shigella species were found to be most sensitive to azithromycin (83.3%), followed by amikacin (50%). Enterobacter species showed an equal sensitivity to both amikacin and gatifloxacin (66.6%) whereas Citrobacter species showed an equal sensitivity to amikacin, ceftriaxone and cefotaxime. In comparison with other isolates, E. coli showed maximum resistance to azithromycin (85.4%) [Table 1].

On determination of the MIC of azithromycin by the agar dilution method among 96 strains of E. coli , 13(13.54%) strains showed an MIC <8µg/mL whereas 42 (43.75%) strains had an MIC ≥256µg/mL. Similarly, when tested for Klebsiella species, 21 (46.6%) strains showed an MIC <8µg/ mL whereas 3 (6.66%) strains had an MIC ≥256µg/mL.

The Salmonella and Shigella species showed an 83.3% and 66.6% sensitivity, respectively, but the Enterobacter and Citrobacter species were showed a 100% resistance (i.e., MIC >8µg/mL).

When the MIC of azithromycin was determined by the E-test method, 18 (18.7%) strains of E. coli were found to be in the susceptible range (i.e., <8µg/mL) and 22 (22.9%) strains had an MIC >128µg/mL. Among the 45 strains of Klebsiella species isolated, 29 (64.44%) strains had an MIC in the susceptible range. The Salmonella and Shigella species showed a 100% sensitivity to azithromycin. The Enterobacter and Citrobacter species showed a 66.6% and 33.3% sensitivity to azithromycin, respectively.

A comparison of the MIC of azithromycin by agar dilution, E-test and disk diffusion methods was made. It was found that 18.75% strains of E. coli were susceptible by E-test in comparison with 14.58% and 13.54% by disk diffusion and agar dilution, respectively. For the Klebsiella species, 64.44% strains showed an MIC in the susceptible range by E-test in comparison with 48.8% and 46.6% by disk diffusion and agar dilution, respectively. The Salmonella and Shigella species showed a 100% sensitivity to azithromycin the by E-test but an 83.33% sensitivity by the disk diffusion method. The MIC of Salmonella to azithromycin by agar dilution method was same as that of disk diffusion, but for Shigella species it was 66.66%. Enterobacter and Citrobacter species showed a 100% resistance to azithromycin by the agar dilution method but a 33.33% susceptibility by the disk diffusion method. Although Enterobacter showed a 66.66% susceptibility by the E-test, the Citrobacter species showed only a 33.33% susceptibility by the E-test method [Table 2].

The percentage of agreement between the different test methods show a 96.8% agreement between disk diffusion and agar dilution, an 88% agreement between agar dilution and E-test and a 91.2% agreement between disk diffusion and E-test methods [Table 3].


 ~ Discussion Top


Azithromycin penetrates into cells effectively and this intracellular penetration explains the therapeutic efficacy of this drug against the predominantly intracellular pathogens like S. typhi . The ability of azithromycin to achieve intracellular concentration in monocytes is 231 times and in polymorphonuclear leucocytes is 83 times greater than that of serum concentration. [8],[9] It has a long half life of 2-3 days. The intracellular concentration appears to be essential for its therapeutic activity in typhoid fever. The availability of a paediatric suspension of azithromycin provides an opportunity to examine the efficacy and safety of this drug in young children with typhoid fever. The in vitro resistance of azithromycin to different clinical isolates was reported to be 12% by Butler et al . [10]

In the present study, the antibiotic susceptibility pattern of 159 clinical isolates belonging to the family Enterobacteriaceae was studied. Among the 159 clinical isolates, 96 were E. coli (60.37%), followed by Klebsiella spps. 45 (28.31%). The majority of these isolates were obtained from urine samples. On studying the antibiotic susceptibility patterns of these clinical isolates by the disk diffusion method, it was found that amikacin was the most sensitive drug for all the isolates of the family Enterobacteriaceae, except Salmonella and Shigella species, where azithromycin was found to be more sensitive. Although previous studies showed an excellent in vitro activity of azithromycin against the most common enteric bacterial pathogens, [11] in our study, an increasing resistance of azithromycin to all the enteric bacterial pathogens except Salmonella and Shigella species was obtained. Thus, it was found to be the most effective antimicrobial agent for these two species.

On comparison of the MIC by the different test methods, it was found that MICs obtained using the E-test was lower than those by agar dilution regardless of the species of organism tested. The correlation between the E-test and the agar dilution MICs varied greatly depending on the antimicrobial agent tested. [12] If the performance of these test methods is ranked by the percentage agreement of the interpretive results with the consensus interpretive results (highest agreement ranked best), the rank order would be E-test>disk diffusion>agar dilution. [13] Our study also showed a similar type of result. In our study, the percentage of agreement between agar dilution and E-test is 88%, between disk diffusion and agar dilution is 96.8% and between disk diffusion and E-test is 91.2%.

Emergence of antimicrobial drug resistance in S. typhi is a global problem [14],[15] and was the compelling reason to undertake the trial of a new alternative antibiotic for typhoid fever. This study showed an excellent in vitro activity of azithromycin against Salmonella and Shigella species . Because of its good intracellular activity and relatively low MICs for enteropathogens, this drug may become an important addition to our antimicrobial strategies for the treatment of typhoid fever and also for bacillary dysentery. Further studies to define its role in the management of enteric bacterial infections may be required.

 
 ~ References Top

1.Murray BE. Resistance of Shigella , Salmonella , and other selected enteric pathogens to antimicrobial agents. Rev Infect Dis 1986;8:S172-81.  Back to cited text no. 1  [PUBMED]  
2.Murray BE. New aspects of antimicrobial resistance and the resulting therapeutic dilemmas. J Infect Dis 1991;163:1185-94.  Back to cited text no. 2    
3.Andremont A., Raibaud P, Tancrede C. Effect of erythromycin on microbial antagonisms: A study in gnotobiotic mice associated with a human fecal flora. J Infect Dis 1983;148:579-87.  Back to cited text no. 3    
4.Gordillo ME, Singh KV, Murray BE. In vitro activity of azithromycin against bacterial enteric pathogens. Antimicrob Agents Chemother 1993;37:1203-5.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Butler T, Girard AE. Comparative efficacies of azithromycin and Ciprofloxacin against experimental Salmonella typhimurium infection in mice. J Antimicrob Chemother 1993;31:313-9.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Betty AF, Sahm DF, Weissfeld AS, editors. Laboratory cultivation and isolation of bacteria. Chapter 10. In: Bailey and Scott's diagnostic microbiology. 11 th ed. St. Louis: Mosby Company; 2002. p. 133-47.  Back to cited text no. 6    
7.Betty AF, Sahm DF, Weissfeld AS, editors. Overview of conventional methods for bacterial identification. Chapter 11. In: Bailey and Scott's diagnostic microbiology 11 th ed. Mosby Company; St. Louis: 2002. p. 148-61.  Back to cited text no. 7    
8.Wildfeuer A, Laufen H, Zimmermann T. Uptake of azithromycin by various cells and its intracellular activity under in-vivo conditions. Antimicrob Agents Chemother 1996;40:75-9.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Pascual A, Conejo MC, Garcνa I, Perea EJ. Factors affecting the intracellular accumulation and activity of azithromycin. J Antimicrob Chemother 1995;35:85-93.  Back to cited text no. 9    
10.Butler T, Sridhar CB, Daga MK, Pathak K, Pandit RB, Khakhria R, et al . Treatment of typhoid fever with azithromycin versus chloramphenicol in a randomized multicentre trial in India. J Antimicrob Chemother 1999;44:243-50.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.Gordillo ME, Singh KV, Murray BE. In vitro activity of azithromycin against bacterial enteric pathogens. Antimicrob Agents Chemother 1993;37:1203-5.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]
12.Beilei GE, Bodeis S, Walker RD, White DG, Zhao S, McDermott PF, et al . Comparison of the E-test and agar dilution for in vitro antimicrobial susceptibility testing of Campylobacter. J Antimicrob Chemother 2002;50:487-94.  Back to cited text no. 12    
13.Steward CD, Stocker SA, Swenson JM, O'Hara CM, Edwards JR, Gaynes RP, et al . Comparison of agar dilution, disk diffusion, microscan, and vitek antimicrobial susceptibility testing methods to broth microdilution for detection of fluroquinolone: Resistant isolates of the family enterobacteriaceae. J Clin Microbiol 1999;37:544-7.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Mirza SH, Beeching NJ, Hart CA. Multi-drug resistant typhoid: A global problem. J Med Microbiol 1996;44:317-9.  Back to cited text no. 14  [PUBMED]  
15.Coovadia YM, Gathiram V, Bhamjee A, Garratt, RM, Mlisana K, Pillay N, et al . An outbreak of multiresistant Salmonella typhi in South Africa. Q J Med 1992;82:91-100.  Back to cited text no. 15    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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