Cefazolin

Cefazolin resistance was observed in the disc diffusion method of antibiotic susceptibility testing of Salmonella typhi strains isolated from January to June 1999. Eighty six multi drug resistant (MDR) isolates were studied. Of these, 46 (53.5%) were found resistant to cefazolin by the disc diffusion test. The minimum inhibitory concentration (MIC) of cefazolin was determined for the 86 isolates of S. typhi by the agar dilution method. MIC of these strains ranged from 1.0 to 64.0 pg/ml. In comparison, 25 MDR S. typhi strains isolated between 1994-1998 (5 per year) also tested by the same method, gave MIC values of 0.5 to 4.0 itg/ml. Our findings indicate a definite upward shift in MIC of cefazolin to MDR S. typhi since 1999. This needs to be monitored since the second, third and fourth generation cephalosporins are therapeutic options for typhoid fever.

Key words Cefazolin MIC - Salmonella typhi

Multidrug resistant (MDR) Salmonella typhi has been prevalent in India since 19891%2. These strains were resistant to the three commonly used antibiotics viz., chloramphenicol, ampicillin and trimethoprimsulphamethoxazole (TMP-SMZ), so fluroquinolones were tested, found suitable and used widely to treat patients with MDR typhoid fever. During 1996 we noticed an upward shift in ciprofloxacin MIC values, although all strains of S. typhi tested so far are still sensitive to ciprofloxacin according to the National Committee for Clinical Laboratory Standards (NCCLS)4. This observation of an upward shift in MIC coincided with an observation by clinicians that there was a delayed response in some patients with confirmed typoid fever, who were on ciprofloxacin therapy. Although they were subjectively better, there was a delayed defervescence of fever even up to 7 days, whereas in 1989, this occurred in 3 days. In such patients, the therapeutic option was often cephalosporins of second or third generation. From January 1999, cefazolin resistant isolates have been observed among the MDR S. typhi which was not so in those isolated earlier. We have tested cefazolin MIC for all MDR S. typhi isolated from January to June 1999 and have observed an increasing trend in these values.

Material & Methods

All 86 isolates of MDR S.typhi isolated from January to June 1999 were tested for cefazolin susceptibility by the disc diffusion test and interpreted using standard methods. MIC was determined by the agar dilution method using Mueller Hinton Agar medium (Hi Media, India) using an inoculum of 105,6 organisms/ml followed by 24 h incubation in air and interpreted using standard protocol. Control strain used was ATCC Staphylococcus aureus 29213 with a known cefazolin MIC of 0.25 - 1.0 gg/ml. Twenty five strains of MDR S. typhi isolated between 1994-1998 (5 strains each year, randomly selected) were also similarly tested.

Twenty five isolates with cefazolin MICs in the resistant range were tested for extended spectrum beta lactamase (ESBL) production. This was done by determining the MIC for the isolates of cefazolin and clavulanic acid incorporated at a concentration of 4 gg/ml, using agar dilution method as above.

Results & Discussion

Forty six of the 86 (53%) MDR S. typhi isolates were found resistant to cefazolin by the disc diffusion test; the readings were such that colonies of S. typhi were present inside the zone of inhibition for some and there was no zone of inhibition for other isolates. The cefazolin MIC values as determined by the agar dilution method ranged between 1.0 and 64.0 gg/ml. During the earlier years 1994-1998 (5 isolates from each year), the MIC ranged between 0.5 and 4.0 gg/ml. Thus there is an obvious upward shift in MICs in 1999; also in January to June 1999, isolates showing MIC of 16.0 ltg/ml and above had increased in numbers. Of the 46 isolates resistant by disc diffusion method, 4 had a MIC of 64.0 gg/ml, I I a MIC of 32.0, 11 a MIC of 16.0, and 12 a MIC of 8.0 (mu)g/ml; the remaining 8 isolates were sensitive i.e., MICs

This indicates that there has been an upward shift in the MIC of MDR S. typhi to cefazolin and the MIC has slowly progressed towards the resistant range (as per NCCLS recommendations :5 8[ig/ml is considered sensitive and >! 32.0 gg/ml resistant).

With the addition of clavulanic acid, the MICs reduced to 0.5 gg/ml. This substantial drop in MIC of cefazolin when clavulanic acid was incorporated suggests that the cephalosporin resistance in MDR S. typhi, could be due to the production of an extended spectrum 0 lactamase (ESBL). We have already demonstrated the presence of TEMA P lactamase in MDR S. typhi isolates conferring ampicillin resistance'. The drop in MIC that we observe when clavulanic acid is added could be due to the TEM 1 effect or due to ESBL production. The former is a possibility since the isolates tested are still sensitive to the third generation cephalosporins. However, further study by hydrolysis assays and other methods will determine ESBL production in these isolates.

Currently 50 per cent of S. typhi strains from blood cultures are MDR (unpublished data). Quinolones are being used as the first line of treatment for MDR typhoid fever. If the patient does not respond to ciprofloxacin, a second (cefuroxime) or third generation cephalosporin is being used. Since MDR S. typhi has already shown resistance to cefazolin which is a first generation cephalosporin, it is a possibility that this resistance may extend -to the second and third generation cephalosporins and further reduce the therapeutic options available to treat MDR typhoid fever.

References

1. Anand AC, Kataria VK, Singh W, Chatterjed Sk. Epidemic multiresistant enteric fever in eastern India. Lancet 1990; 335:352.

2. Jesudason MV, John T.J. Multiresistant Salmonella typhi in India. Lancet 1990; 336: 252.

3. Jesudason MV, Malathy B, John TJ. Trend of increasing levels of minimum inhibitory concentration of ciprofloxacin to Salmonella typhi. Indian J Med Res 103; 1996: 247-9.

4. National Committee for Clinical Laboratory Standards, Table 2. Zone diameter interpretive standards and equivalent minimum inhibitory concentration (MIC) breakpoints for organisms other than Haemophilus sps. N. gonorrhoeae and S. pneumoniae:

vol 14, No. 16:8, Wayne, Pennsylvania, USA: National Committee for Clinical Laboratory Standards; 1994.

5. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility test: Approved standard; 7th ed. NCCLS document M2-A7 Wayne, Pennsylvania, USA: National Committee for Clinical Laboratory Standards; 2000 p. 15.

6. Barry AL. Procedures for testing antibiotics in agar media: Theoretical considerations. In: Larian V, editor. Antibiotics in laboratory medicine. 2nd ed. Baltimore: Williams & Wilkins, 1986 p. 69.

7. Shanahan PM, Jesudason MV, Thomson CJ, Amyes SGB. Molecular analysis of and identification of antibiotic resistant genes in clinical isolates of Salmonella typhi from India. J Clin Microbiol 1998; 36:1595-1600.

Mary Jesudason & L.K. Jeyaraj

Department of Clinical Microbiology, Christian Medical College & Hospital, Vellore

Accepted September 26, 2000

Reprint requests: Dr Mary Jesudason, Professo & Head, Department of Clinical Microbiology Christian Medical College & Hospital, Vellore 632004

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