ABSTRACT 
Background: Development of ciprofloxacin resistance among multidrug resistant (MDR) Salmonella enterica serovar Typhi (S. typhi resistant to ampicillin, chloramphenicol, cotrimoxazole) leaves cephalosporins of third generation in the treatment of typhoid fever. Combination effect of antibiotics is of particular interest in order to combat drug resistance in bacterial strains. 
Aims: To assess the in vitro efficacy of ciprofloxacin in combination with cefazolin against S. typhi isolates with decreased susceptibility to ciprofloxacin.
Material and Methods: Minimum inhibitory concentration (MIC) values of cefazolin for 16 S. typhi isolates were determined by agar dilution method. Combined effects of ciprofloxacin and cefazolin against the isolates were studied following agar dilution method, and time-kill curves. Fractional inhibitory concentration (FIC) index were calculated, and the results were analyzed statistically. 
Results: In combination, by agar dilution checkerboard method, the 16 S. typhi isolates showed FICs 0.075-0.25 and 1.25-2.5 µg/ml, respectively for ciprofloxacin and cefazolin. The combination effects between the two antibiotics were additive for 9(56.25%) isolates (FIC index 0.512-0.916) and synergistic for 7(43.75%) isolates (FIC index 0.208-0.5). The synergy between ciprofloxacin and cefazolin was also confirmed, employing one cefazolin resistant isolate, by time-kill method. 
Conclusion: Results suggest that ciprofloxacin in combination with cefazolin may be the important regimen in combating antibiotic resistance among S. typhi. However, the combination warrants further evaluation for potential usefulness as a clinical regimen.
Key words: S. typhi, Ciprofloxacin-cefazolin combination, Synergism

INTRODUCTION
Salmonella enterica serovar Typhi (S. typhi) is a well-recognized cause of enteric fever both in developed and developing countries. The most important characteristic of S. typhi is its resistance to multiple antibiotics, including the most common conventional antityphoid agents: chloramphenicol, cotrimoxazole and ampicillin (1). Resistance to ciprofloxacin, the effective fluoroquinolone drug for typhoid fever, among the multidrug resistant (MDR) S. typhi has also been reported from different parts of the world in an increasing rate (2, 3). In the era of MDR typhoid fever, cephalosporins of third generation are the choice of antibiotic against S. typhi infection (4). But after a course of monotherapy, ciprofloxacin-resistant enteric fever becomes unresponsive also to the cephalosporins (5). In such a background, the management of MDR S. typhi infection becomes a great challenge for the clinicians because of the lack of suitable effective alternative regimens. In the present study, in order to combating drug resistance, we evaluated the activity of ciprofloxacin in vitro against S. typhi isolates in combination with cefazolin, an antibiotic that alone is ineffective in typhoid fever.

MATERIAL AND METHODS
Bacterial strains
A total of 16 blood culture S. typhi isolates, which did not respond to ciprofloxacin (MICs of 0.5-1.25 µg/ml) during treatment (1999-2001), were studied. 
Determination of cefazolin MICs

The MICs of cefazolin for the isolates were determined by agar dilution method following the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (6) using Mueller-Hinton agar (MHA) [Hi-Media, Mumbai, India] plates containing different concentration of cefazolin ranging from 0.25-75 µg/ml. Each of the antibiotic mixed agar plates was divided into 16 equal sectors and inoculated with approximately 10⁴ CFU/spot. The plates were then incubated at 37⁰ C for 24 h. The MIC was defined as the lowest concentration of antibiotic at which there was no visible growth. Hazy growth and one or two colonies on the spot were ignored.

Cefazolin-ciprofloxacin combination
The study of combined antimicrobial activity of ciprofloxacin and cefazolin against 16 S. typhi isolates was carried out by checkerboard agar dilution method (7). This method utilized an inoculum of approximately 10⁴ CFU/spot on MHA plates mixed with the two antibiotics in combination at eight different concentrations, which were 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2 and 0.25 µg/ml for ciprofloxacin, and 0.125, 0.25, 0.5, 1, 1.25, 2, 2.5 and 3 µg/ml for cefazolin. The FICs were derived from the lowest concentration of antibiotic combination permitting no visible growth of the test organisms on MHA plates after an incubation for 24 h at 37⁰ C, and the FIC indices were calculated. Synergy was defined as the FIC index = 0.5 and addition as an FIC index 0.5-4 (8).

For one isolate (B225), for which synergistic interaction with the ciprofloxacin-cefazolin combination has been proved by checkerboard agar dilution method, bacterial killing studies were also carried out to measure changes over different times. The combination of one-quarter the MIC was applied: 0.1875 µg/ml for ciprofloxacin and 15 µg/ml for cefazolin. Overnight grown bacterial suspension was diluted to approximately 5 × 10⁵ CFU/ml in fresh Mueller-Hinton broth [Hi-Media, Mumbai, India] containing antibiotics (in combination or alone) or no antibiotic. The samples were removed at 0, 3, 6 and 24 h and were diluted and plated on MHA. The agar plates were incubated at 37⁰ C for 24 h and CFU were counted. Synergism was defined as a =2 log10 decrease in CFU/ml between the combination and its most active compound (8). 

Statistical analysis
Correlation coefficient between the activity of ciprofloxacin by itself and in combination with cefazolin against S. typhi isolates was assessed with a simple regression analysis program. Test of significant difference from zero of the correlation coefficient was calculated at 5% level for 14 degrees of freedom using t-test and the difference was considered significant if the calculated value of t was greater than table value of t.

RESULTS
The MICs and FICs of ciprofloxacin and cefazolin, and FIC index for 16 S. typhi isolates are represented in the Table 1. Fourteen, out of 16 isolates, were sensitive to cefazolin showing MICs of 2.5-25 µg/ml; the remaining two were resistant (MIC value 45 and 60 µg/ml). FICs of ciprofloxacin for the isolates, which showed ciprofloxacin MICs of 0.5-1.25 µg/ml, ranged 0.075-0.25 µg/ml. The two cefazolin resistant isolates showed FICs of 2.5 µg/ml, while that of the sensitive isolates were 1.25-2.5 µg/ml. FIC index for 9 isolates ranged 0.208-0.5; 0.512-0.916 for the remaining 7 isolates. Correlation coefficient between combined ciprofloxacin-cefazolin and cefazolin alone was 0.312. The equation for regression: FIC of ciprofloxacin = 0.0956 + 0.085 × MIC of ciprofloxacin.
The results of the Time-kill studies are shown in Fig. 1. The combination of ciprofloxacin and cefazolin was inhibitory after 3 h of incubation. After an incubation for 24 h the bacterial cell count was reduced by 2.329 log10 CFU/ml. Ciprofloxacin (0.1875 µg/ml) and cefazolin (15 µg/ml) did not show bactericidal activity against the isolates, when used alone.

DISCUSSION
Several authors studied the combination effect of quinolone and non-quinolone antibiotics against many bacterial isolates excluding S. typhi, and reported the results in terms of FIC index (9, 10). Ciprofloxacin was found more active in combination with amoxycillin within the clinically achievable concentrations against S. typhi in vitro, and the combinations showed additive effects (11). In this communication, we completed an evaluation of the activity of a combination between ciprofloxacin and cefazolin against blood culture isolates of S. typhi isolates showing high MICs (0.5-1.25 µg/ml) for ciprofloxacin. This combination resulted both synergistic and additive effects. For isolates with high MICs of cefazolin synergy was demonstrated exerting very low FICs of cefazolin, and FIC index = 0.5. For those isolates showing low cefazolin MICs the combination showed additive effects.

Jesudason et al. (12) showed an increasing trend of MICs of cefazolin for S. typhi strains isolated during 1999. Saha et al. (5) reported about the emergence of S. typhi isolates showing resistance to third generation cephalosporins. Thus, in S. typhi there has been the possibility of extending cephalosporin resistance from the first to third generation. Moreover, ciprofloxacin treatment failure of typhoid fever due to the infection of MDR S. typhi is a common phenomenon (2, 3). The acquisition and spread of multidrug resistance among S. typhi isolates constitute a major threat in modern medicine. Therefore, to study the effect of antibiotic usage in combination on bacterial populations is a most important experiment in the field of medical practice and public health in combating resistance to newly introduced antibiotics to the bacterial populations. Thus, the present findings suggest for the introduction of combined chemotherapy in the treatment of MDR typhoid fever. But until the underlying mechanisms responsible for the synergistic interactions are elucidated, the information of the present study may be useful for the empiric clinical management of typhoid fever patients.

Reference:
1. Mandal S, Deb Mandal M, Pal N K. Antibiotic resistance pattern of Salmonella typhi isolates in Kolkata, India during 1991-2001: a retrospective study. Jpn J Infect Dis 2002; 55: 58-59.
2. Murdoch D A, Banatvala N A, Bone A, Shoismatulloev B I, Ward L R, Threlfall E J. Epidemic ciprofloxacin resistant Salmonella typhi in Tajikistan. Lancet 1998; 351: 339. 
3. Lathi N, Sudarsana J. Changing sensitivity pattern of Salmonella typhi in Calicut. Calicut Medical Journal 2004; 2(1): e2. URL: http://www.calicutmedicaljournal.org/2004/2/1/e2/index.html  
4. Christopher M, Parry M B, Hien T T, Dougan G, White N J, Farrar J J. Typhoid Fever. New Eng J Med 2002; 347: 1770-1782.
5. Saha S K, Talukder S Y, Islam M, Saha S. A highly ceftriaxone-resistant Salmonella typhi in Bangladesh. Pediatr Infect Dis J 1999; 18: 387.
6. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically; approved standards-fourth edition. M7-A4. Wayne (PA): The Committee; 1997.
7. Krogstad D J, Moellering R C. Combinations of antibiotics, mechanisms of interaction against bacteria. In: Lorian V, editor. Antibiotics in Laboratory Medicine. Baltimore: Williams & Wilkins; 1980. p. 298-341.
8. Leclercq R, Bingen E., Su Q H, Lambert-Zechovski N, Courvalin P, Duval J. Effects of combinations of ß-lactams, daptomycin, gentamycin and glycopeptides against glycopeptide resistant Enterococci. Antimicrob Agents Chemther 1991; 35: 92-98.
9. Huovinen P, Wolfson J S, Hooper D C. Synergism of trimethoprim and ciprofloxacin against clinical bacterial isolates. Eur J Clin Microbiol Infect Dis 1992; 11: 255-257.
10. Neu H C. Synergy of fluoroquinolones with other antimicrobial agents. Rev Infect Dis 1989; 2 (Suppl. 5): S1025-S1035.
11. Mandal S, Deb Mandal M, Pal N K. In vitro efficacy of ciprofloxacin alone and in combination with amoxycillin against Salmonella typhi isolates. Indian J Exp Biol 2003; 41: 360-362. 
12. Jesudason M, Jeyaraj L K. 2000 Increasing minimum inhibitory concentration of cefazolin for Salmonella typhi. Indian J Med Res 2000; 112: 183-185.



Table 1. Effect of ciprofloxacin (Ci)-cefazolin (Cz) combination against S. typhi isolates.

MIC, Minimum inhibitory concentration; FIC, Fractional inhibitory concentration.

Figure legend:

Fig. 1. Time-kill curves for a clinical isolate of S. typhi with ciprofloxacin (Ci) and cefazolin (Cz). The control was antibiotic-free medium.

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