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The rate of resistance against sulfonamides among community acquired uropathogens has increased in recent years. Therefore, there is a need of monitoring the resistant rates of commonly used sulfonamides for the treatment of urinary tract infections. In the present study, antibiotic resistance patterns of two commonly used sulfonamides antibiotics; sufisoxazole and TMP-SXT; were determined in order to evaluate the emergence of antibiotic resistance among uropathogens. It was observed E. coli displayed lower resistance rates against sulfisoxazole (3.3%) and trimethoprim-sulfamethoxazole (TMP-SXT) (2.6%) and only 2% (1/51) isolates of K. pneumoniae were resistant to trimethoprim-sulfamethoxazole. In case of Gram positive bacteria 4.4% (3/68) isolates of S. saprophyticus were resistant to sulfisoxazole and 1.5% (1/68) isolates of S. saprophyticus were resistant to trimethoprim-sulfamethoxazole.
Sufisoxazole, trimethoprim-sulfamethoxazole (TMP-SXT), K. pneumoniae, S. saprophyticus, E. coli.
The sulfonamides, sulfa drugs, are synthetic antimicrobial agents that contain sulfonamide group. These act as enzyme inhibitors and exhibit a bacteriostatic effect (Nester et al., 2004). The representatives of this group of antibiotic used in the present study were sulfisoxazole and trimethoprim-sulfamethoxazole (TMP-SMX). Treatment of acute uncomplicated UTI with first-line agents has traditionally involved a 3-day regimen of trimethoprim-sulfamethaxazol (TMP-SMX) or trimethoprim (TMP) alone for patients with sulfa allergies (Warren et al., 1997). However, trimethoprim itself can cause hypersensitivity and rashes that are erroneously ascribed to sulfa (Alonso et al., 1992).
Trimethoprim-sulfamethoxazole is also the drug of choice for the treatment of UTI in women during breastfeeding (Scholar and Pratt, 2000). In the southeastern and western United States, including southern California, resistance to TMP-SMX has become widespread and is detected up to 18% in pathogens isolated from urine samples of women with acute cystitis (Manges et al., 2006). Concentration of this drug in the urinary tract is excellent. However, use of this drug in pregnancy and neonates under one month of age is contraindicated (Iravani, 1991). The TMP-SMX has occupied a central role for the treatment of various infections. However, changing resistance patterns have led to the need of carefully redefined the appropriate use of this drug. Although, the additional role of the TMP-SMX has been modified by increasing resistance, it remains a highly useful alternative to the new generation of expended spectrum agents. Trimethoprim-sulfamethoxazole continues to be the drug of choice for the several clinical indications (Masters et al., 2003). The rate of resistance to TMP-SMX among community acquired uropathogens has increased in many years. It is thought that increase in TMP-SMX resistance is associated with poor bacteriological and clinical outcomes when it is used for the therapy (Gupta et al., 2001).
MATERIALS AND METHODS
Mueller-Hinton agar (MHA) (Merck) was used as antibiotic susceptibility test medium and Mueller-Hinton broth (MHB) (Merck) was used for preparation of inoculum.
Preparation of plates
The plates of 100 mm diameter were used for antibiotic susceptibility test. MHA (20 ml) was poured into sterile petriplates to get a depth of 4-6 mm. All the plates were incubated for 24 hours to check sterility.
Different antibiotic discs (Table 1) were used for antibiotic susceptibility test.
Preparation of 0.5 McFarland Nephelometer Standard
McFarland tube number 0.5 was prepared by mixing 0.5 ml 1.175% barium chloride solution and 99.5 ml 1% sulphuric acid solution.
Four to five colonies from pure growth of organisms were transferred to 5 ml MHB. The broth was incubated at 37oC for 18 - 24 hours. The turbidity of the culture was compared to 0.5 McFarland turbidity standard. The standardized inoculum was inoculated within 15 - 20 minutes.
Inoculation of medium
A sterile cotton swab was immersed into the standardized inoculum. Excess broth was drained off by pressing and rotating the swab against the wall of tube. It was streaked evenly in three directions on the surface of agar plate. A final circular motion was made around the agar rim with the cotton swab. These plates were allowed to dry for 3-5 minutes.
Antibiotics discs were placed on the surface of inoculated plates by using a sterile forceps. After placement the discs were pressed gently to the agar surface. The inoculated plates with discs were incubated at 35-37oC for 18-24 hours.
Inhibition zone diameters were measured in mm and the susceptibility or resistance of the organisms were interpreted on the basis of criteria mentioned in Table 1.
RESULTS AND DISCUSSION
Results of the resistance rates of Gram negative and Gram positive bacteria to sulfisoxazole and TMP-SXT are shown in Table 2. The rate of resistance against sulfisoxazole was high (12/534; 2.2%) as compared to TMP-SXT (9/534; 1.7%). In contrary, in a study the resistance patterns of Gram-negative bacilli were evaluated by Kim et al., (2008). It was found that 73.9% isolates of Gram-negative bacilli were susceptible to TMP-SXT. In another study, TMP-SXT was found to be effective against Gram-negative organisms showing 55 % efficacy (Gul et al., 2004). The data about resistance pattern of urinary pathogens against sufisoxazole is lacking in the literature.
In the present study, sulfisoxazole and TMP-SXT were found effective against most of the Gram negative as well as Gram positive isolates. It was observed from the antibiotic resistance pattern of E. coli that among sulfonamides i.e. sulfisoxazole and TMP-SXT displayed lower resistance rates as 3.3% and 2.6% respectively. These results are not in correlation with the findings of Kim et al. (2008) who reported resistance rate against TMP-SXT i.e. 37.9% in 2002 and 26.1% in 2006. In some other studies resistance to TMP-SXT against E. coli was 92.8% (Sahm et al., 2001), 66.1% (Astal et al., 2002), 14.8% to 17% from 1995 to 2001 (Kawlowsky et al., 2002), >25% (Raka et al., 2004), 63.9% (Elmanama et al., 2006) and 23.3% (Anatoliotaki et al., 2007). While among Gram positive bacteria, only 4.4% (3/68) isolates of S. saprophyticus were resistant to sulfisoxazole. Next to this antibiotic was TMP-SXT, against which 1.5% (1/68) isolates of S. saprophyticus were resistant.
The emergence of antibiotic resistance in the management of urinary tract infections is an important public health issue (Mordi and Erah, 2006). Instead of using narrow spectrum antibiotics, the use of broad spectrum antibiotics is a reason for increasing resistance of bacteria to antibiotics (Akram et al., 2007). There are considerable geographioc variations and bacterial patterns and resistance properties depending on local antimicrobial prescription practices (Yuksel et al., 2006). Trends of increase antibiotic resistance and dissemination of antibiotic resistant strains of uropathogens have shown the necessity of keeping up the monitoring of antibiotic resistance. By keeping in view these facts, antibiotic resistance patterns of commonly used antibiotics; sufisoxazole and TMP-SXT; were determined in order to evaluate the emergence of antibiotic resistance among uropathogens.
TABLE 1: CRITERIA FOR THE INTERPRETATION OF ANTIBIOTIC RESISTANCE/SUSCEPTIBILITY
Antibiotics disc code potency Inhibition zone diameter in mm
(g) Resistant Intermediate Susceptible
Sulfisoxazole S 300 ≤ 12 13-16 ≥ 17
TMP-SXT TS 5 ≤ 10 11-15 ≥ 16
TABLE 2: ANTIBIOTIC RESISTANCE PATTERN OF BACTERIA
Organisms No. of isolates Percentage of isolates resistant to
E. coli 270 3.3 (9) 2.6 (7)
K. pneumoniae 51 0 (0) 2.0 (1)
K. ozaenae 03 0 (0) 0 (0)
P. aeruginosa 10 0 (0) 0 (0)
P. mirabilis 05 0 (0) 0 (0)
S. marcescens 02 0 (0) 0 (0)
S. typhi 01 0 (0) 0 (0)
S. paratyphi A 02 0 (0) 0 (0)
S. paratyphi B 01 0 (0) 0 (0)
S. aureus 87 0 (0) 0 (0)
S. saprophyticus 68 4.4 (3) 1.5 (1)
S. haemolyticus 08 0 (0) 0 (0)
M. varians 13 0 (0) 0 (0)
M. lylae 07 0 (0) 0 (0)
M. roseus 03 0 (0) 0 (0)
M. sedentarius 02 0 (0) 0 (0)
M. halobius 01 0 (0) 0 (0)
Total 534 2.2 (12) 1.7 (9)