Abstract: In this study, the sensitivity of 100 P. aeruginosa isolates to ciprofloxacin has been investigated by determining minimum bactericidal concentration. The susceptibilities of isolates to other antibiotics were tested using agar disk diffusion method. The isolates contains 67 isolate from urine, 19 isolate from wound, 10 isolate from sputum, two isolate from blood, one isolate from stool and one isolate from ear. Thirty-five percent of isolates showed resistance to 10 antibiotics and also to ciprofloxacin and were sensitive only to imipenem. Thirty-five antibiotypes were recognized for all the isolates. The rates of resistances were determined to antibiotics as follows: gentamicin 49%, ticarcillin 100%, ceftizoxime 78%, co-trimoxazole 97%, amikacin 35%, carbenicillin 65%, ceftriaxone 65%, piperacillin 53%, imipenem 2%, kanamycin 65% and ofloxacin 71%. Fifty-five percent of isolates were resistant to ciprofloxacin. In conclusion, ciprofloxacin-resistant P. aeruginosa has been frequent in our clinical isolates, this data remind the worldwide emerging resistance against ciprofloxacin and this is a serious problem in therapeutic management of P. aeuroginosa infections and has a local and worldwide concern.
INTRODUCTION
Pseudomonas aeruginosa is an opportunistic gram negative bacilli and one of the most important causes of nosocomial infections especially in patients with burns, cystic fibrosis and neutropenia. P. aeruginosa shows high resistance to different classes of antimicrobial agents (Algun et al., 2004).
P. aeruginosa is an opportunistic pathogen found along with other Pseudomonas sp. as part of the normal flora of the human skin (Larson et al., 2002). When the host is immunocompromised, this opportunistic bacterium can quickly colonize and infect the burn and wound sites. Since P. aeruginosa can rapidly disseminate from the wounds into other organs via the bloodstream and can produce a number of virulence factors, the clinical outcome in these patients can lead to sepsis which is often fatal. In case studies of burn patients who developed P. aeruginosa septicemia, the mortality rate was more than 75% (Holder, 1985; Wurtz et al., 1995). Antibiotics, with the exceptions of the fluoroquinolones, are generally ineffective against most serious infections by P. aeruginosa. The introduction of fluoroquinolones offers a promising effective therapy of these types of infections caused by P. aeruginosa but unfortunately, the numbers of isolates of P. aeruginosa which have developed drug resistance to fluoroquinolones have increased rapidly in recent years (Dale et al., 2004).
Ciprofloxacin is a commonly used antibiotic in clinical practice (Chaudhry et al., 1999). It is a broad-spectrum fluoroquinolone with coverage against most gram-positive and gram-negative organisms, including Pseudomonas aeruginosa (Chaudhry et al., 1998; Snyder and Katz, 1992; Knauf et al., 1996; Kunimoto et al., 1999). This study was conducted to examine the susceptibility of clinical isolates of P. aeruginosa to ciprofloxacin.
MATERIALS AND METHODS
Bacterial strains and culture media: A total of 100 isolates were collected from Clinical specimens (wound, urine, sputum, blood, stool and ear) submitted to hospital diagnostic laboratories in Urmia/Iran from June to September 2005. The isolates were further processed by the standard methods to identify as P. aeruginosa. Isolated bacteria were maintained for long storage on skimmed milk medium (BBL) by adding 10% glycerol in -60° C, cultures were maintained for daily use on Nutrient agar slants on 4°C.
Preparation of ciprofloxacin powder: Ciprofloxacin powder was kindly provided by Exir pharmaceutical company, Tehran, Iran. The pure content of active ciprofloxacin was 96% in the provided powder.
Determination of antimicrobial activity of ciprofloxacin: For determining of the bacterial isolates sensitivity to ciprofloxacin, classic broth dilution susceptibility test were used (Sahm and Weissfeld, 2002). Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of isolates to ciprofloxacin were determined. The initial concentration of antibiotic in the first tube was 52 μg mL-1, this solution was diluted serially in 8 steps. 1.5x108 inoculums of the isolates were added to each concentration of ciprofloxacin in Muller Hinton Broth (MHB). A tube containing growth medium without ciprofloxacin and an un-inoculated tube were used as a positive and negative growth control respectively. Antibacterial activity was measured by determining MBC by culturing on MHA medium in a sterile Petri dish. Five microlitre of each tube streaked on MHA plates, the highest dilution that inhibits bacterial growth on MHA after overnight incubation was taken as MBC (Sahm and Weissfeld, 2002). In vitro resistance was defined as MBC of 4 or more μg mL-1 for isolates of P. aeruginosa (Chaudhry et al., 1999).
Determination of the isolates sensitivity to antibiotics: The susceptibilities of isolates to different antibiotics were tested using agar disk diffusion method (Bauer et al., 1966) with NCCLS breakpoints. P. aeruginosa ATCC27853 was used as reference strain. To represents the different classes of antimicrobial agents commonly used for the treatment of P. aeruginosa infections, we used Piperacillin (100 mcg), gentamicin (10 mcg), ofloxacin (5 mcg), ticarcillin (75 mcg), kanamycin (30 mcg), imipenem (10 mcg), amikacin (30 mcg), co-trimoxazole (1.25/23.75 mcg), ceftizoxime (30 mcg), ceftriaxone (30 mcg), carbenicillin (100 mcg) (Hi-media, Mombay, India).
RESULTS
A total of 100 P. aeruginosa isolates were collected from clinical specimens submitted to the hospital clinical microbiology laboratories of selected hospitals in Urmia, Iran. The frequency of isolates from each kind of clinical specimen has been shown in Table 1.
Sensitivity of bacterial isolates to ciprofloxacin: As shown 50 isolates (50% of all isolates) were resistant and the other isolates were sensitive to ciprofloxacin (Table 2).
| Table 1: | Types of specimens obtained from patients with P. aeruginosa infections |
| Table 2: | The average of Minimum bactericidal concentrations (MBC)* of ciprofloxacin for 100 isolates of P. aeruginosa |
| *: MIC amount for each isolate was equivalent to MBC, R: Resistant, S: Sensitive | |
| Table 3: | The rates of resistance to different antibiotics for 100 clinical isolates of P. aeruginosa |
| Ti: Ticarcillin, Ck: Ceftizoxime, Co: Co-trimoxazole, Ak: Amikacin, Cb: Carbenicillin, Cf: Ceftriaxone, Pip: piperacillin, I: Imipenem, K: Kanamycin, Of: Ofloxacin | |
Sensitivity of bacterial isolates to antibiotics: Thirty-five percent of isolates showed resistance to 10 antibiotics in adding to ciprofloxacin and were sensitive only to imipenem. Thirty five antibiotypes were recognized for all the isolates. The rates of resistances were showed in Table 3.
DISCUSSION
The data obtained from this research indicate that the prevalence of resistance of P. aeruginosa isolates to tested antibiotics was relatively high. Increasing resistance to the various anti-pseudomonas agents has been reported worldwide and this poses a serious problem in therapeutic management of P. aeuroginosa infections (Carmeli et al., 1999; Obritsch et al., 2004). Approximately 65% of P. aeruginosa isolates in this study were multi-drug resistant strains, however the prevalence of multi-drug resistance isolates of P. aeruginosa in the other studies are much lower than our study (Ohara et al., 2007).
Ciprofloxacin is a bactericidal, rapidly acting antimicrobial agent with a wide spectrum and is very effective against many gram negative bacterial pathogens, its effect against gram negative bacilli, including P. aeruginosa, is one of the most important features of this antibiotic (Algun et al., 2004).
In the present study, 50% of this isolates were resistant to ciprofloxacin (Table 2).
In another study has been done on 2067 clinical isolates of P. aeruginosa in United kingdom the resistance amount of isolates to ciprofloxacin were 7.3% (Lambert, 2002). In another survey has been done on Pseudomonas aeruginosa isolated from burn patients at two hospitals of Tehran, Iran in 2003, the resistance amount of isolates to ciprofloxacin were 86.7% (Shahcheraghi et al., 2003), however the higher resistance of burn isolates must be considered.
Chaudhry et al. (1999) reviewed in vitro sensitivities of ocular isolates of P. aeruginosa between July 1991 and September 1998. They found that only nine of 423 (0.021%) ocular isolates of P. aeruginosa were resistant to ciprofloxacin.
Reports from the United States have shown dramatic increases in antimicrobial resistance to ciprofloxacin (from 15 to 32%) however the overall incidence of ciprofloxacin resistance among P. aeruginosa isolates has been reported to range between 30 and 40% (Jones et al., 2002; Van Eldere, 2003).
In conclusion the present study is important from a practical point of view. Ciprofloxacin-resistant P. aeruginosa has been frequent in our clinical isolates, however there is not any burn isolates in our research (Table 1). This data and the previously reported by others (Kunimoto et al., 1999) remind the worldwide emerging resistance against ciprofloxacin. If the present trend continues, clinicians may encounter more frequent ciprofloxacin resistance among common P. aeruginosa isolates and this has a local and worldwide concern.
ACKNOWLEDGMENTS
This study has been supported by a research grant from Urmia Medical Sciences University. We thank Dr. Shahram Shahabi and Mrs. Minoo Zartoshti for their kindly helps.