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35 Antibiotic Resistant patterns in MRSA Tanaffos (2004) 3(11), 37-44 2004 NRITLD, National Research Institute of Tuberculosis and Lung Disease, Iran Antibiotic Resistant Patterns in MRSA Isolates from
Patients Admitted in ICU and Infectious Ward
Parviz Vahdani 1, Mahnaz Saifi 2, Mohammad Mehdi Aslani 2, Ahmad Ali Asarian 1, Kamran Sharafi 1
1 Department of Infectious Diseases, Loghman Hakim Hospital, Shaheed Beheshti University of Medical Sciences and Health Services, 2 Department of Microbiology, Pasteur Institute of Iran, TEHRAN-IRAN ABSTRACT Background: Methicillin- Resistant Staphylococcus Aureus (MRSA) has become one of the highest – ranking hospital
acquired pathogens throughout the world, capable of causing a wide range of hospital infections. Staphylococcus aureus is a major nosocomial pathogen that causes a range of diseases, including endocarditis, osteomyelitis, pneumonia, toxic shock syndrome, food poisoning, carbuncles, and boils. Materials and Methods: One hundred S.aureus isolates recovered from patients in Loghman Hakim hospital were included
in this study. Minimum inhibitory concentration (MIC) of strains for methicillin was determined by broth macrodilution method as recommended by NCCLS. Antibiotic susceptibility was tested by using the “disk diffusion technique on Mueller-Hinton Agar”. Nineteen antibiotics were tested including Ampicillin, Penicillin, Cephalexin, Cefepime, Gentamicin, Doxycycline, Erythromycin, Chloramphenicol, Tetracycline, Nitrofurantoin, Kanamycin, Amikacine, Cefotaxime, Clindamycin, Cefazolin, Amoxicillin, Sulfamethoxazole-trimethoprim, Vancomycin, and Ciprofloxacin. Results: The MIC range for methicillin was from 1µg/ml to 1024µg/ml. Ninety percent of the isolated strains had methicillin
MIC≥ 16µg/ml and were designated as resistant. Vancomycin and Chloramphenicol were the most effective antibiotics and only 7% and 14% of the isolates were resistant respectively. Forty-four percent hospital acquired MRSA strains were resistant to Co-trimoxazole. The high antibiotic resistance among MRSA strains could be originated due to widespread use of Conclusion: Out of 90 MRSA isolates characterized in this study, approximately half of them displayed resistance to one or
more antimicrobial agents, including Penicillin, Cephalosporins, Tetracycline and aminoglycosides. These data are in accord with previous study suggesting use of these drugs was important in the emergence of antimicrobial resistance in MRSA. In addition, 66% of MRSA isolates were sensitive to Trimethoprim-Sulfamethoxazole (Co-Trimoxazole). Since this drug combination is recommended for treating a range of human infections, S.aureus isolates should be monitored for further emergence of Co-Trimoxazole resistance. (Tanaffos 2004;3(11): 37-44)
Key words: Staphylococcus aureus, MRSA, (Methicillin Resistant Staphylococcus Aureus), Antibiotic Resistant

Correspondence to: Vahdani P
Tel: +98-9123277456
Email address: parvizvahdani@yahoo.com

38 Antibiotic Resistant Patterns in MRSA Staphylococcus aureus is a major nosocomial MRSA has emerged as a significant cause of both pathogen that causes a range of diseases, including nosocomial and community acquired infections in endocarditis, osteomyelitis, pneumonia, toxic shock Iran now. In a recent study in Shiraz, Iran 37.7% of syndrome, food poisoning, carbuncles and boils (1). the isolates were methicillin – resistant and In the early 1950s acquisition and spread of beta – resistance to vancomycin or rifampin was not seen lactamase producing plasmids thwarted the (8). The aim of this study was to determine effectiveness of penicillin for treating S.aureus methicillin – resistant phenotype in isolated S.aureus infections. In the year 1959, methicillin, a synthetic and also to ascertain the susceptibility pattern of Since first reported by Jevons in 1961, methicilin- resistant staphylococcus aureus (MRSA) has been implicated as a pathogen in hospital-acquired The Staphylococcal infection was confirmed by infections causing endemic and epidemic infections clinical and paraclinical conditions. All strains were in health care centers world wide (2). The proportion isolated from patients in whom S.aureus was the sole of nosocomial infections caused by MRSA increased or predominant causative infectious agent. Skin, substantially, and MRSA is now a leading cause of wounds, sputum, and external ear were the potential sites for contamination; therefore, only the isolates Recent studies suggest that the epidemiology of from these sites were accepted where the S.aureus MRSA may be changing, as isolation of MRSA is no was the dominant pathogen. The isolates were from longer limited to hospitalized persons or persons with different parts of body. The number and percentage predisposing risk factors. MRSA infections as of isolated strains from different sites of body was emerging pathogens are responsible for substantial diseases and death (5, 6). While no satisfactory Isolated strains were identified by standard biochemical test. In brief, gram-positive cocci that explanation exists for the recent proliferation of were catalase and coagulase positive were identified MRSA, expanded use of antimicrobial drugs in outside the hospitals has been suggested as a major Antibiotic susceptibility was tested by using the contributor in emerging resistance in community (7). disk diffusion technique on Mueller-Hinton agar, Health care workers and infection control according to the procedures established by the personnel depend on the laboratory for the reliable “National Committee for Clinical Laboratory detection of MRSA in clinical specimens. This has Standards” (NCCLS). Plates were incubated at 37ºc implications for treatment, invasive infections, for 18h for antibiotics (9). The antibiotics tested were perioperative prophylaxis, and infection control Ampicillin, Penicillin, Cephalexin, Cefepime, procedures. Surveillance of MRSA locally, Gentamicin, Doxycycline, Erythromycin, nationally and globally depends on accurate Chloramphenicol, Tetracycline, Nitrofurantoin, laboratory reporting. Nosocomial MRSA strains in Kanamycin, Amikacine, Cefotaxime, Clindamycin, the community including nursing homes and other Cefazolin, Amoxicillin, Sulfamethoxazole– care facilities, may be transmitted by discharged Trimethoprim, Vancomycin, and Ciprofloxacin (B.BL, Becton Dickenson Microbiology system). Mueller-Hinton agar supplemented with 2% NaCl The MIC range for methicillin was from 1µg/ml and Methicillin (5µg/ml) (Sigma Co.St.Louis, USA) to 1024µg/ml. The MIC range was shown in table 2. was used for screening of MRSA. In this method, the Nearly 90% of the isolated strains had methicillin inoculum’s suspension was prepared by MIC≥ 16µg/ml and were designated as resistant. Ten microdilution method and inoculated with 104 cfu/ml percent of strains were methicillin–sensitive S.aureus strains. After 24 hours incubation at 35ºc the test plates were examined for any evidence of growth. Isolates were defined as resistant or sensitive The MIC at which 50% of isolates are inhibited, according to detecting “growth” or “no growth” on The MIC50 and MIC90 were 256 µg/ml and 16 µg/ml respectively. Otherwise, results obtained from plate Minimum inhibitory concentration (MIC) of method demonstrated 65 (72%) strains that had no strains for methicillin was determined by broth growth on methicillin plate. Thus, disk diffusion macrodilution method as recommended by NCCLS. method alone without MIC is not reliable. S.aureus ATCC 29213 was used as the control strain The antibiotic susceptibility patterns of MSSA and MRSA to nineteen antibiotics tested are shown in figure-1 and figure 2, respectively. Analyzing the The isolates were from different parts of body. antibiotic susceptibilities to the nineteen antibiotics The number and percentage of isolated strains from tested with the 90 isolates of MRSA showed 100% different sites of body was shown in Table 1. resistant to penicillin, 92% to ampicillin, and 93% to cefotaxime. Comparison of antimicribial resistance Table 1. The number and percentage of isolated strains from different
frequencies for S.aureus is shown in figure 2. Vancomycin and Chloramphenicol were the most effective antibiotics and only 7% and 14% of isolates Body sites
were resistant respectively. Nitrofurantoin, gentamycin, amikacine, ciprofloxacin and other cephalosporins like cefepim and cefazolin were at the second row. These antibiotics represented the second most effective agents. Our study showed that 44% of hospital acquired MRSA strains were resistant to co-
Table 2. The MIC range of methicillin – among isolated strains of methicillin resistant S.aureus. MRSA had MIC 16≥ µg/ml.
1024 512 256 128 64 32 16 8 4 2 1 0.5 0.25 - 40 Antibiotic Resistant Patterns in MRSA
Figure 1. Comparison of antimicrobial resistance frequencies for S. aureus.
P: Penicillin, E: Erythromycin, C: Chloramphenicol, Te: Tetracycline, FM: Nitrofurantoin, DO: Doxycycline, AM: Ampicillin, CN: Cephalexin, GM: Gentamicin, Va: Vancomycin, K: Kanamycin, AN: Amikacine, CTX: Cefotaxime, FEP: Cefepime, AMC: Amoxicillin, Clinda: Clindamycin, CZ: Cefazolin, SXT: Sulfamethoxazole- Figure 2. Comparison of antimicrobial resistance frequencies for Methicillin Resistant S. aureus.
P: Penicillin , E: Erythromycin, C: Chloramphenicol, Te: Tetracycline, FM: Nitrofurantoin, DO: Doxycycline, AM: Ampicillin, CN: Cephalexin, GM: Gentamicin, Va:
Vancomycin, K: Kanamycin, AN: Amikacine, CTX: Cefotaxime, FEP: Cefepime, AMC: Amoxicillin, Clinda: Clindamycin, CZ: Cefazolin, SXT: Sulfamethoxazole- co-trimoxazole versus intravenous vancomycin in During the past few years, news on MRSA have 101 cases of severe S.aureus infection, the authors usually been discouraging and clinicians and reported 100% cure rates for either drug in MRSA infection control practitioners appear to have lost infections, including bacteremia (15). More recently, confidence in their capability to control the hospital Stein et al. showed varying degrees of success in acquired spread of this pathogen. The number of treating with co-trimoxazole orthopedic implant papers focusing on the over whelming spread of infections caused by S.aureus. Unfortunately, this MRSA is increasing, whereas those addressing study did not distinguish MRSA from MSSA strains successful efforts of control or stating that hospital acquired spread of MRSA can and should be As we can see in this study, there is a significant controlled are few (1, 10, 11). A number of usefulness of chloramphenicol against MRSA. researchers debating the control of MRSA have However, it seems that by passing time and questioned whether controlling this microorganism is introduction of new drugs, this antibiotic is forgotten. reasonable, feasible or justified and whether the Soon after chloramphenicol was released in the tracing of colonized people are justified or not. There United States in 1949, reports linked this highly is a report from a university hospital and a medical- effective agent with aplastic anemia, and it quickly district-wide control policy for MRSA on the fell into disfavor. The increased awareness of the elimination of MRSA after the outbreak (12). pathogenicity of anaerobic organisms and the From the 90 MRSA isolates characterized in this development of ampicillin-resistant H. influenzae study, approximately half of them displayed accounted for a brief resurgence. However, the resistance to one or more antimicrobial agents, availability of other agents has dramatically reduced including penicillin, cephalosporins, tetracycline and the need for this antibiotic. Since it is effective, aminoglycosides. These data are in accord with readily available (often over the counter), and previous study suggesting use of these drugs has inexpensive, it is still used as first-line therapy for been a key factor in the emergence of antimicrobial enteric fever and other infections in many parts of the resistance in MRSA (8). In addition, 66% of MRSA world. In the United States and other developed nations, chloramphenicol remains as a useful sulfamethoxazole (co-trimoxazole). Since this drug antibiotic, but only as alternative therapy in seriously combination is recommended for treating a range of ill patients or for patients infected with highly human infections, S.aureus isolates should be antibiotic-resistant organisms. But unfortunately monitored for further dissemination of co- there is not any perfect and suitable study in this regard, and from this matter, we can conceive that Eventually, our data may favor the use of Co- MRSA must be resistant to chloramphenicol as well. Trimoxazole as a potentially cost effective antimicrobial drug for treating MRSA infections. Co- In this study, we report infections due to MRSA trimoxazole has been shown to be effective against strains with reduced susceptibility to vancomycin. In MRSA both invitro and invivo in mice, as well as in a study conducted in Shiraz, 100 percent of isolates clinical reports on meningitis, septicemia and were sensitive to vancomycin (8). In our study 7% of isolates were resistant to vancomycin. This report is In a controlled comparative trial of intravenous an early warning that S.aureus strains with full 42 Antibiotic Resistant Patterns in MRSA resistance to vancomycin might emerge in future. and epidemiologic observations. N Engl J Med 1968; 279
MRSA strains with reduced susceptibility to vancomycin in Japan and in the United States were 3. Emori TG, Gaynes RP. An overview of nosocomial isolated after prolonged exposure to antibiotics. infections, including the role of the microbiology laboratory. These strains were isolated from a patient treated Clin Microbiol Rev 1993; 6 (4): 428- 42.
with vancomycin for 75 days with teicoplanin for 3 4. Fluit AC, Wielders CL, Verhoef J, Schmitz FJ. days and/or the strains were isolated after 14 days of Epidemiology and susceptibility of 3,051 Staphylococcus vancomycin therapy (17, 18). In some cases, the aureus isolates from 25 university hospitals participating in strains might have been transmitted to the patient the European SENTRY study. J Clin Microbiol 2001; 39
from another patient who had undergone treatment for MRSA infections. Since in our study, the patient 5. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, does not have any history of antibiotic treatment with Gaskin RE, Boyle-Vavra S, Leitch CD, Daum RS. vancomycin or related antibiotics, these strains might Community-acquired methicillin-resistant Staphylococcus have been transmitted from other patients who were aureus in children with no identified predisposing risk. infected with vancomycin – resistant MRSA strains. JAMA 1998; 279 (8): 593- 8.
The high antibiotic resistance among MRSA strains 6. Naimi TS, LeDell KH, Boxrud DJ, Groom AV, Steward CD, could be originated due to widespread use of Johnson SK, Besser JM, O'Boyle C, Danila RN, Cheek JE, Osterholm MT, Moore KA, Smith KE. Epidemiology and Further phenotypic and genotypic studies are clonality of community-acquired methicillin-resistant needed to establish and clarify the genetic Staphylococcus aureus in Minnesota, 1996-1998. Clin Infect
mechanism behind reduced susceptibilities to Dis 2001; 33 (7): 990-6. Epub 2001 Sep 5.
7. Tenover FC. Development and spread of bacterial resistance to antimicrobial agents: an overview. Clin Infect Dis 2001;
ACKNOWLEDGMENT
8. Alborzi A, Pourabbas Ba, Salehi H, Pourabbas Bh, Oboodi supported by the Shaheed Beheshti University of B, Panjehshahin MR. Prevalence and pattern of Antibiotic sensitivity os methicillin-sensitive and methicillin-resistant Medical Sciences and Health Services and staphylococcus aureus in shiraz-Iran. Iranian J of Medical
coordinated by Microbiology Department of Pasteur Science 2000; Vol 25. Nos 1 & 2: 1-9.
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