Published by : PROFESSIONAL MEDICAL PUBLICATIONS
October - December 2008 (Part-I)
Study of methicillin resistance in staphylococcus aureus
and species of coagulase negative staphylococci
isolated from various clinical specimens
Manijeh Mehdinejad1, Ahmad Farajzadeh Sheikh2, Abbas Jolodar3
Objective: To identify the Methicillin Resistant Coagulase Negative Staphylococci (MRCONS) and Methicillin Resistant Staphylococcus aureus (MRSA) from clinical specimens and their antimicrobial susceptibility patterns.
Methodology: All strains were recovered from clinical specimens according to 0.5 McFarland standard inoculated on Muller Hinton agar. Antibacterial susceptibility was tested by disks diffusion method.
Results: Of 346 Staphylococci, 250 (72.25%) were identified as methicillin resistance. From these strains, ninety three (37.2%) were MRSA and 157 (62.8%) identified as MRCONS. The highest rate of MRCONS identified in S. haemolyticus and the lowest rate of MRCONS belonged to S. intermedius and S. schleiferi. The susceptibility pattern of MRSA and MRCONS against other antimicrobial agents revealed that the lowest resistance was for vancomycin.
Conclusions: The rate of MRCONS was more than MRSA and vancomycin was the most effective antibiotic against those organisms. As such, this antibiotic is the drug of choice for treatment in such cases.
KEY WORDS: Methicillin Resistance Staphylococcus aureus, Methicillin Resistant Coagulase Negative Staphylococci, Vancomycin.
Pak J Med Sci October - December 2008 (Part-I) Vol. 24 No. 5 719-724
How to cite this article:
Mehdinejad M, Sheikh AF, Jolodar A. Study of methicillin resistance in staphylococcus aureus and species of coagulase negative staphylococci isolated from various clinical specimens. Pak J Med Sci 2008;24(5):719-24.
1. Manijeh Mehdinejad,
Dept. of Microbiology,
School of Medicine,
Ahwaz Jondi Shapour University of Medical Sciences,
2. Ahmad Farajzadeh Sheikh,
Dept. of Microbiology,
School of Medicine,
Ahwaz Jondi Shapour University of Medical Sciences,
3. Abbas Jolodar
Faculty of Veterinary Medicine,
Shahid Chamran University of Ahwaz,
Dept. of Microbiology, School of Medicine,
Ahwaz Jondishapour University of Medical Sciences,
* Received for Publication: July 17, 2007
* Revision Received: March 17, 2008
* 2nd Revision Received: July 8, 2008
* Final Revision Accepted: July 12, 2008
Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Resistant Coagulase Negative Staphylococci (MRCONS) are prevalent worldwide and are an important cause of nosocomial infection, resulting in increased morbidity, mortality, length of hospital stay, and health care costs.1-3 As many as 60-70% of all clinical Coagulase Negative Staphylococci (CONS) and proximally 25% of S. aureus isolated from nosocomial infections are resistant to methicllin.4 As of early 2005, the number of deaths in the United Kingdom attributed to MRSA has been reported from various sources.5 The MRSA prevalence in Saudi Arabia has been reported as 26% among healthy adults.6 Its first epidemic was detected during outbreak in the Bellvitge hospital in Barcelona, Spain.7 CDC (Centers for Disease Control and prevention) has reported that health-care providers should be aware that MRSA can cause skin infections among healthy newborns.8 In the US hospitals, the prevalence of MRSA has increased from 2.4% in 1975 to 29% in 1991.9 Hospital connection was the single most important risk factor for the acquisition of MRSA.10
Some investigators have reported that 20-62% of patients colonized with the MRSA organism were detected within 48-72 hours after admission indicating a possible carrier state.11 It has also been reported that MRSA strains and MRCONS, share the same habitats and permanently or transiently colonize the anterior nares and further regions of skin and mucous membranes which may act as sources of subsequent bacteremia and other infections.12
Mechanism of methicillin resistance in S. aureus and CONS is due to production of an additional nonnative penicillin binding protein (PBP2a) which is encoded by the mecA gene.13 MRSA and MRCONS are cross resistance to other lactam antibiotics such as cephalosporins. These organisms are often resistant to other antibacterial agents such as: macrolides, lincosamides, aminoglycosides, fluroquinolos except vancomycin.14,15 In this study, MRSA and MRCONS species from clinical specimens were identified and their susceptibility patterns against some antimicrobial agents tested.
From August 2004 to December 2006 a total of 346 Staphylococci were recovered from various clinical sources (blood, pus, urine, wounds and thraco tube) of patients who were hospitalized and admitted at several wards to the Golestan Hospital in Ahwaz, Iran. All samples were cultured on blood agar and MacConkey’s agar plate (Himedia, India & Merck, Germany). These plates were incubated at 37oC for 24 hours; the isolates S. aureus were identified via colonial morphology on blood agar plates, gram stain characteristics, Catalase test, Coagulase Positive test, Mannitol fermentation, and the Coagulase Negative Staphylococci were identified for with using other necessary biochemical tests (produce acid from various sugar, susceptible to novobiocin, resistant to polymyxin B, and bacitracin test and etc.) according to standard microbiological procedures as various Staphylococci.16,17
Antibiotic susceptibility test:Antimicrobial susceptibility testing was done on Mueller-Hinton agar (Merck, Germany) using Disk diffusion (Kirby Bauer’s) technique, according to NCCLS guidelines.18,19 Suspension of each Staphylococcus was made to turbidity of 0.5 McFarland standards, and cultivated on Muller Hinton agar. The potency of methicilin was 5µg/disk and the zone of growth inhibition at least lower than 9 mm in diameter.19
Finally, the following drugs and concentrations (in brackets) were used to determine the antibiogram of the strains: Vancomycin (30µg). Cephalexi (30µg), Clindamycin (2µg), Erythromycin (15µg), Tetracycline (30µg), Streptomycin (10µg) and Penicillin (10µg), (Padtan Teb, Iran and Haimedia, India Company). All tests were performed on Muller-Hinton agar (Oxoid Co., Hampshire, UK) and were interpreted after incubation for 24 hours at 37oC. No supplemental NaCl was added to the agar medium. The zone diameters measured around each disk were interpreted on the basis of guidelines published by the NCCLS.18
Based on identification method, a total of 346 Staphylococci including 123 (35.54%) S. aureus and 223 (64.46%) as sixteen CONS species were identified. Table-I, represents the various strains MRCONS according to clinical specimens identified. The present study showed S. haemolyticus as the most prevalent MRCONS strains isolated from clinical specimens, while we could not find any S. intermedius or S. schleiferi strain to be resistant to methicillin.
The majority of MRSA (35.48%) belonged to specimens isolated from tracheo tube, whereas the majority of MRCONS (57.96%) was isolated from urine samples Table-II. The highest frequency of MRSA (39%) and MRCONS (29%) were seen among patients with age of 21-40 years.
The prevalence of MRSA and MRCONS among male patients was 55.91% and 56.68%, respectively. The majority of MRSA were seen in neurosurgery (30.10%) and outpatient (23.65%) wards. However, the highest rates of MRCONS were isolated from outpatient (24.84%), pediatric (15.92%) and neurosurgery (14.01%) wards. Moreover, we determined susceptibility of MRCONS and MRSA to other antibacterial agents. The highest resistance among MRCONS and MRSA were identified against penicillin the lowest resistance of MRSA (7.5%) and MRCONS (21.6%) were determined against vancomycin Table-III.
Based on antibiotic disk susceptibility pattern against methicillin on 346 Staphylococci including S. aureus and CONS (Distributed among 16 species), 250 (72.25%) were identified as methicillin resistance staphylococci (MRS). This included 37.2% S. aureus (MRSA) and 62.8% CONS (MRCONS). The percentage of MRSA and MRCONS reported in different countries in France (MRSA 31.5; MRCONS 71.0), Germany (MRSA 17.2; MRCONS 67.4), Greece (MRSA 36.6; MRCONS 83.3), Italy (MRSA 38.8; MRCONS 82.4) and Turkey (MRSA 30.9; MRCONS 74.4).20 This study revealed that the CONS are more resistance to methicillin than S. aureus. Our data related to MRSA percentages are in accordance with data from Greece and Italy. However, data which belongs to MRCONS are lower than from other countries. Isolation of MRCONS (70 to 80%) was reported by another investigator in central Europe which is entirely compatible with our data.21 The frequency rate of methicillin resistance among staphylococci in Sweden was 42.5%.20 Nevertheless, this rate was low in comparison with our study. Our data is in agreement with the Japoni et al., statement that acknowledged: "If the strategy for antibiotic usage is not changed, we should expect more than 60% of staphylococci isolated from patients be methicillin-resistant in the near future".22
In our study, the highest rate of MRSA and MRCONS were isolated from the neurosurgery, outpatients and pediatric wards. We suggested that the administrators must consider this issue and necessary measures relating to these wards should be taken.
The majority of MRCONS were S. haemolyticus, S. saprophyticus, S. epidermides, and followed by S. cohnii.spp S. capitis.spp, S. lugdunensis and S. hominis and the minority of them were S. intermedius and S. schleiferi.spp. However, Zilevica et al., reported that the majority of the MRCONS belonged to S. epidermidis, S. saprophyticus, S. haemolyticus, followed by S. hominis, S. warneri, S. capitis.23 In our data, except S. capitis, all MRCONS species had lower percentage than data reported by Caierao et al in 2004.24 The majority of MRSA were isolated from tracheo tube, whereas the majority of MRCONS belonged to specimens isolated from urine. Therefore, administration of methicillin antibiotic in patients who had urinary tract infections (UTI) with CONS should be reconsidered.
Since 1996, vancomycin-resistant MRSA strains have also been found.25 However, 0% resistance of MRSA and MRCONS was reported in the studies conducted by Alborzi et al.,26 Al-Haodad et al.27 and Zilevica.28 Methicilin Resistance of S. aureus and CONS strains may have an effect on the resistance of these strains against other antibiotics. As we have shown, the highest resistance rates of MRSA and MRCONS to other antibacterial agents were against penicillin. On the other side, the lowest resistance of MRSA and MRCONS was against vancomycin. In this study, the resistance of MRCONS against vancomycin (21.6%) was more than MRSA (7.5%). Perwaiz et al estimated the resistance rate of MRSA against penicillin, clindamycin, cephalexin, erythromycin over 90% that this percentage except for penicillin, is more than our data. However, our data regarding vancomycin was confirmed by this study.29 Several reports have stated that aminoglycoside resistance is closely related to methicillin-resistance.30,31 In our study the resistance rate of MRSA against tetracycline, streptomycin and erythromycin was lower than those reported by Al-Haodad.27 In another study, the resistance rate for these organisms against streptomycin (0%) and penicillin (87.2%) were reported.32
The early identification of methicillin resistance in Staphylococci, especially from blood isolates, could curtail unnecessary use of vancomycin and allow earlier optimal therapy of infections in few cases related to critically ill patients under the strict clinician supervision.
In this study, the rate of MRCONS was lower than other countries and vancomycin was the most effective antibiotic against MRCONS and MRSA.
This study was supported by a grant (321) from Jondi Shapour University of Medical Sciences, Ahwaz, Iran. The authors wish to thank Dr. Azar D. Khosravi, Dept. of Microbiology, School of Medicine, Ahwaz Jondi Shapour University of Medical Sciences, Ahwaz, Iran, for her help in the review.
1. Crossley K, Loesch D, Landesman B, Mead K, Chern M, Strate R. An outbreak of infection caused by strains of Staphylococcus aureus resistant to methicillin and aminoglycosides. I. Clinical studies. J Infect Dis 1979;139:273-9.
2. Rubin RJ, Harrington CA, Poon A, Dietrich K, Greene JA, Moiduddin A. The economic impact of Staphylococcus aureus infection in New York City hospitals. Emerg Infect Dis 1999;5:9-17.
3. Cosgrove SE, Sakoulas G, Perencevich EN, Schwaber MJ, KarchmerAW, Carmeli Y. Comparison of mortality associated with methicillin-resistant and methicillin- susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin Infect Dis 2003;36:53-9.
4. Chambers HF. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev1997;10:781-91.
5. Johnson AP, Pearson A, Duckworth G. "Surveillance and epidemiology of MRSA bacteraemia in the UK". J Antimicrob Chemother 2005;56(3):455-62.
6. Algaithy AA, Bilal NE, Gedebou M, Weily AH. Nasal carriage and antibiotic resistance of Staphylococcus aureus isolated from hospital and non-hospital personnel in Abba Saudi Arabia. Trans R Soc Trop Med Hyg 2000;94(5):504-7.
7. Dominguez MA, de Lencastre H, Linares J, Tomasz A. Spread and maintenance of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone during an outbreak of MRSA disease in a Spanish hospital. J Clin Microbiol 1994;32:2081-7.
8. From the Centers for Disease Control and Prevention: Morbidity and Mortality Weekly Report Community-Associated Methicillin-Resistant Staphylococcus aureus Infection among Healthy Newborns-Chicago and Los Angeles County, 2004. JAMA 2006;296(1):36-8.
9. Panlilio AL, Culver DH, Gaynes RP, Banerjee S, Henderson TS, Tolson JS, et al. Methicillin-resistant Staphylococcus aureus in U.S hospitals 1975-1991. Infect Control Hosp Epidemiol 1992;13:582-6.
10. Warshawsky B, Hussain Z, Gregson DB, Alder R, Austin M, Brucksch D, et al. Hospital and community-based surveillance of methicillin-resistant Staphylococcus aureus: previous hospitalization is the major risk factor. Infect Control Hosp Epidemical 2000;21:724-7.
11. Layton MC, Hierholzer WJ, Patterson JE. The evolving epidemiology of methicillin-resistant Staphylococcus aureus at a university hospital. Infect Control Hosp Epidemiol 1995;16:12-7.
12. Costa SF, Miceli MH, Anaissie EJ. Mucosa or skin as source of coagulase-negative staphylococcal bacteraemia. Lancet Infect Dis 2004;4:278-86.
13. Hackbarth C, Chambers HF. Methicillin-resistant staphylococci: genetics and mechanisms of resistance. Antimicrob Agents Chemother 1989;33:991-4.
14. Lyon BR. Skurray R. Antimicrobial resistance of Staphylococcus aureus: genetic basis. Microbiol Rev 1987;51:88-134.
15. Grubb WB. Genetics of MRSA. Rev Med Microbiol 1998;9:153-62.
16. Jorgensen JH, Turnidge JD, Washington JA. Antibacterial susceptibility tests: dilution and disk diffusion methods. In: Murray PR, Baron E J, Pfaller MA, Tenover FC, Yolken R H, editors. Manual of clinical microbiology. 7th ed. Washington, D.C: American Society for Microbiology; 1999;1526–43 and 2005;221-36.
17. Koneman EW, Alln SD, Janda MW, Schreckberger PC, Washington C.Winn, Jr. Colour Atlas and text book of Diagnostic Microbiology. 5th Ed. Philadelphia, New York, 1997;539-66.
18. Finegold SM, Martin WJ. Bailey & Scott’s Diagnostic Microbiology. Sixth ed. St. Louis: Mosby, 1982;545-9.
19. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard. 5th ed. NCCLS document M7-A5. National Committee for Clinical Laboratory Standards 2000;20(2):1-32.
20. Sader HS, Watters AA, Fritsche1 TR, Ronald N J. Daptomycin antimicrobial activity tested against methicillin-resistant staphylococci and vancomycin-resistant enterococci isolated in European medical centers 2005. BMC Infectious Diseases 2007;7:29.
21. Diekema DJ, Pfaller MA, Schmitz F J, Smayevsky J, Bell J, Jones R N, et al. Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997-1999. Clin Infect Dis 2001;32:114-32.
22. Japoni A, Alborzi AV, Rasouli M, Pourabbas B. Modified DNA Extraction for Rapid PCR Detection of Methicillin - Resistant Staphylococci. Iranian Biomedical J 2004;8(3):161-5.
23. Zilevica A, Tračevska T, Viesturs U. Biotechnological Methods for Precise Diagnosis of Methicillin Resistance in Staphylococci. Bioautomation 2005;2:54-64.
24. Caierao J, Musskopf M, Superti S, Roesch E, Dias1 C G, d’Azevedo PA. Evaluation of phenotypic methods for methicillin resistance characterization in coagulase-negative staphylococci (CNS) J Med Microbiology 2004;53:1195-9.
25. Centers for Disease control and Prevention [CDC]. Laboratory detection of: vancomycin-intermediate/resistant Staphylococcus aureus (VISA/VRSA) [online].CDC; 2005Feb. Available at: http://www.cdc.gov/ncidod/dhqp/ar_visavrsa_lab FAQ.html. Accessed 17 Apr 2006.
26. Alborzi A, Pourabbas B, Oboodi B, Panjehshahin M. Prevalence and pattern of antibiotic sensitivity of Methicillin-sensitive and methicillin-resistant Staphylococcus aureus in Shiraz-Iran. Iranian J Med Sci 2000;25:1-2.
27. AL- Haddad AM, Udo EE, Mokadas EM, Sanyal SC, Grubb WB. Persistence of a clone of methicillin - resistant Staphylococcus aureus in a burns unit. J Med Microbial 2001;50:558-64.
28. Zilevica A, Tracevska T, Vingre I, Paberza R. Identification of Methicillin Resistance in Staphylococci. Proc. of LU (LU Zin tniskie raksti), Medicine 2004;668:183-91.
29. Perwaiz S, Barakzi Q, Farooqi B J, Khursheed N, Sabir N. Antimicrobial susceptibility pattern of clinical isolates of Methicillin Resistant Staphylococcus aureus. J Pak Med Assoc 2007;57(1):2-4.
30. Busch SC, Frimodt MN, Miller GH, Espersen F. Aminoglycosid resistance among Danish blood culture isolates of coagulase-negative staphylococci. APMIS 1996;104:873-80.
31. Kim HB, Kim T, Lee BB, Kim US, Park SW, Shin JW, et al. Frequency of resistance to aminoglycoside antibiotics in Staphylococcus aureus isolates from tertiary hospitals. Korean J Infect Dis 2002;34:39-46.
32. Beth E, Domaracki, Evans AM, Richard A, Venezia. Vancomycin and Oxacillin Synergy for Methicillin-Resistant Staphylococci. Antimicrob Agents Chemother 2000;44:1394-6.
HOME | SEARCH | CURRENT ISSUE | PAST ISSUES
Room No. 522, 5th Floor, Panorama Centre
Building No. 2, P.O. Box 8766, Saddar, Karachi - Pakistan.
Phones : 5688791, 5689285 Fax : 5689860