Antimicrobial susceptibility, virulence factors and biofilm formation among Staphylococcus aureus strains from hospital infections in Kerman, Iran

Öz

Amaç: Bu çalışma hastane kaynaklı Staphylococcus aureus suşlarının antimicrobial duyarlılık, virülans faktör ve biyofilm oluşturma özelliklerini belirlemeyi hedeflemektedir. Metotlar: İran, Kerman’da 30 adet, tekrarlamayan S. aureus suşu hastanelerden izole edildi. Antimicrobial duyarlılıklar disk diffüzyon metoduyla CLSI rehberinin sınırdeğerlerine göre çalışıldı. Vankomisin ve metisilin için minimum inhibitor konsantrasyonu (MİK) sıvı mikrodilüsyon ve E-test prosedürleri ile belirlendi. Bu suşlarla birlikte olan virülans faktörleri (proteaz, DNaz, lesitinaz, kapsül ve hemoliz) çalışıldı. Biyofilm değerlendirmesi mikrotitre tekniği ile miktarı belirleme şeklinde yapıldı. Bulgular: Toplam olarak 14 (% 46, 7) S. aureus suşu alt solunum yollarından, altı suş (% 20,0) üriner sistemden ve kalan 10 suş (% 33,3) yaralar, kan ve ortopedi hastalarından izole edildi. Bütün suşlar tigesikline duyarlıydı, sekiz suş (% 26,7) metisiline dirençli (MRSA) bulundu ve dört suş (% 13,3) vankomisine azalmış duyarlılık gösterdi. Suşların hiç biri vankomisine dirençli değildi (p≤0.05). MİK sonuçları; vankomisin için suşlardan dördünde (% 13,3) MİK 4 µg/mL olarak bulunurken beşinde (% 16,6) metisilin için MİK 32 µg/mL olarak bulundu. Suşlar aynı zamanda amoksisilin/klavulanik asit, tekrasiklin ve tobramisine dirençli idiler. MSSA suşları ile karşılaştırıldığında MRSA suşlarından 6’si (% 75,0) lesitinaz, yedisi (% 96,7) proteaz ve DNaz aktivitesi gösterdi. Yirmi suş (% 66,7) kuvvetli, yedi suş (% 23,3) orta ve üç suş (% 10,0) zayıf biyofilm yapmaktaydı.Sonuç: Bu sonuçlardan bu enfeksiyonlarda tedavi seçeneklerinin sınırlı olduğu anlaşılmaktadır. Bundan dolayı vankomisine duyarlılığı azalmış MRSA suşlarının etken olduğu enfeksiyonların izlenmesi ve yönetimi bu suşların hastane ortamına yayılmasının kontrolüyle yapılmalıdır

Anahtar Kelimeler

• Staphylococcus aureus, metisilin direnci, MİK, virülans faktörleri, biyofilm

Antimicrobial susceptibility, virulence factors and biofilm formation among Staphylococcus aureus isolates from hospital infections in Kerman, Iran

Abstract

Objective: The aims of present study were to determine the antimicrobial susceptibility, virulence factors and biofilm formation among MRSA hospital isolates of Staphylococcus aureus. Methods: Thirty non-repetitive strains of S. aureus isolated from three hospitals in Kerman, Iran. Antimicrobial sus­ceptibility was determined by disk diffusion breakpoints method according to CLSI guideline. The minimum inhibitory concentration (MIC) of vancomycin and methicillin were measured by the broth microdilution and E-test procedures. Virulence factors (protease, DNase, lecithinase, capsule and hemolysis) associated with the above isolates was studied. Biofilm was quantified by microtiter technique. Results: In total, 14 (46.7%) S. aureus were isolated from lower respiratory tract, six (20.0%) from urinary tract and re­maining 10 (33.3%) were recovered from wounds, blood and orthopedic patients. All of the isolates were susceptible to tigecycline, eight (26.7%) were found to be resistant to methicillin (MRSA) and 4 (13.3%) showed reduced susceptibility to vancomycin. No any vancomycin resistant isolate was detected (p≤0.05). MIC results showed that four of the isolates (13.3%) exhibited MIC 4 μg/mL to vancomycin while, five (16.6%) demonstrated MIC 32 μg/mL to methicillin. The iso­lates were also resistant to amoxicillin/clavulanic acid, tetracycline and tobramycin. It was found that, six (75 %) of MRSA strains produced lecithinase, seven (96.7%) demonstrated protease and DNase activities as compared to MSSA isolates. Biofilm analysis revealed that twenty (66.7%) isolates formed strong, seven (23.3%) formed moderate and three (10.0%) had weak biofilm. Conclusion: From the results, it can be concluded that, treatment options available for these infections are limited; therefore, monitoring, and management of infections due to MRSA with reduced susceptibility to vancomycin, must be done in order to control spread of these strains in the hospital environment.

Keywords

• Staphylococcus aureus
• methicillin resistance
• MIC
• virulence factor
• biofilm

Kaynakça

1. Rubinstein E, Kollef MH, Nathwani D. Pneumonia caused by methicillin-resistant Staphylococcus aureus. Clin Infect Dis 2008; 46:378-385.
2. David MZ, Daum RS. Community associated methicillin resis- tant Staphylococcus aureus: epidemiology and clinical con- sequences of an emerging epidemic. Clinic Microbiol Rev 2010; 23:616-687.
3. National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2003, issued August 2003. Am J Infect Control 2003; 31: 481-498.
4. Klevens RM, Morrison MA, Nadle J, et al. Invasive methicil- lin-resistant Staphylococcus aureus infections in the United States. JAMA 2007; 298:1763-1771.
5. Fischbach MA, Walsh CT. Antibiotics for emerging pathogens. Science 2009; 325:1089-1093.
6. Rodvold KA and McConeghy KW. Methicillin resistant Staphy- lococcus aureus therapy: past, present, and future. Clin In- fect Dis 2014; 58: S20-S27.
7. Weinstein RA and Fridkin SK. Vancomycin intermediate and resistant Staphylococcus aureus: What the infectious dis- ease specialist needs to know. Clin Infect Dis 2001; 32 (1): 108-115.
8. Hiramatsu K, Hanaki H, Ino T, et al. Methicillin-resistant Staph- ylococcus aureus clinical strain with reduced vancomycin susceptibility. Antimicrob Chemother 1997; 40: 135-136.

9. Brumfitt W, Hamilton-Miler JM. The worldwide problem of methicillin resistant Staphylococcus aureus. Drugs Exp Clin Res 1990; 16: 205-14.
10. Noskin GA, Rubin RJ, Schentag JJ, et al. The burden of Staphylococcus aureus infections on hospitals in the United States. Arch Intern Med 2005; 65:1756-1761.
11. Azimian A, Najar-pirayeh S, Mirab-Samiee S, et al. Occur- rence of methicillin resistant Staphylococcus aureus (MRSA) among clinical samples in Tehran-Iran and its correlation with polymorphism of specific accessory gene regulator (agr) groups. Braz J Microbiol 2012; 43:779-785.
12. Hasani A, Sheikhalizadeh V, Hasani A, et al. Methicillin re- sistant and susceptible Staphylococcus aureus: Appraising therapeutic approaches in the Northwest of Iran. Iran J Mi- crobiol 2013; 5:56-62.
13. Melzer M, Eykyn SJ, Gransden WR, Chinn S. Is methicillin- resistant Staphylococcus aureus more virulent than methi- cillin-susceptible S. aureus? A comparative cohort study of British patients with nosocomial infection and bacteremia. Clin Infect Dis 2003; 37: 1453-1460.
14. Blot S, Vandewoude KH, Hoste EA, Colardyn FA. Patients with bacteremia involving methicillin susceptible and meth- icillin-resistant Staphylococcus aureus. Arch Intern Med 2002; 162:2229-2235.
15. Cabrera-Contreras R, Morelos-Ramírez R, Galicia-Macho N, et al. Antibiotic resistance and biofilm production in Staphylo- coccus epidermidis strains, isolated from a tertiary care hos- pital in Mexico City. SRN Microbiology 2013; http: //dx.doi. org/ 10.1155/2013/918921.
16. Shikh-Bardsiri H, and Shakibaie MR. Antibiotic resistance pattern among biofilm producing and non-producing Proteus strains isolated from hospitalized patients; Matter of Hospital Hygiene and Antimicrobial Stewardship. Pak J Biol Scienc 2013; 16 (22):1496-1502.
17. Kateete D, Kimani C, Katabazi F, et al. Identification of Staph- ylococcus aureus: DNase and manitol salt agar improve the efficiency of the tube coagulase test. Ann Clinic Microbiol and Antimicrob 2010; 9:23-28.
18. Clinical Laboratory Standards Institute (CLSI) (2012) Guide- lines. Performance Standards for antimicrobials disk sus- ceptibility tests. Approved standard. 11th ed. CLSI document M100-S22. Wayne, PA: CLSI.
19. Bjorklind A, and Arvids S. Occurrence of an extracellular ser- ine proteinase among Staphylococcus aureus strains. Acta Pathol Microbiol Scand 1977; 5:277-280.
20. Matos JE, Harmon RJ, Langlois BE: Lecithinase reaction of Staphylococcus aureus strains of different origin on Baird- Parker medium. Lett Appl Microbiol 1995; 21:334-335.
21. Welch PG, Fattom A, Moore J, et al. Safety and immuno- genicity of Staphylococcus aureus type 5 capsular polysac- charide Pseudomonas aeruginosa a recombinant exoprotein a conjugate vaccine in patients on hemodialysis. Am Soc Nephrol 1996; 7:247-253.
22. Shakibaie M, Forootanfar H, Yaser Golkari Y, et al. Anti-bio- film activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis. Journal of Trace Elements in Medicine and Biology 2015; 29: 235-241. doi:10.1016/j.jtemb.2014.07.020
23. Stepanovic S, Vukovic D, Hola V, et al. Quantification of bio- film in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm pro- duction by Staphylococci. APMIS 2007; 115: 891-899.
24. Fatholahzadeh B, Emaneini M, Gilbert G, et al. Staphylo- coccal Cassette Chromosome mec (SCCmec) Analysis and Antimicrobial Susceptibility Patterns of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates in Tehran, Iran. Mi- crob Drug Resist 2008; 14: 217-220.
25. Japoni A, Alborzi A, Orafa F, et al. Distribution Patterns of Methicillin Resistance genes (mecA) in Staphylococcus au- reus isolated from clinical specimens. Iranian Biomedical Journal 2004; 8: 173-178.
26. Shakibaie MR, Mansouri S, Hakak S. Plasmid Pattern of an- tibiotic resistance in β-lactamase producing Staphylococcus aureus isolated from hospitals in Kerman, Iran. Archive of Iranian Medicine 1999; 2:93-97.
27. Lin SY, Chen TC, Chen FJ, et al. Molecular epidemiology and clinical characteristics of hetero-resistant vancomycin intermediate Staphylococcus aureus bacteremia in a Taiwan medical center. Microbiol Immunol Infect 2012; 45:435-441.
28. Golan Y, Baez-Giangreco C, O’Sullivan C, et al. Trends in vancomycin susceptibility among consecutive MRSA iso- lates. In: Abstracts of the Forty-fourth Annual Meeting of the Infectious Diseases Society of America, Toronto, Ontario, Canada, 2006. Abstract LB-11, p. 238. Infectious Diseases Society of America, Alexandria, VA, USA.
29. Soriano A, Martínez JA, Mensa J, et al. Pathogenic signifi- cance of methicillin resistance for patients with Staphylococ- cus aureus bacteremia. Clin Infect Dis 2000; 30:368-73.
30. Chen YH, Liu CU, Ko WC, et al. Trends in the susceptibility of methicillin-resistant Staphylococcus aureus to nine antimi- crobial agents, including ceftobiprole, nemonoxacin, and ty- rothricin: results from the Tigecycline In Vitro Surveillance in Taiwan (TIST) study, 2006-2010. Eur J Clin Microbiol Infect Dis 2014; 33:233-239.
31. Rezaei M, Moniri R, Mousavi SA et al. Prevalence of bio- film formation among methicillin resistance Staphylococcus aureus iso lated from nasal carriers. Jundishapur Journal of Microbiology 2013; 6(6): e9601. DOI: 10.5812/jjm.9601