Koagülaz Negatif Stafilokoklarda Biyofilm Oluşumunun Çeşitli Kongo Kırmızısı Besiyerlerinde Değerlendirimi

Amaç: Bu çalışmada koagülaz negatif stafilokok (KNS) izolatlarının biyofilm oluşturma özelliklerinin saptanmasında kullanılan Kongo kırmızısı agar (KKA) besiyeri ve modifikasyonlarının performanslarının mikroplak yöntemi ile karşılaştırılarak değerlendirilmesi amaçlanmıştır. Gereç ve Yöntem: Çalışmada 24’ü (%30) kateter kolonizanı, 13’ü (%16,25) kan dolaşım enfeksiyonu (KDE) etkeni, 20’si (%25) kateter ilişkili kan dolaşım enfeksiyonu (KİKDE) etkeni ve 23’ü (%28,75) hastane çalışanlarının burun boşluklarından elde edilen olmak üzere toplam 80 KNS izolatı değerlendirilmiştir. Biyofilm özelliklerinin incelenmesinde KKA besiyerleri [KKA G (%2 glukoz ilaveli), KKA GN (%2 glukoz ve %1,5 NaCl ilaveli), KKA GNV (%2 glukoz, %1,5 NaCl 0,5 µg/mL vankomisin ilaveli)] ve mikroplak yöntemi kullanılmıştır. Mikroplak yöntemi ile kıyaslanarak KKA besiyerleri için duyarlılık, özgüllük, pozitif ve negatif prediktif değerleri hesaplanmıştır. Bulgular: İzolatların %36,25’i Staphylococcus hominis, %35’i Staphylococcus epidermidis, %13,75’i Staphylococcus haemolyticus, %11,25’i Staphylococcus capitis, %2,5’i Staphylococcus saprophyticus ve %1,25’i Staphylococcus warnerii olarak tiplendirilmiştir. KKA yöntemi ile 34’ü (%42,5), mikroplak yöntemi ile 57’si (%71,25) [18’i (%22,5) zayıf, 24’ü (%30) orta kuvvette ve 15’i (%18,75) kuvvetli], biyofilm pozitif olarak saptanmıştır. KDE etkeni olan KNS’lerin 6’sı (%7,5), KİKDE etkeni olan KNS’lerin 20’si (%25), kateter kolonizanı olanların 18’i (%22,5), burun boşluklarından izole edilenlerin 13’ü (%16,25) çeşitli kuvvetlerde biyofilm oluşturmuştur. KKA, KKA G ve KKA GN ve KKA GNV yöntemlerinin duyarlılıkları sırasıyla %59,6, %59,6, %59,6 ve %58,2, özgüllükleri ise %100’dür. Sonuç: Stafilokok türlerinde biyofilm oluşumu çeşitli çevresel koşullardan ve besiyeri içeriğinden etkilenmektedir. Çalışmamızda stafilokok izolatlarında biyofilm oluşumunu değerlendirmek için, modifiye KKA besiyerlerinin orijinal KKA besiyerine göre üstünlüğü saptanmamıştır. Biyofilm üreten bakterilerin saptanmasında KKA ve modifiye formlarına kıyasla, mikroplak yönteminin kullanılması daha uygundur.

Evaluation of Biofilm Formation in Coagulase Negative Staphylococci on Various Congo Red Agar Medium

Objectives: In this study, it was aimed to evaluate the performance of different Congo red agar (CRA) media and its modifications in comparison with the microplate method in the demonstration of biofilm formation in coagulase negative staphylococcus (CoNS) isolates. Materials and Methods: A total of 80 CoNS isolates, including 24 (30%) catheter colonizers, 13 (16.3%) bloodstream infection (BSI) agents, 20 (25%) catheter-related bloodstream infection (CR-BSI) agents and 23 (28.8%) obtained from the nasal cavities of hospital staff, were evaluated in the study. In the determination of biofilm formation, CRA mediums (CRA G (2% glucose added), CRA GN (2% glucose and 1.5% NaCl added), CRA GNV (2% glucose, 1.5% NaCl 0.5 µg/mL vancomycin added) and microplate method were used. Sensitivity, specificity, positive predictive and negative predictive values were calculated by comparing with the microplate method. Results: Staphylococcus hominis was the most frequently detected species (36.25%), followed by Staphylococcus epidermidis (35%), Staphylococcus haemolyticus (13.75%), Staphylococcus capitis (11.25%), Staphylococcus saprophyticus (2.5%), and Staphylococcus warneri (1.25%). Thirty-four (42.5%) isolates were detected by CRA method, 57 (71.25%) [18 (22.5%) weak, 24 (30%) medium strength, 15 (18.75%) strong biofilm] isolates were detected by microplate method. Six (7.5%) of CoNS agents of BSI, 20 (25%) of CoNS agents of CRBSI, 18 (22.5%) of those with catheter colonization, 13 (16.3%) of those isolated from nasal cavities formed a biofilm of varying strengths. The sensitivities of the CRA, CRA G, CRA GN and CRA GNV methods were 59.6%, 59.6%, 59.6% and 58.2%, and their specificities were 100%, respectively. Conclusion: Biofilm formation in staphylococcal species is affected by various environmental conditions and media content. To assess biofilm formation in all staphylococcal isolates, no superiority of modified CRA media was found over the original CRA. Microplate method can be recommended as a general screening method for the detection of biofilm-producing bacteria, compared to CRA and its modified forms.

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Bannerman TL, Peacock SJ Staphylococcus, Micrococcus, and other catalase-positive cocci. In Murray PR, Baron EJ, Jorgensen JH, Landry ML, and Pfaller MA (Eds.), Manual of clinical microbiology 9th ed. Vol. 1. ASM Press. Washington, DC.; 2007. s.390-411.
Götz F. Staphylococcus and biofilms. Mol Microbiol. 2002;43(6):1367–1378.
Halton KA, Cook DA, Whitby M, et al. Cost effectiveness of antimicrobial catheters in the intensive care unit: Addressing uncertainty in the decision. Crit Care. 2009;13:R35.
Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science. 1999;284:5427–5433.
Watnick P, Kolter R. Biyofilm city of microbes. Minireview. J Bacteriol 2000; 182:2675-2679.
Christeen GD, Simpson WA, Bisno AL, et al. Adherence of slime- producing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun. 1982; 37:318-326.
Christeen GD, Simpson WA, Younger JJ, et al. Adherence of coagulase negative staphylocci to plastic tissue culture plates: a quantitative modelfor the adherence of staphylococi to medical devices. J Clin Microbiol. 1985;22: 996-1006.
Vasudevan P, Nair MK, Annamalai T, et al. Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation. Vet Microbiol. 2003;92:179-85.
Seo YS, Lee DY, Rayamahji N, et al. Biofilm-forming associated genotypic and phenotypic characteristic of Staphylococcus spp. isolated from animals and air. Res Vet Sci. 2008;85:433-438.
Montanaro L, Campoccia D, Pirini V. Antibiotic multiresistance strictly associated with IS256 and ica genes in Staphylococcus epidermidis strains from implant orthopedic infections. J Biomed Mater Res A. 2007;83:813- 818.
Temel A, Eraç B. Bakteriyel biyofilmler: Saptama yöntemleri ve antibiyotik direncindeki rolü. Türk Mikrobiyol Cem Derg. 2018;48:1-13.
Idrees M, Sawant S, Karodia N, Rahman A. Staphylococcus aureus biofilm: Morphology, genetics, pathogenesis and treatment strategies. Int J Environ Res Public Health. 2021;18:7602.
Zmantar T, Kouidhi B, Miladi H, et al. A microtiter plate assay for Staphylococcus aureus biofilm quantification at various pH levels and hydrogen peroxide supplementation. New Microbiol. 2010;33:137-145.
Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol. 1989;42:872- 874
Kaiser TD, Pereira EM, Dos santos KR, et al. Modification of the Congo red agar method to detect biofilm production by Staphylococcus epidermidis. Diagn Microbiol Infect Dis. 2013;75:235-239.
Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol. 2010;8:623–633.
Fleming D, Rumbaugh KP. Approaches to dispersing medical biofilms Microorganisms. 2017;5:15.
Molnar C, Hevessy Z, Rozgonyi F, et al. Pathogenicity and virulence of coagulase negative staphylococci in relation to adherence, hydrophobicity, and toxin production in vitro. J Clin Pathol. 1994;47:743–748.
Manandhar S, Singh A, Varma A, et al. Evaluation of methods to detect in vitro biofilm formation by staphylococcal clinical isolates. BMC Res Notes. 2018;11:714.
Hassan A, Usman J, Kaleem F, et al. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis. 2011;15:305-311.
Shrestha LB, Bhattarai NR, Khanal B. Antibiotic resistance and bioflm formation among coagulase-negative staphylococci isolated from clinical samples at a tertiary care hospital of eastern Nepal. Antimicrob Resist infect Control. 2017;6:89.
Zhou S, Chao X, Fei M, et al. Analysis of S.epidermidis icaA and icaD genes by polymerase chain reaction and slime production: A case control study, BMC Infections Diseases. 2013;13:242.
Öcal DN, Dolapçı İ, Karahan ZC, Tekeli A. Stafilokok izolatlarının biyofilm oluşturma özelliklerinin araştırılması. Mikrobiyol Bul. 2017;51:10-19.
Kitti T, Seng R, Thummeepak R, et al. Biofilm formation of methicillin resistant coagulase-negative staphylococci isolated from clinical samples in Northern Thailand. J Glob Infect Dis. 2019;11:112-117.
Kırmusaoğlu S. Staphylococcus epidermidis ve Staphylococcus aureus antibiyotik direncine sebep olan biyofilm oluşumunun belirlenmesi için kullanılan metotların kıyaslanması. Ortadoğu Tıp Dergisi. 2017;9:28-33.
Mathur T, Singhal S, Khan S, et al. Detection of biofilm formation among the clinical isolates of staphylococci: An evaluation of three different screening methods. Indian J. Med. Microbiol. 2006;24:25-29.
Kord M, Ardebili A, Jamalan M, et al. Evaluation of biofilm formation and presence of ica genes in Staphylococcus epidermidis clinical isolates. Osong Public Health Res Perspect. 2018;9:160-166.
Manandhar S, Singh A, Varma A, et al. Phenotypic and genotypic characterization of biofilm producing clinical coagulase negative staphylococci from Nepal and their antibiotic susceptibility pattern. Ann Clin Microbiol Antimicrob. 2021;20:41.
Terki IK, Hassaine H, Oufrid S, et al. Detection of icaA and icaD genes and biofilm formation in Staphylococcus spp. isolated from urinary catheters at the University Hospital of Tlemcen (Algeria) African Journal of Microbiology Research. 2013;7:5350-5357.
Rachid S, Ohlsen K, Witte W, et al. Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis. Antimicrob Agents Chemother. 2000;44:3357–3363.
Ziebuhr W, Krimmer V, Rachid S, et al. A novel mechanism of phase variation of virulance in Staphylococcus epidermidis: Evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol. 1999;32:345-356.
Mónzon M, Oteiza C, Leiva J, et al. Biofilm testing of Staphylococcus epidermidis clinical isolates: Low performance of vancomycin in relation to othersantibiotics. Diagn Microbiol Infect Dis. 2002;44:319–324.
Cargill JS, Upton M. Low concentrations of vancomycin stimulates biofilm formation insome clinical isolates of Staphylococcus epidermidis. J Clin Pathol. 2009;62:1112–1116.
Jain A, Agarwal A. Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci. J Microbiol Methods. 2009;76:88- 92.
Cafiso V, Bertuccio T, Santagati M, et al. Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production. Clin Microbiol Infect. 2004;10:1081-1088.