Citlivosť kmeňov Staphylococcus aureus rastúcich v biofilme na vankomycín, gentamicín a rifampicín

Susceptibility of Staphylococcus aureus Biofilms to Vancomycin, Gentamicin and Rifampin

Study objectives:
To detect biofilm formation in Staphylococcus aureus strains and to determine the minimal biofilm inhibition concentrations (MBIC) and the minimal biofilm eradicating concentrations (MBEC) of vancomycin, gentamicin and rifampin. To compare the MBIC and MBEC with the minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) data for planktonic Staphylococcus aureus forms that are commonly used in antimicrobial susceptibility testing for the purposes of individualized therapy.

Patients and Methods:
Fifteen S. aureus strains isolated from central venous catheters, intratracheal tubes and wound drainage tubes from the patients of the University Hospital, Bratislava-Staré Mesto were included in the study. Selected virulence factors were characterized. The biofilm formation potential was measured by a modified crystal violet micro-assay. The presence of viable cells biofilm in was tested using 3-(4,5-dimethylthiazol--2‑yl)-2,5-diphenyl tetrazolium bromide (MTT). The MIC and MBC of vancomycin, gentamicin and rifampin was tested in planktonic S. aureus forms by the broth microdilution method. The MBIC and MBEC of these antimicrobial drugs for biofilm S. aureus forms were determined by a modified microdilution method. Student’s t-test was used for statistical analysis of the results.

Results:
All of the study strains formed biofilm, with only two of them having a low biofilm formation potential. MTT revealed moderate to high metabolic activity of bacteria biofilm in Vancomycin MICs and MBICs were identical in 80 % of the study strains. Vancomycin MBECs are higher than MBCs in all the study strains, are interpreted as resistance according to the criteria of the Clinical and Laboratory Standards Institute (CLSI) and make the drug unsuitable for use in the treatment. In vitro gentamicin MBICs indicated susceptibility according to the CLSI criteria but gentamicin MBECs were interpreted as gentamicin resistance. Rifampin MICs and MBICs of the study strains revealed susceptibility. Rifampin MBCs were interpreted as susceptibility, but based on MBECs, 13 % of the study strains were considered as resistant and 13 % of the study strains showed intermediate susceptibility. The differences between gentamicin and rifampin MICs and MBICs and those between MBCs and MBECs of all antimicrobials tested were statistically significant.

Conclusion:
The tested biofilm S. aureus forms showed high MBECs of vancomycin, gentamicin and rifampin, with rifampin only being suitable for therapeutic use.

To provide reliable results for individualized antibiotic therapy, it will be needed to test in vitro biofilm formation, to determine MBIC and MBEC of antimicrobial drugs using a standardized method, to interpret the test results in relation to biofilm S. aureus forms and to establish the interpretation criteria for MBIC and MBEC similarly to MIC and MBC.

Key words:
S. aureus – biofilm – susceptibility to antimicrobial drugs – vancomycin – gentamicin – rifampin.

Autori: D. Kotulová; L. Slobodníková
Pôsobisko autorov: Mikrobiologický ústav LFUK a FNsP, Bratislava, Slovenská republika
Vyšlo v časopise: Epidemiol. Mikrobiol. Imunol. 59, 2010, č. 2, s. 80-87

Súhrn

Cieľ práce.
Detekovať tvorbu biofilmu u kmeňov Staphylococcus aureus a určiť koncentrácie vankomycínu, gentamicínu a rifampicínu, ktoré zabránia množeniu S. aureus v biofilme (minimálne biofilm inhibujúce koncentrácie - MBIC), alebo vedú k jeho eradikácii (minimálne biofilm-eradikujúce koncentrácie – MBEC). Porovnať tieto údaje s hodnotami minimálnej inhibičnej koncentrácie (MIC) a minimálnej baktericídnej koncentrácie (MBC) u identických kmeňov baktérií, rastúcich v planktonickej forme, v ktorej sa bežne testuje v laboratóriu citlivosť na antiinfekčné liečivá pre potreby individualizovanej terapie.

Pacienti a etódy:
Do testovaného súboru baktérií sa zaradilo 15 kmeňov S. aureus, izolovaných z entrálnych venóznych katétrov, intratracheálnych kanýl a anových drénov od pacientov FNsP Bratislava-Staré Mesto. Charakterizovali sa ich vybrané faktory virulencie. Schopnosť tvoriť biofilm sa určila modifikovanou mikrometódou s ryštalickou violeťou (KV). Prítomnosť živých buniek v biofilme sa určovala s použitím 3-(4,5-dimetyltiazol-2-yl)-2,5-difenyl tetrazólium bromidu (MTT). MIC a BC vankomycínu, gentamicínu a ifampicínu sa testovala v planktonickej forme rastu kmeňov S. aureus bujónovou mikrodilučnou metódou. Účinok týchto antibiotík na inhibíciu a radikáciu živých baktérií v iofilme sa testoval modifikovanou mikrodilučnou metódou. Výsledky sa štatisticky hodnotili Studentovým t‑testom.

Výsledky:
Podľa našich výsledkov všetky kmene tvorili biofilm a ba dva z ich ho tvorili slabo. Pri hodnotení metabolickej aktivity baktérií v iofilme (MTT) sa zistila u ich stredná alebo vysoká aktivita. Pri sledovaní účinku vankomycínu in vitro sa zistilo, že hodnoty MIC a BIC u ankomycínu sú u 0 % kmeňov identické. Hodnoty MBEC vankomycínu sú však vyššie ako MBC u šetkých vyšetrovaných kmeňov, ich hodnota patrí podľa kritérií Clinical and Laboratory Standards Institute (CLSI) do kategórie rezistencie a oto antibiotikum už nie je použiteľné v erapii. Pri hodnotení výsledkov gentamicínu sa ukázalo, že in vitro inhibuje biofilm v oncentráciách, ktoré sa zaraďujú podľa CLSI do kategórie citlivý, avšak hodnoty, potrebné na eradikáciu, sú interpretované ako rezistencia kmeňa. Pri testovaní rifampicínu sa hodnoty MIC aj MBIC pohybovali v ategórii citlivý. Hodnoty MBC sa interpretovali v ategórii citlivý, ale MBEC už v 3 % ako rezistentný a 3 % ako intermediárne citlivý. Rozdiely v ameraných hodnotách MIC a BIC gentamicínu a ifampicínu a BC a BEC všetkých testovaných antibiotík boli štatisticky signifikantné.

Záver:
Nami sledované kmene S. aureus sú v iofilme eradikované iba vyššími koncentráciami vankomycínu, gentamicínu a ifampicínu, pričom hodnoty MBEC použiteľné v erapii dosahoval iba ifampicín.

Z ľadiska poskytovania spoľahlivých výsledkov pre voľbu terapie antiinfekčnými liečivami bude perspektívne potrebné zisťovať in vitro tvorbu biofilmu, estovať MBIC a BEC liečiv štandardizovanou metódou, interpretovať výsledky vo vzťahu k aktériám rastúcim v iofilme a tanoviť hraničné koncentrácie MBIC a BEC obdobne ako je to pri kritériách pre hodnotenie MIC a BC.

Kľúčové slová:
S. aureus – biofilm – citlivosť na antimikrobiálne liečivá – vankomycín – gentamicín – rifampicín.

Projekt bol finančne podporovaný grantom MŠ SR VEGA 1/4253/07.

Ďakujeme za technickú spoluprácu V. Augustovičovej, G. Skýpalovej a Ing. J. Havelovej.

Prof. MUDr. Daniela Kotulová, PhD.
Mikrobiologický ústav LF UK a FBsP
Sasinkova 4/II.
811 08 Bratislava
Slovenská republika
e-mail: daniela.kotulova@fmed.uniba.sk

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