Pseudomonas aeruginosa, an opportunistic pathogen, has been found to cause several chronic and acute infections in human. Moreover, it often shows drug-tolerance and poses a severe threat to public healthcare through biofilm formation. In this scenario, two molecules, namely, cuminaldehyde and tobramycin, were used separately and in combination for the efficient management of biofilm challenge. The minimum inhibitory concentration (MIC) of cuminaldehyde and tobramycin was found to be 150 μg/mL and 1 μg/mL, respectively, against Pseudomonas aeruginosa. The checkerboard assay revealed that the fractional inhibitory concentration (FIC) index of cuminaldehyde and tobramycin was 0.36 suggesting a synergistic association between them. The sub-MIC dose of cuminaldehyde (60 μg/mL) or tobramycin (0.06 μg/mL) individually did not show any effect on the microbial growth curve. However, the same combinations could affect microbial growth curve of Pseudomonas aeruginosa efficiently. In connection to biofilm management, it was observed that the synergistic interaction between cuminaldehyde and tobramycin could inhibit biofilm formation more efficiently than their single use (p < 0.01). Further investigation revealed that the combinations of cuminaldehyde and tobramycin could generate reactive oxygen species (ROS) that resulted in the increase of membrane permeability of bacterial cells leading to the efficient inhibition of microbial biofilm formation. Besides, the synergistic interaction between cuminaldehyde (20 μg/mL) and tobramycin (0.03 μg/mL) also showed significant biofilm dispersal of the test microorganism (p < 0.01). Hence, the results suggested that synergistic action of cuminaldehyde and tobramycin could be applied for the efficient management of microbial biofilm.
- Klíčová slova
- Tobi Podhaler,
- MeSH
- aplikace inhalační MeSH
- cystická fibróza farmakoterapie genetika komplikace MeSH
- dítě MeSH
- infekce dýchací soustavy farmakoterapie imunologie komplikace MeSH
- lidé MeSH
- nebulizátory a vaporizátory MeSH
- Pseudomonas aeruginosa patogenita účinky léků MeSH
- tobramycin aplikace a dávkování farmakologie MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
Vyhodnotenie účinku subinhibičných koncentrácií (sub-MICs) amikacínu, tobramycínu a kolistínu na tvorbu biofilmu, na povrchovú hydrofobicitu, lipázu a odpoveď na oxidačný stres u dvoch klinických kmeňov K. pneumoniae. Metodika: Tvorbu biofilmu sme kvantitatívne vyhodnotili testom absorpcie kryštálovej violete, povrchovú hydrofobicitu adherenciou na xylén, lipázu spektrofotometrickou metódou s Tweenom 20 ako substrátom, oxidačný stres ako zónu vyčírenia obklopujúcu disk nasiaknutý peroxidom vodíka. Výsledky: Antibiotiká výrazne znížili tvorbu bakteriálneho biofilmu v priamej závislosti na koncentrácii. Najúčinnejšie boli v koncentráciách 1/2 a 1/4 MICs. Biofilm po účinku 1/2 sub-MICs amikacínu bol inhibovaný na 21,2 % (kmeň 39) a 22,6 % (kmeň 61/P), po tobramycíne na 25,1 % (39) a 19,5 % (61/P), po kolistíne na 7,4 % (39) a 19,1 % (61/P) kontrolných hodnôt (bez antibiotika). Podobne 1/4 MICs amikacínu zredukovala produkciu biofilmu na 28,6 % (39) a 28,9 % (61/P), tobramycínu na 35,3 % (39) a 20,5 % (61/P) a kolistínu na 8,7 % (39) a 20,4 % (61/P) kontrolných hladín. Biofilm bol najmenej redukovaný po kultivácii kmeňov s 1/16 MICs antibiotík. V týchto prípadoch jeho hodnota bola znížená na 80,4 % (39) a 97,7 % (61/P) kontrolných hodnôt po amikacíne, na 69,4 % (39) a 64,4 % (61/P) po tobramycíne, na 61,3 % (39) a 74,7 % (61/P) po kolistíne. Testované kmene boli silne hydrofilné a zmeny v povrchovej hydrofobicite účinkom antibiotík boli veľmi nepatrné. Kmene po pôsobení antibiotík vo väčšine prípadov vykazovali mierne zvýšenú citlivosť na oxidačný stres a zníženú aktivitu lipázy (s výnimkou kolistínu u kmeňa 39). Záver: Amikacín, tobramycín a kolistín v sub-MICs u kmeňov K. pneumoniae výrazne znížili tvorbu biofilmu, vo väčšine prípadov mierne zvýšili citlivost na oxidačný stres, znížili aktivitu lipázy ale adherenciu na xylén prakticky neovplyvnili.
To evaluate the effect of subinhibitory concentrations (sub-MICs) of amikacin, tobramycin and colistin on biofilm formation, surface hydrophobicity, lipase activity and response to oxidative stress in two clinical K. pneumoniae strains. METHODS: Biofilm formation was quantitatively determined by a crystal violet absorption assay, surface hydrophobicity was measured by adherence of bacteria to xylene, lipase activity was determined by the spectrophotometric method with Tween-20 as a substrate and oxidative stress was visualized as a zone of clearing around the disc soaked with hydrogen peroxide. RESULTS: The antibiotics significantly reduced bacterial biofilm formation in a dose-dependent manner. They were most effective at concentrations of 1/2 and 1/4 MIC. Biofilm formation was inhibited by 1/2 MICs of amikacin to 21.2% (strain 39) and 22.6% (61/P), of tobramycin to 25.1% (39) and 19.5% (61/P) and of colistin to 7.4% (39) and 19.1% (61/P) of the control values (no antibiotic). Similarly, 1/4 MICs reduced biofilm formation to 28.6% (39) and 28.9% (61/P) of the control levels for amikacin, to 35.3% (39) and 20.5% (61/P) for tobramycin and to 8.7% (39) and 20.4% (61/P) for colistin. Cultivation of the strains with the antibiotics at 1/16 MICs was least effective in inhibiting biofilm formation. It was reduced to 80.4% (39) and 97.7% (61/P) of the control levels for amikacin, to 69.4% (39) and 64.4% (61/P) for tobramycin and to 61.3% (39) and 74.7% (61/P) for colistin. The tested strains were strongly hydrophilic and changes in surface hydrophobicity caused by antibiotics were negligible. Most antibiotic treated strains showed mildly increased sensitivity to oxidative stress and decreased lipase activity (with the exception of colistin in strain 39). CONCLUSION: Amikacin, tobramycin and colistin at sub-MICs considerably reduced biofilm formation K. pneumoniae strains, in most mildly increased sensitivity to oxidative stress, decreased lipase activity but practically did not affect adherence to xylene.
Sledovali sme potlačenie bakteriálneho rastu siedmich kmeňov Acinetohacter spp. po 30 min účinku tobramycínu v suprainhibičných koncentráciách (postantibiotický účinok - PAE) a v supra-subinhibičných koncentráciách (postantibiotický účinok subinhibičných koncentrácií - PA SME) ako aj zmeny v povrchovej hydrofobicite, v produkcii lipázy a histamínu u týchto exponovaných kmeňov. Sledované farmakodynamické parametre (PAE a PA SME) ako aj zmeny v testovaných vlastnostiach Študovaných kmeňov boli závislé na koncentrácii antibiotika a na kmeni. Potlačenie bakteriálneho rastu po účinku tobramycínu v koncentrácii 2x MIC sa pohybovalo v rozsahu od 0,6-4,5 h, vyššia koncentrácia (4x MIC) indukovala dlhšie PAE (1,9-5,4 h). Tobramycín v supra-subinhibičných koncentráciách (2x MIC + 0,2x MIC a 4x MIC + 0,2x MIC) spôsobil úplnú supresiu bakteriálneho rastu. U väčšiny testovaných kmeňov sa po účinku tobramycínu zistilo zvýšenie hydrofobicity hodnotenej adherenciou baktérií na xylén, ako aj zvýšenie lipolytickej aktivity. Tobramycín v testovaných koncentráciách neovplyvnil produkciu histamínu.
Suppression of bacterial growth in seven strains of the Acinetobacter species after 30 min treatment with tobramycin at suprainhibitory concentrations (postantibiotic effect - PAE) and at supra-subinhibitory concentrations (postantibiotic effect of subinhibitory concentrations - PA SME) as well as changes in surface hydrophobicity and in the production of lipase and histamine in the exposed strains were studied. Pharmacodynamic parameters (PAE and PA SME) as well as changes in bacterial characteristics tested were dependent on antibiotic concentration and on the strain used. Suppression of bacterial growth after treatment with tobramycin at 2x MIC was in a range of 0.6-4.5 h, a higher concentration (4x MIC) induced a longer PAE (1.9-5.4 h). Tobramycin at supra-subinhibitory concentratinos (2x MIC + 0.2x MIC and 4x MIC + 0.2x MIC) caused total suppression of bacterial growth. In the majority of the tobramycin-treated strains, an increase in hydrophobicity manifested by adherence of bacteria to xylene as well as an increase in hpolytic activity was observed. Tobramycin at the concentrations tested did not affect the production of histamine.
