Effect of daptomycin and vancomycin on Staphylococcus epidermidis biofilms: An in vitro assessment using fluorescence in situ hybridization

Abstract

Colonization of in-dwelling catheters by microbial biofilms is a major concern in patient health eventually leading to catheter-related blood stream infections. Biofilms are less susceptible to standard antibiotic therapies that are effective against planktonic bacteria. Standard procedure for the detection of microorganisms on the catheter tip is culture. However, viable but non-culturable cells (VBNCs) may be missed. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) as an indicator to visualize and quantify the effect of the antibiotics daptomycin and vancomycin on biofilms in situ . We established an in vitro catheter biofilm model of Staphylococcus epidermidis biofilms on polyurethane catheters. Biofilm activity was measured by FISH and correlated to colony forming units (CFU) data. Digital image analysis was used for quantification of total biofilm mass and the area of the FISH positive biofilm cells. FISH showed a pronounced effect of both antibiotics on the biofilms, with daptomycin having a significantly stronger effect in terms of both reduction of biofilm mass and number of FISH-positive cells. This supports the anti-biofilm capacity of daptomycin. Interestingly, neither antibiotic was able to eradicate all of the FISH-positive cells. In summary, FISH succeeded in visualization, quantification, and localization of antibiotic activity on biofilms. This technique adds a new tool to the arsenal of test systems for anti-biofilm compounds. FISH is a valuable complementary technique to CFU since it can be highly standardized and provides information on biofilm architecture and quantity and localization of survivor cells.

Aerococcus urinae – A potent biofilm builder in endocarditis

“…While this matrix is composed of only bacterial products in vitro, in biofilm-associated infections it is also formed by the host's own substances such as fibrin, blood components, and tissue components [27]. Although FISH is unable to analyze the matrix in which the bacteria are located, it is still able to visualize the typical biofilm property of structural organization of the bacteria: the fluorescence signal intensity of FISH correlates with the bacterial ribosome content and therefore demonstrates activity of the bacteria [31,32]. The cases presented here display areas with a higher ribosome content Fixed cultures of Aerococcus sanguinicola and A. urinae, as well as heart valve tissue were simultaneously hybridized with the probes EUB338 (FITC), AURI (Cy3) and NONEUB (Cy5).…”

Cutibacterium avidum resists surgical skin antisepsis in the groin—a potential risk factor for periprosthetic joint infection: a quality control study

“…Ribosomes are highly abundant in actively replicating bacterial cells. The FISH signal intensity correlates with the ribosomal content and therefore can be used as activity marker [ 29 – 32 ]. However, since the ribosome content may vary between bacteria, the absence of a FISH signal cannot differentiate between a dead or resting state.…”

“…These single bacteria are visualized according to size and morphology and frequently located in the subcutaneous fat tissue. In one case, we detected FISH positive bacteria, indicating ribosome-rich and therefore presumably active bacteria [ 29 – 32 ]. Using the specific C. avidum/ A. acidipropionici/ A. propionicum/ A. jensenii/ A. thoeni - probe, we did not find any FISH-positive bacteria in the lumen of the sweat glands.…”

Fluorescence in situ Hybridization (FISH) in the Microbiological Diagnostic of Deep Sternal Wound Infection (DSWI)

“…DAPI-stained microorganisms not visualized by FISH probably correspond to dead or metabolically inactive bacteria. 20 , 21 In addition, culture is not able to discriminate between infection and contamination with skin flora upon sampling. DSWIs regularly show a wide variety of different bacteria supporting the persistence of a wound healing disorder.…”