Catheter-related infections continue to be a significant source of morbidity and mortality in patients requiring catheterization [41
] and increase medical expenses by prolonging hospitalization. One of the most common etiologies of catheter infections are staphylococci, either coagulase-negative staphylococci or S. aureus
, and P. aeruginosa
. There are a lot of studies trying to demonstrate the efficiency of different substances as anti-biofilm-coated agents in reducing the incidence of catheter-associated biofilm infections (i.e., cefazolin, teicoplanin, vancomycin, silver sulfadiazine, chlorhexidine-silver sulfadiazine, minocycline-rifampin, lysostaphin, ciprofloxacin, and protamine sulfate combinations). There are a lot of studies reporting the efficacy of antibiotic-bonded catheters in preventing microbial biofilms from developing. It was demonstrated that the immersion of central venous catheters and arterial catheters in a 50 mg/mL cefazolin solution reduced the catheter colonization with Staphylococcus epidermidis
from 40% to 2%, proving that antibiotic bonding is an efficient, safe, and cost-effective method of reducing intravascular catheter infections in patients who are in intensive care units [42
]. Also, other research teams demonstrated that catheter coating with lysostaphin might be more suitable than antimicrobial bonding, due to the rapid coating time of catheters with minimal on-site catheter preparation, and the rapidity of kill would eradicate adherent bacteria within a very short amount of time, eliminating the risk of infections [44
]. Nano-silver coatings have been applied to several medical devices, of which catheters, drains, and wound dressings are the most prominent [15
Previous studies have demonstrated that the synergism between ciprofloxacin and protamine sulfate significantly enhanced the efficacy of ciprofloxacin against planktonic and biofilm-grown P. aeruginosa
]. In our study, concerning P. aeruginosa
biofilms, catheter coating by nanoparticles alone proved to be significantly more prone to bacterial colonization (p
0.0001) at 24 h than the standard catheter and the catheter sections either immersed in the antimicrobial solution or coated with the nanoparticles loaded with the newly synthesized compounds (Figure ). The compounds incorporated in nanoparticles (1a
) and (1b
) proved to be more efficient than the nanoparticles alone against P. aeruginosa
biofilm development at 24 h (p
0.0001). It is to be noticed that at 72 h, the compounds incorporated in nanoparticles (1a
) exhibited a very significant improvement of the anti-biofilm activity as compared with the catheter sections immersed in the soluble compound (1a
Concerning S. aureus
biofilms, at 24 h, as for the case of P. aeruginosa
biofilms, the catheters coated only with nanoparticles were significantly more colonized as compared with the uncoated catheter (p
0.0001; Figure ). However, compound (1a
) exhibited a strong anti-biofilm activity, the results being very significant when comparing either the uncoated catheter versus
the catheter immersed in the soluble compound (p
0.0001) or the catheter coated with nanoparticles versus
the catheter coated with nanoparticles and compound (1a
At 48 h, both compounds (1a
) and (1b
) in soluble form exhibited protective activity against S. aureus
mature biofilm development (p
0.0001). Only compound (1b
) significantly improved the anti-biofilm of the catheter surface in the presence of nanoparticles (p
0.0001). Very statistically significant results have been obtained at 72 h when a strong inhibitory effect of S. aureus
biofilm development was obtained for the catheter coating constituted of nanoparticles loaded with compounds (1a
) and (1b
), as compared with the results obtained for the catheter sections immersed in the compound solution alone or coated only with nanoparticles (p
Our results are demonstrating that the nanoparticle layer alone is not protective against microbial colonization, probably affecting the roughness and the electric charge of the catheter surface, favoring the interaction with the microbial surfaces. On the other hand, our study is clearly proving that the nanoparticle layer is interacting differently with the incorporated substances, influencing their release time in active forms and their antimicrobial activity.
The coating system represented by nanoparticles loaded with (1a
) proved to be efficient in preventing both the initial formation as well the development of mature microbial biofilms formed by S. aureus
and P. aeruginosa
, demonstrating the efficiency of the nanoparticle coating in the delivery of the chemical compound in active forms for a long period of time. These results are also proving that the obtained nanostructured coating agent is not only preventing bacteria to adhere to the catheter surface, but also acting as a biofilm dispersal agent. The molecular mechanisms of bacterial biofilm dispersal are only beginning to be elucidated; however, biofilm dispersal is a promising area of research that may lead to the development of novel agents that inhibit biofilm formation or promote biofilm cell detachment. Such agents may be useful for the prevention and treatment of biofilms in a variety of industrial and clinical settings [46
Scanning electron microscopy (SEM) was used for the evaluation of catheter surface, detection of biofilm, and studying the effect of the coating agents on biofilm development. This technique provides excellent visualization of glycocalyx, which is one of the most prominent features of biofilms and a crucial research subject in searching for alternative antimicrobial and anti-adherent agent treatment [47
]. After 24, 48, and 72 h of incubation, the samples were removed from the plastic wells, washed three times with PBS, fixed with cold methanol, and dried before microscopic examination. The samples were visualized using a HITACHI S2600N electron microscope (Chiyoda-ku, Japan), at 25 keV, in primary electron fascicle, on samples covered with a thin silver layer. The culture-based findings were substantiated by the SEM studies of colonized catheter samples, showing the gradual decrease of microbial colonization from the uncoated catheter to the catheter pieces immersed in the compound solution to the catheter pelliculized with nanofluids (nanoparticles and compound solution; Figures , , and ).
SEM images showing the uncoated catheter surface colonized with S. aureus harvested at 48 h. The images show a mature biofilm with a rich matrix (left, ×2,500; right, ×5,000).
SEM images showing compound (1b)-coated catheter surface colonized with S. aureus harvested at 48 h. The images show a dense biofilm (left, ×2,500; right, ×10,000).
SEM images showing nanofluid-coated catheter surface colonized with S. aureus harvested at 48 h. The images show a reduced biofilm with rare bacterial cells (left, ×1,500; right, ×10,000).
When HEp-2 cells were grown on slides coated with the obtained nanofluid, containing nanoparticles and compounds (1a) and (1b), no changes were observed in their morphology (Figure ). The analysis of the cell cycle of HEp-2 cells grown on slides coated with nanofluid showed no significant changes of cell cycle phases (Figure ). Only unsignificant cell death rate was induced by a concentration of 50 μg/mL (Figure ).
Inverted microscope images of HEp-2 cells grown on slides treated with the obtained nanofluid. Phase contrast microscopy, ×200; left, (1a); right, (1b).
HCT8 cell cycle analysis after 24-h development on microscopic slides coated with the obtained nanofluid. From left to right: (1a), (1b), and control.
The effects of the obtained nanofluids on HCT8 cell viability. IF, ×200; left, (1a); right, (1b).