Abstract
Central line associated bloodstream infections (CLABSI) are commonly induced due to bacterial colonization of medical devices such as central venous catheters (CVCs) and is leading cause of concern due to increasing hospitalization duration, costs, and morbidity. This study evaluated the efficacy of boron carbon nitride (BCN) nano-coatings on CVC for antimicrobial activity. RF magnetron sputtering technique was utilized to deposit nano-coatings of BCN on CVCs. For comparison purposes, RF magnetron sputtered TiO2 nano-coatings were also investigated. Antimicrobial activity of nano-coatings was tested against gram-positive Bacillus cereus and gram-negative Escherichia coli bacterial cells. Nanoparticle coated and uncoated catheter surfaces were studied using FE-SEM and AFM to determine if the surface characteristics correlated with anti-adhesive effects of the bacteria. Biofilm formation on uncoated and BCN coated catheters was quantified using absorbance spectrophotometry.
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CLABSI remain one of the leading concerns in healthcare facilities with significant contributions to morbidity, costs and mortality.1–6 Annually over 250,000 CLABSIs occur,7 with an attributable cost per infection estimated at US $34,508–$56,000.8 The annual cost of caring for patients infected with CLABSI lie between $296 million to $2.3 billion.9 CLABSIs are the most common complication of CVCs. However, CVCs are imperative in the delivery of fluids and medications and to monitor patient health. After insertion, the catheter surfaces are rapidly covered by extracellular host proteins that create an environment conducive to microbial attachment and growth, leading to biofilm formation.10 Leading pathogens responsible for these infections include gram-positive bacteria (such as Bacillus cereus) and gram-negative bacteria (such as Escherichia coli).7,11,12 Biofilm protects these pathogens from host's immune defenses and antibiotics, often necessitating removal of the devices to avert life threatening complications.13 As bacterial biofilms make antibiotic therapies less effective, one of the promising ways of preventing infections is the development of catheter surfaces or materials that work against antimicrobial adhesion. Deposition of metal and metal oxide coatings such as Ag, Ti, TiO2 to inhibit microbial adhesion and reduce biofilm formation on catheters are reported in the literature.14–16
Boron carbon nitride (BCN) compounds have gained attention due to their unique properties such as low dielectric constant, wide bandgap, excellent thermal and mechanical strength, transparency in the optical and UV range.17–20 BCN compounds are known to combine excellent properties of boron carbide (B4C) and boron nitride (BN) with their properties adjustable depending on the composition and structure. As graphite is semi-metallic and h-BN is insulating, hybrid BCN between graphite and h-BN exhibits semiconducting properties.21 BCN has proven to be a multi-functional material finding applications in supercapacitor,22,23 UV detectors,20 anti-wear and protective coatings,24,25 and energy storage applications.26,27 Recently, BCN has been implemented for applications in water purification,28 nano-biotechnology and nanomedicine field.29,30 Although BCN has been reported as a novel surface for biological applications, the effectiveness of BCN coatings on inhibiting bacterial growth on medical devices has not been examined.
In this study, we investigated the inhibition of bacterial colonies on BCN coated CVCs for the first time. RF magnetron sputtering technique was used to deposit nano-coatings of BCN and TiO2 on CVCs. BCN coated and uncoated standard catheters were exposed to live cultures of Escherichia coli (E. coli) and Bacillus cereus (B. cereus) and colony forming assays were performed. As TiO2 is more commonly used for medical device coatings, this study provided a comparison between bacterial inhibition on BCN films and TiO2 films. Colony forming units (CFUs) for uncoated, TiO2 coated and BCN coated catheters were examined and compared. For the rest of the article uncoated catheters, TiO2 nano-coating catheters and BCN nano-coating catheters will be referred as U-C, TiO2-C and BCN-C respectively.
Materials and Methods
BCN and TiO2 nano-coatings
Nano-coatings were deposited using RF magnetron sputtering technique. This technique directs highly energetic Ar ions to bombard the surface of high purity source material to be deposited, which causes the ejection of source target atoms by momentum transfer. Since the sputtering is performed in high vacuum ambiance, ejected source material atoms are deposited onto the substrate surface, subsequently forming the required thin film. In this study, the source material was boron carbide (B4C) target and the deposition was performed in presence of inert argon and reactive nitrogen gas.
CVCs were cut into 7 cm long segments and were coated with either BCN or TiO2 respectively using an AJA ultra- high vacuum 3-gun sputtering system. For BCN films, the source material was a B4C target with 99.9% purity. The base pressure of deposition chamber was 4 × 10−7 Torr. BCN nano-coatings were deposited at a fixed pressure of 5mTorr in presence of N2/Ar (0.25) while maintaining the total gas flow at 20 sccm. RF power to the B4C target was set at 200 W. For TiO2 nano-coatings on CVCs, 99.9% purity Ti target was used as source material in presence of O2/Ar (0.25) gas. The working pressure was set as 10mTorr at 50 W RF power to the Ti target. The depositions were performed at room temperature for both BCN and TiO2 nano-coatings.
Surface characterization of the nano-coatings
Thickness of the BCN and TiO2 coatings was measured using Dektak 150 stylus profilometer (Veeco). After coating the CVCs with BCN and TiO2 layers, field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) were used for surface characterization. The FEI NOVA 430 SEM was used to observe morphological features on the coated and uncoated CVCs. Surface roughness of U-C, BCN-C and TiO2-C was determine using the SPM/AFM dimension 3100 in non-contact tapping mode. Catheter surface area of 100 μm2 were scanned for comparison. Separately, random 10 μm2 surface scans were obtained to assess peak and valley gradients.
Bacterial growth on catheters
- i.Serial dilutions of E. coli K-12 and B. cereus bacterial cultures were made with Luria broth to acquire a cell density of 5 × 104 cells ml−1. Luria broth is used for maintaining and propagating cultures of E. coli and other bacteria for microbiology procedures. One liter of this sterile medium consists of 10 grams of tryptone, 5 grams of yeast, and 10 grams of sodium chloride, with a pH of 7.0.
- ii.U-C, BCN-C and TiO2-C segments (1 cm long) were immersed in 5 ml culture in culture tubes with sufficient aeration. Culture tubes were incubated at temperatures of 37 °C and 30 °C for E. coli and B. cereus respectively for 6 h.
- iii.Using sterile forceps, catheter segments were removed from culture tubes and rinsed in tubes of sterile water, then rolled onto the surface of nutrient agar plates for E. coli K-12 and blood agar plates for B. cereus (see Figs. 2a–2f). This technique is known as the roll plate method, and is described in detail elsewhere.3,31
- iv.Culture plates were covered and incubated for 24 h at 37 °C and 30 °C for E. coli and B. cereus respectively. Number of colonies per plate were counted manually and recorded. Experimental values were obtained in triplicates for both E. coli K-12 and B. cereus.
Biofilm formation and analysis
Biofilm growth
- i.Serial dilutions of E. coli K-12 and B. cereus bacterial cultures were made with Luria broth to acquire a cell density of 5 × 106 cells ml−1.
- ii.U-C and BCN-C (1 cm long) were immersed in 5 ml culture in culture tubes with sufficient aeration. Culture tubes were incubated at 37 °C for 6 h. All experiments were performed in triplicates.
- iii.Catheter segments were removed and rinsed in sterile water. Rinsed catheters were inserted into culture tubes containing 2 ml of Luria broth and incubated at temperatures of 37 °C and 30 °C for E. coli and B. cereus respectively for 24 h.
Staining the biofilm
- i.Catheter segments were removed and rinsed in sterile water.
- ii.U-C and BCN-C segments were immersed in tubes containing 0.1% crystal violent solution for 5 min.
- iii.Crystal violet stained catheter segments were rinsed in sterile water and transferred to separate wells of a 96 well microtiter plate.
Quantifying the biofilm
- i.125 μl of 30% acetic acid was dripped on to the surface of each catheter, allowing for the adhered biofilm to solubilize into their respective wells.
- ii.In order to quantify the biofilm, absorbance was performed at 550 nm using SpectraMax iD3 and iD5 multi-mode microplate readers (Molecular Devices). Optical densities of E. coli and B. cereus biofilms on U-C and BCN-C were recorded. 30% acetic acid was used as the blank to record optical density.
Statistical analysis
The data presented in Fig. 2 is the average of three independent experiments. One tailed student t-test analysis was performed and the probability value (p-value) is reported. Significant difference with p < 0.05 are marked with * and highly significant difference with p < 0.01 are marked with **.
Results and Discussion
Surface characterization of nano-coatings
The film thickness of BCN and TiO2 coatings ranged between 80–90 nm. Surface morphology of the deposited nano-coatings was determined before the evaluation of antimicrobial activity. Figures 1a–1c shows the FESEM pictures of U-C, TiO2-C and BCN-C surfaces respectively. U-C exhibited smooth surface morphology. Compared to U-C and TiO2-C, BCN-C surface displayed significant nano-protrusions. Given the BCN-C surface differences observed in FESEM, non-contact AFM characterization was performed to evaluate surface roughness of U-C and BCN-C. Figures 1d, 1e shows the AFM scans of U-C and BCN-C conducted on catheter surfaces. The surface roughness of U-C was recorded to be 41.7 nm whereas BCN-C exhibited surface roughness of 90.7 nm. In separate 10 μm2 surface scans, BCN-C exhibited 15-fold higher peak gradient difference as compared to U-C.
Figure 1. FESEM images of (a) UC, (b) TiO2C and (c) BCN-C. AFM surface topography of (d) UC and (e) BCN-C.
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Standard image High-resolution imageInhibitory effects of bacterial growth on BCN-C
Table I shows the CFUs of E. coli bacteria for UC, TiO2-C and BCN-C recorded after 24 h of incubation. Results of three individual experiments are displayed. The uncoated catheters demonstrated the highest number of bacterial CFUs. TiO2-C demonstrated slightly lower CFUs compared to U-C, however, this difference was not significant implying that TiO2 coating did not inhibit colonization by E. coli. BCN-C demonstrated 81.14% reduction in E. coli CFUs when compared to the U-C, thus confirming the anti-bacterial activity of BCN coatings.
Table I. CFUs of E. coli on UC, TiO2-C and BCN-C.
| Trial | Number of CFUs | Average % Decrease in CFU | |||
|---|---|---|---|---|---|
| U-C | TiO2-C | BCN-C | TiO2-C | BCN-C | |
| 1 | 29 | 24 | 3 | 19.19 | 81.14 |
| 2 | 26 | 20 | 4 | ||
| 3 | 34 | 28 | 10 | ||
| Average | 29.7 | 24.0 | 5.6 | ||
Table II shows the CFUs of B. cereus bacteria recorded for U-C, TiO2-C and BCN-C after incubation. Experimental results of three individual experiments are displayed. It can be seen that the average CFUs of U-C was 104.3 which reduced to 84.3 for TiO2-C, and 3 for BCN-C. Although TiO2-C exhibited only 19.17% decrease in bacterial colonies, BCN-C exhibited an excellent 97.12% decrease in B. cereus bacterial colonization. This reduction observed in bacterial activity of E. coli and B. cereus on BCN-C could be attributed to high surface roughness of BCN-C. The correlation between bacterial adhesion and surface roughness has been investigated.32 A surface under magnification appears as a series of peaks and valleys with varying height and spacing. Surface roughness is determined by the height variation between these peaks and valleys on the surface of interest. It is commonly accepted that nanorough surfaces have a lower probability of bacterial adhesion when compared to smooth surfaces.33 Similar trend in surface roughness of coatings and bacterial adhesion has been reported.34–37 Figures 2a–2c shows E. coli bacterial colonies after rolling the catheters onto the surface of nutrient agar plates. Figures 2d–2f shows B. cereus bacterial colonies after rolling the catheters onto the surface of blood agar plates. Figures 2g, 2h shows the average number of CFUs plotted for the three types of catheters examined in this study. Reduction in bacterial CFUs on BCN-C was statistically significant with p value of 0.0008 for E. coli. Similarly, BCN-C displayed excellent reduction in bacterial CFUs for B. cereus with highly significant p value of 0.00004.
Table II. CFUs of B. cereus on U-C, TiO2-C and BCN-C.
| Trial | Number of CFUs | Average % Decrease in CFUs | |||
|---|---|---|---|---|---|
| U-C | TiO2-C | BCN-C | TiO2-C | BCN-C | |
| 1 | 96 | 67 | 1 | 19.17 | 97.12 |
| 2 | 101 | 88 | 2 | ||
| 3 | 116 | 98 | 6 | ||
| Average | 104.3 | 84.3 | 3.0 | ||
Figure 2. Microbial activity of (a) U-C, (b) TiO2-C, and (c) BCN-C after rolling the catheters onto the surface of nutrient agar plate for E. coli. Microbial activity of (d) U-C, (e) TiO2-C, and (f) BCN-C after rolling the catheters onto the surface of blood agar plates for B. cereus. Bar diagram demonstrating bacterial inhibition of E. coli (g) and B. cereus (h) for all the catheters. * represents a statistically significant difference of p < 0.05 when compared to the control sample which is U-C. ** represents p < 0.01.
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Biofilm formation
BCN-C exhibited excellent bacterial inhibition when compared to TiO2. To further study the strong inhibition of E. coli and B. cereus on BCN nanocoated catheters, biofilm formation was quantified using absorbance spectrophotometry. Table III displays the optical density values of the stained biofilms formed by E. coli and B. cereus. BCN-C reduced biofilm formation by 62.13% for E. coli and 75.52% for B. cereus. This biofilm inhibitory property of BCN nano-coatings can be attributed to the surface roughness which impacts the surface topography. As BCN-C surface topographic features are in nanometric range, which is much smaller than the microbial cell size, there is a significant decrease in the contact area between bacterial cell and the nanocoated surface. This may influence the activation of bacterial adhesion genes and genes that secrete extracellular matrix, thereby affecting cell attachment and subsequent biofilm formation on BCN-C. There is increasing evidence that bacterial adhesion and subsequent biofilm formation are greatly impacted by the surface topography.32,38,39 These results indicate that BCN nano-coatings demonstrates antibiofilm activity which will reduce colonization rates on CVCs and thereby could reduce the incidence of CLABSIs, which will reduce antibiotic use and deter the development of antibiotic resistant organisms.
Table III. Optical density (OD) of E. coli and B. cereus of BCN-C in comparison with U-C.
| Trial | OD of E. coli Stained Catheters | % Decrease in E. coli OD | OD of B. cereus Stained Catheters | % Decrease in B. cereus OD | ||
|---|---|---|---|---|---|---|
| U-C | BCN-C | U-C | BCN-C | |||
| 1 | 0.4868 | 0.1839 | 62.13 | 0.6398 | 0.1451 | 75.52 |
| 2 | 0.5006 | 0.1613 | 0.5308 | 0.1271 | ||
| 3 | 0.4680 | 0.2070 | 0.5964 | 0.1589 | ||
| Average | 0.4851 | 0.1837 | 0.5880 | 0.1439 | ||
Conclusions
For the first time, this study has discovered the inhibitory effects of BCN nano-coatings on bacterial growth and biofilm formation on CVCs. BCN nano-coatings were synthesized using RF sputtering technique. BCN-C reduced bacterial CFUs by 81.14% for E. coli and 97.12% for B. cereus. TiO2-C was used in this study for comparison and reduced bacterial CFUs by only 19% for both E. coli and B. cereus. BCN nano-coatings suppressed E. coli biofilm formation by 62.13% and B. cereus biofilm formation by 75.52%. The bacterial inhibition and antibiofilm ability of BCN-C maybe attributed to the surface roughness of CVCs. These observed properties of BCN nano-coating should be explored further to reduce catheter and other medical device associated infections.