UV sterilization of biofilm contaminated polymer tubes
We have demonstrated that UVC light from LEDs can be launched into and transmitted through tubes made of teflon, polyurethane and silicone. We show that the light transmitted through the tubes is attenuated exponentially. It shown experimentally that the teflon material is very suitable for UVC light guidance because of its low refractive index as compared to the other tube materials. The disinfection efficiency of the LED based light source is demonstrated on silicone catheter and teflon tubes contaminated at the inner lumen surface with a Pseudomonas aeruginosa biofilm produced in a flow system.
Contaminating the tubes
A peristaltic pump was used to maintain a constant flow through tubes placed in parallel. The flow rate was maintained at 20 ml/min during all biofilm growth experiments in all tubes (control and UVC treatment). A Pseudomonas aeruginosa culture (clinical strain, isolate from a patient with urinary infection, 5 ml 105 CFU/ml in 5% serum bouillon) was diluted in 100 ml nutrient (serum bouillon). This solution was next further diluted in one liter of 0.9% saline solution. During the contamination procedure the pump system was run in four half hour time intervals (total 2 hours duration daily) during three days at a constant temperature (37º C). With a parallel (or serial) set-up tubes for control measurement and UVC treatments comparable levels of biofilm could be obtained.
UVC light propagation set-up
Figure 1 shows the experimental set-up for measuring the transmittance, T = Iout/Iin, through all the tube samples. Exactly the same set-up is used for the later disinfection experiments on Pseudomonas aeruginosa contaminated tubes. The tubes are placed and fixed on a small table. The UVC LED light source is fixed in a housing for positioning and making electrical contact to a power supply (6 V).
A ball lens placed on top of LED diode focuses the light into a small spot 1.5-2 mm in diameter at with a focal length of a few millimeters. The angle of the light cone launched into the tube openings is approximately 6 degrees. The measured total UVC output was 0.25 mW.
UVC treatment of tubes with Pseudomonas aeruginosa biofilm and culturing
After biofilm formation the tubes were flushed with sterile water in order to remove the residual bacteria solution from the tube lumen. Next the tubes were filled with a sodium chloride solution (20 % wt. NaCl). The tubes for UVC treatment were then placed in the set-up shown in figure 1. UVC doses ranging from 15 to 300 minutes were applied to the tubes. After UVC treatment tubes (pairs of control and UVC treated) were flushed with sterile water before biofilm was sampled. A brush (pipet brush) was used to remove biofilm from both UVC treated and control tubes. The collected biofilm sample was flushed into a test tube and suspended into 5 ml of nutrient broth and shaken in a Whirley mixer for 2 min. Ten-fold dilutions of each sample were repeatedly made in 0.9 % saline solutions (0.5 ml from the first suspension to 4.5 ml saline). 0.2 ml of each of these dilutions were spread on blood agar plates and incubated aerobically at 37 C for 24 hours. After incubation, the number of colony forming units (CFUs) was determined for the UVC-treated biofilm samples and controls. The counted number of CFUs ranged from < 5 CFU/ml (detection limit – first dilution) to a total of 1.3 × 109 CFUs/ml.
UVC Transmittance of teflon and catheter tubes
Tubes made of different polymer materials are not expected to transmit the UVC light with equal efficiency. The measured transmittance data expressed as –ln(T) for the various tube materials and lengths is plotted in figure 2. It is observed from the figure that the attenuation, expressed as ‘-ln(T)= a×L’ is linear. The loss through the tubes is therefore well described by an exponential function. The attenuation of the light at distance ‘L’ through the tube can then be expressed as: I = I0 exp[-a×L]. Here ‘I0’ is the input intensity after the stop and ‘I’ is the intensity before leaving the last stop (detector reading corrected for the loss through the last stop). The constant ‘a’ is a damping parameter which takes into account all possible loss mechanisms such as: absorption in the tube material, light scattering due to the roughness of the tube surfaces and the relative refractive index of the tube and saline solution. If biofilm is present on the inner surface intensity loss due to absorption and scattering from biofilm and cellular components is expected too. It is apparent form the plot that the low refractive material FEP teflon performs much better than the real catheter material made of silicone and PUR. In the specific case where both tubes are filled with a 20 % NaCl solution the teflon material transmits 39.4 % and 6.5 % is transmitted through a silicone/PUR tube with equal length (10 cm). The attenuation of the UVC light through the silicone and PUR tubes seems to be comparable (same slope).
The influence of the sodium chloride concentration i.e. the higher refractive index of the solution relative to that of the tube material can be demonstrated at visible wavelengths. Figure 3 depicts the light distribution and transmission of blue laser light in a FEP teflon tube filled with a 20 % NaCl solution (refractive index n ~ 1.37) (photo shown to the left) and in the same tube filled with pure water shown at right (n=1.33). The FEP teflon has a refractive index of 1.338 at visible wavelengths. It is seen that parts of the light launched into the tube filled with pure water is lost (transmitted through the polymer wall) close to the point where it enters the tube. In comparison the light is transmitted and distributed much more evenly an efficiently downwards inside the tube containing the solution with the high refractive index.
Disinfection of tube lumens with UVC LED exposure
The germicidal effect of the UVC light on Pseudomonas aeruginosa biofilm in contaminated tubes is shown in table I. The number of CFU/ml found on the control tubes varies up to four orders of magnitude ranging from 5×105 – 1.3×109 CFU/ml. Different numbers of bacteria at start and slightly different conditions for growth during the contamination procedure with the flow system are probably the main reason for the differences in number of CFU on the control samples. Varying treatment times (15 – 300 min) corresponding to doses in the range 0.1 – 2.1 J (15 × 60 s × 117×10-6 J/s = 0.1 J) were applied. The efficiency of the UVC light was tested at two different tube lengths (10 and 20 cm) . It is observed that relative low doses are required to effectively kill the Pseudomonas aeruginosa in 10 cm teflon tubes (logCFU reduction = 6.78 for a 15 min dose). It is observed that a substantially higher dose is required to obtain the same killing rate for a 20 cm teflon tube (300 min). This result is supported by the exponential attenuation of the UVC light towards the distal of the tube. From figure 2 it is seen, that the intensity at the end of the 20 cm teflon tube (15.5 %) is approximately a factor of 2.5 weaker than at the end of the 10 cm tube (39.5 %). It is expected then that the exposure time should be increased by the same factor in order to obtain the same disinfection rates as those obtained for the 10 cm tubes (i.e. at least 45 min). Finally a part (10 cm) from a peritoneal catheter made of silicone was contaminated and UVC treated. A reasonable disinfection rate is obtained for this high refractive index material (logCFU reduction = 4.00 i.e. 99.99 %). The applied dose is, however, substantially higher compared to what was necessary for the 10 cm teflon tubes and the start CFU are smallest of the numbers found on all the tubes. From figure 3 it is found that the difference in output intensity between the two tube materials of equal lengths (10 cm) is approximately a factor of 6 (exp[-0.094 ×10]/exp[-0.273×10]). We therefore expect that the treatment times should be at least a factor of 6 longer for the silicone/PUR tubes compared to the teflon tubes in order to obtain comparable disinfection rates.
|
Material
(length/cm) |
Teflon
(10) |
Teflon
(10) |
Teflon
(10) |
Teflon
(10) |
Teflon
(20) |
Teflon
(20) |
Silicone
(10) |
|
CFU before
(control) |
3´108 |
7.5´106 |
1.5´106 |
1.5´108 |
1.5´108 |
1.3´109 |
5´105 |
|
UVC dose (min) |
15 |
30 |
80 |
300 |
30 |
300 |
300 |
|
CFU after
UVC exposure |
50 |
<5 |
<5 |
<5 |
5.5´106 |
<5 |
50 |
|
Disinfection rate (%) |
99.99 |
100 |
100 |
100 |
96 |
100 |
99.99 |
Doses for disinfection and eradication of early Pseudomonas aeruginosa biofilm
As can be seen from table I it is possible with at treatment time of 15 min to obtain the high disinfection efficiency with a contamination from start at 108 CFU/ml. The average UVC power entering the tubes was measured to be ~ 110 microwatt. The total inner surface area is 2×pi×R×L = 2×pi×2mm×100mm = 1257 mm2 ~12.6 cm2. The total dose to obtain 99.99 % killing in the teflon tubes contaminated with Pseudomonas is then found to be: Dose = 110-6 J s-1×15×60 s/12.6×10-4 m2 = 78.6 J m-2 = 7.86 mJ/cm-2. This dose is a factor of 250 less than that required for disinfection of the mature biofilm formed on the inner surface of urinary catheters. The Pseudomonas aeruginosa biofilm do not contain any other major scattering and absorbing components other than the bacterial cells. Therefore, high disinfection efficiency due to a negligible loss of power in the pure culture Pseudomonas biofilm could be the main reason for this difference. The tabulated value for 99.9 % killing of Pseudomonas in the planktonic state (105 J m-2 = 10.5 mJ/cm2, Bacteria Destruction Chart. UVP Ultraviolet Products. http://www.uvp.com) is close to what is found for the biofilm we had grown on the teflon tubes. One explanation for this similarity in lethal doses could be that the biofilm formed on the polymer tubes is more or less transparent in the UVC region allowing the germicidal rays to penetrate the entire biofilm. Furthermore, it is difficult to compare the effective doses per unit area delivered through the tube opening by the LED with those given on the urinary catheter pieces by the mercury lamp reported earlier. The effective lethal dose experienced by the microorganisms hosting the tube is expected to be higher because internal reflections mostly due to the light guide principle causes the light ray to be recycled several times before it is absorbed and lost in the polymer surface. In the urinary catheter experiments with the mercury lamp reported earlier the UVC rays were orthogonal to the catheter samples. Only in these cases where the rays were impinging onto the cells possible killing could occur. Otherwise the light was absorbed immediately in the polymer material beneath the biofilm. If it is assumed that the Pseudomonas biofilm generated in our experiments is close to the biofilm formed in newly placed patient CVC catheters the LED light source could have a potential for use on newly inserted catheters. If the UVC doses are administered frequently from start it may be possible to avoid or at least disinfect thin biofilm within relative short treatment times (~ 30-60 min). The number of CFU/ml generated in our experimental set-up is comparable those obtained in other test devices and observed on real patient catheters. The total CFU on our 10 cm tubes vary between 5×105 – 3×108 CFU/ml. The biofilm samples were suspended in 5 ml of sterile water which gives a total CFU between 2.5×106 – 1.5×109 CFU for the entire tubes. The numbers are then 2×105 – 1.2×108 CFU/cm2 (inner surface area = 12. 6 cm2).
References
Bak, J.; Ladefoged, S.; Tvede, M.; Begovic, T.; Gregersen, A.; Disinection of Pseudomonas aeruginosa biofilm contaminated tube lumens with ultraviolet C light emitting diodes Biofouling Vol. 26 , No. 1 (2010) 31-38.
Bak, J.; Spanget-Larsen, J., Molecular, vibrational structure of the extracellular bacterial signal compound N-butyryl-homoserine lactone (C4-HSL). Vibrational Spectroscopy 49 (2009) 237-241
Bak, J.; Ladefoged, S.;Tvede, M.; Begovic, T.; Gregersen, A.; Dose requirements for UVC disinfection of catheter biofilms. Biofouling Vol. 25 , No.4 (2009) 289-296