Efficacy of Radiations against Bacterial Biofilms

Abstract

A biofilm has been defined as a community of bacteria living in organized structures at a liquid interface. Biofilms can colonize a wide range of domains, including essentially industrial sectors, different natural environments, and also biomedical environments. Bacteria in biofilms are generally well protected against environmental stresses and, as a consequence, are extremely difficult to eradicate. The current study was to investigate the efficacy of different radiations against bacterial biofilms on different surfaces. It was established that the majority of available treatments have proven less effective against pathogenic biofilms, compared to planktonic bacteria. Therefore, new biofilm treatment strategies are needed, including physical treatments such as radiations. UV LEDs offer new solutions to prevent biofilm formation on inaccessible surfaces, such as medical and food equipment and, potentially, sanitary facilities, to limit nosocomial infections, compared to continuous UV irradiation treatment. Moreover, the antimicrobial effectiveness of gamma irradiation is therefore guaranteed in the treatment of bacteria associated with a biofilm, compared to planktonic bacteria. However, limited studies have been conducted to evaluate the inactivation effect of low-energy X-rays on more resistant biofilm pathogens on food-contact surfaces.

1. Introduction

Biofilms consist of structured communities of bacteria, embedded in a self-produced polymeric matrix and adherent to inert or living surfaces [1, 2, 3]. Biofilm mode of growth is an approach in microorganisms to survive harsh growth conditions. Most microorganisms such as Pseudomonas aeruginosa [4], Staphylococcus aureus [5], and Escherichia coli [6] favor a way of life where the bacterial population is attached to a support, named sessile state, rather than free and isolated in the environment, named planktonic state. The attachment to a surface is a “survival strategy” that allows the bacteria to settle and colonize an environment. This structure represents the normal way of life of a bacterium [7]. This way of life is of great interest for the bacteria since it gives them a resistance to different sources of stress to which planktonic bacteria are sensitive [1]. In effect, bacteria in biofilms are generally well protected against environmental stresses, antibiotics [8], disinfectants, and the host immune system [9] and as a consequence are extremely difficult to eradicate [10]. Therefore, biofilms constitute a protected mode of growth that allows survival in a hostile environment. This strength is essentially due to the biofilm matrix composed of numerous polysaccharides, proteins, and extracellular DNA (eDNA) which is crucial in biofilm structural integrity [11]. Although it is widely accepted that eDNA is released primarily by cell lysis, several studies have shown that other mechanisms of active secretion may coexist [12]. This implies that eDNA is an interesting target in the control of biofilms. Numerous studies have demonstrated that biofilm formation can be prevented by enzymatic degradation of eDNA by DNase [11]. It was reported for Campylobacter.jejuni biofilm-attached to stainless steel surfaces that degradation of eDNA by exogenous addition of DNase led to rapid biofilm removal and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments, like radiations, antibiotics [13]. For UVC radiation, they target genomic DNA by forming thymidine dimers in RNA and DNA, which can interfere with transcription and replication and thus induce bacterial death [14]. For the extracellular DNA, the formation of thymidine dimers, following exposure to UVC, has no consequence on bacterial multiplication. Therefore, the presence of eDNA in the matrix can only increase the viscosity of the matrix and therefore continues to block the passage of radiation through the biofilm which limits the effectiveness of the radiations.

2. Surfaces colonized by biofilms

Biofilms can colonize a wide range of domains, including essentially industrial sectors, different natural environments (soil, sediment, etc.), and biomedical environments [15]. Many bacteria form clumps at the bottom of the containers. Then, they reach the surface of the liquid-type media. However, some bacteria such as Salmonella [16], E. coli, P. fluorescens, and Vibrio cholera produce rigid or fragile pellicle structures at air-liquid interfaces [17]. Biofilm production by the colonization of the air interface can facilitate and contribute to gas exchange while enabling the acquisition of nutrients and water from the liquid phase. The biofilms at air-liquid interfaces can cause severe problems in industrial water systems [18].

In the medical sector, microbial adhesion resulting in biofilm formation on implanted medical devices is a common occurrence and can lead to serious illness and death [19]. Implanted medical devices like intravascular catheters, urinary catheters, pacemakers, heart valves, stents, and orthopedic implants, normally used for therapeutic purposes, can also be the source of real infectious risks when colonized by bacterial biofilms [20].

3. Biofilms treatment

The majority of available antibacterial treatments have shown their effectiveness against planktonic bacteria. However, these treatments have proven to be ineffective against pathogenic biofilms [21, 22], which are thousands of times more resistant to this type of treatment [23, 24, 25]. It is therefore difficult to eradicate biofilms effectively because of the phenomenon of biofilm recalcitrance [22]. Despite the importance of biofilm treatment either in the medical or environmental sectors, studies into the effectiveness of irradiation on biofilm-associated cells are lacking. Therefore, new biofilm treatment strategies are needed, including physical treatments such as radiations. This review presents an overview of bacterial biofilm development and seeks to highlight the efficacy of radiations against bacterial biofilms.

3.1 Continuous UVC irradiation treatment efficiency on biofilms

Though germicidal UV radiation is widely applied for disinfection of water and food from planktonic bacteria, it may also be used to prevent bacterial growth and colonization on surfaces, as biofilms, within engineered systems [26]. Moreover, the UVC-based method is to be of practical use for disinfection of catheters in the clinic, as they are the major sources of infection [27]. However, higher UV doses would be required to inactivate biofilm-bound bacteria than planktonic bacteria because the biofilm would provide some degree of protection from the effects of UVC irradiation [28].

Torkzadeh et al. [26] have developed an experimental device and method to ensure the growth of biofilms in the presence of UV radiation and to measure the resulting reduction in surface biofilm growth. Under optimal growth conditions and after 48 h of growth, the reduction of the bio-volume of the E. coli surface is about 95% by a UV intensity of 50.5 μW/cm² at 254 nm, compared to the control biofilms. The UV intensity required for biofilm prevention was greater than that expected due to the UV dose–response of tested bacteria and the cumulative doses applied to the tested surfaces. This results indicate that biofilms can form even under irradiation conditions that should inactivate planktonic cells completely. This is probably due to the protective effects of colloidal material and microbial exudates, that form biofilm matrix.

In water and wastewater infrastructure, biofilms pose a real problem for disinfection. Until now, the majority of ultraviolet (UV) disinfection studies focus on planktonic bacteria, with limited attention given to UV irradiation of biofilms. Among the few outstanding studies, the study of Myriam et al. [29] focused on the study of UVC dose/biofilm production relationship for five P.aeruginosa strains, isolated from wastewater. The aim was to evaluate the impact of incremental UVC doses, up to 100 mJ.cm⁻², on the ability of Pseudomonas strains to produce biofilm, knowing that the UV dose equal to 40 mJ.cm⁻² is the dose recommended for the disinfection of water in Europe and America. The results of this study showed that biofilm production presents a progressive increase in function of an increasing of exposure UVC dose until a threshold UV dose. Moreover, the values of threshold UV doses were different in relation with the response of each bacteria strain to UVC dose (dose/response). This may be explained by the fact that intraspecific difference showed in the UV dose/response relationship is probably dependent on several factors: the degree of DNA damage induced by UV, the speed of induction of DNA repair mechanisms for each tested bacteria. On the other hand, beyond the threshold, a progressive decrease in the production of biofilm correlated with the increase of UV dose was noticed. This decrease in biofilm production can be explained by the fact that the bacterial strains have received a lethal UV dose reducing bacterial sustainability by the accumulation of photoproducts surpassing the capability of bacteria DNA repair mechanisms allowing for consequent, a decrease of biofilm formation and the weakening of this resistant structure.

The UV treatment has evolved a lot since the development of UV light sources from the conventional mercury lamp to the light-emitting diode (LED). It was established that pulsed UV can be more effective than a continuous emitting mode to control biofilms. Moreover, adaptable UV LED is promising to control biofilms in the water distribution system, according to the review of [30]. Luo et al. [30] have, recently, demonstrated that pulsed UV can be more effective than a continuous emitting mode to control biofilms, on one side and that a selective combination of UV LED wavelengths allows targeting damaged biofilm components, on the other hand.

In the medical sector, an application of radiation treatment on catheters looks promising. In this context, the study of Jimmy Bak et al. [31], who proposed a method for disinfecting the inner surface of catheters biofilm, has demonstrated that mean killing rates were 89.6% for 0.5 min exposure, 98% for 2 min exposure, and 99% for 60 min exposure. About 99% of the cells were killed with a UVC dose of 15 kJ m⁻². This dose, which is 100 to 1000 times higher than the lethal dose required for planktonic cells, is assumed to be the maximum dose necessary to avoid contamination of newly inserted catheters. The need for high doses to kill mature biofilm and the limited effect of currently available UVC light sources result in a relatively long treatment time of about 60 minutes, hence the need for new UV sources like UV LED.

Recently, Jimmy Bak et al. [31] have tested a newly developed UVC disinfection device, which can be connected to a Luer catheter hub, on polymer tubes contaminated with a wide range of either bacterium, including S. aureus, E. coli, and P. aeruginosa and fungi like Candida albicans. Their results have shown no viable counts after 2 min of radiation for bacteria. Whereas, Killing of C. albicans needs more than 20 minutes to be obtained in a UVC absorbing suspension.

On any type of surface contaminated by biofilm, the effectiveness of UVC light in inactivating biofilm-forming microorganisms is mainly due to the ability of DNA molecules to absorb UV photons between 200 and 300 nm, with an absorption peak at 260 nm, at first. Then, this uptake causes damage to the DNA by altering the pairing of nucleotide bases, creating new bonds between adjacent nucleotides on the same DNA strand. This damage occurs particularly between pyrimidine bases [32]. Therefore, to limit UV damages, bacteria generally possess molecular mechanisms to restore DNA lesions [33], which preserve the irradiated biofilm, from damage due to UVC exposure. This repair mechanism has been shown to be effective up to a threshold dose-related to a maximum accumulation of photoproducts and of reactive oxygen species, which can no longer be managed by this mechanism [29]. Our study in 2016, confirmed the oxidative stress through ROS accumulation, following UVC exposure, and has demonstrated that, in the enzymatic ROS-scavenging pathways, catalase and peroxidase enhancement improved the resistance of P. aeruginosa treated with incremental UV-C doses. However, longer exposure to UV-C rays inhibited SOD activity. This result confirms that SOD cannot efficiently remove superoxide radicals that accumulated in cells of P. aeruginosa at longer irradiation time and further confirms the inability of the repair system besides the ROS-scavenging pathways to deal with photoproducts and ROS accumulation, respectively [34, 35].

We can then conclude that the resistance of bacteria to UVC treatment remains at dose limits. Beyond these doses, there is an exhaustion of the repair system and a sure bacterial death. Hence the need to exceed the dose limits in order to escape bacterial resistance (Figure 1).

Figure 1.

UVC time/biofilm production relationship and DNA repair mechanisms.

3.2 UV LED irradiation treatment efficiency on biofilms

UV LEDs are emerging as competitive light sources because of advantages such as the possible selection of combined-wavelength UV LED [30], adjustable emitting mode, and the designable configuration that facilitate their incorporation into confined spaces. Therefore, UV LEDs offer new solutions to prevent biofilm formation on inaccessible surfaces, such as medical and food equipment and, potentially, sanitary facilities, to limit nosocomial infections. These results imply that surfaces more exposed to bacterial colonization require adequate UVC irradiation to prevent biofilm establishment. Furthermore, continuous surface irradiation may be insufficient as a sole source for biofilm prevention in many circumstances [26]. However, problems with low wall plugs and reliable power supplies still limit the effectiveness of UV LEDs, which further enlightens the prospective of UV in dealing with the biofilm issue in water infrastructure and also in the medical sector.

In this context, the study of Aikaterini et al. [36] on P. aeruginosa biofilms at different growth stages, within 24, 48, and 72 h of growth, was conducted to judge the effectiveness of ultraviolet B (UVB), at 296 nm and ultraviolet C (UVC) irradiation, with central wavelength at 266 nm, two different light-based treatments. The effectiveness of the UVB and UVC irradiations was quantified by counting colony-forming units. For UV exposure, a type of AlGaN light-emitting diodes (LEDs) was used to distribute UV irradiation on the biofilms. For P.aeruginosa biofilms, it appears that UVB irradiation is much more effective than UVC radiation for the inactivation of mature biofilms. The fact that UVB at 296 nm is present in daylight and has such a disinfecting capacity on biofilms opens the way to the treatment of infectious pathologies [36].

In parallel, the study of Gora et al. [37] has demonstrated that UV LED irradiation at 265 nm achieved 1.3 log inactivation of biofilm-bound P. aeruginosa at a UV dose of 8 mJ/cm². This inactivation level is lower than those that have been reported by researchers using UVC LEDs to inactivate planktonic P. aeruginosa, a finding that can be explained by the higher resistance of biofilm-bound bacteria to UV inactivation.

Moreover, the combination of UV LED and Blue laser was tested on S. aureus biofilm and gave the highest biofilm reduction of about 80.57%. It was then demonstrated that it to be the best choice to eradicate more biofilm [38].

Concerning the effect of radiations on biofilm matrix, it is well established that bacteria enclosed in a layer of exopolysaccharides are protected by 13% from UVC radiation. It was also confirmed that absorption of UV light by the alginate, an important matrix molecule, translated into a higher survival rate than observed with planktonic cells, for the same UV dose [39]. In effect, alginate water retention seems to be at the origin of the obvious ability to survive severe environments, like UVC exposure. On the other hand, the effect of UV LED on exopolysaccharides (EPS) has not been extensively studied, but it is predicted to be similar to the effect of continuous UVC on EPS. It is then assumed that following the prolonged exposure to UVC radiation, the production of EPS is stimulated [34]. Moreover, in the framework of the development of a profitable strategy to improve the EPS yield, UV irradiation mutagenesis of Bacillus licheniformis significantly improved the EPS yield. Significantly enhanced yield (>3-folds) of EPS after UVC exposure can only confirm the stimulating effect of UVC radiation on the production of EPS, to ensure better protection against UVC rays and then bacterial survival [40] (Table 1).

Light sources	Microorganisms	UV dose	Inactivation	Reference
UV LED	Biofilm-bound P.aeruginosa	8 mJ/cm^2 (265 nm)	1.3 log reduction	Gora et al. [37]
	Less mature P. aeruginosa biofilms (24 h grown)	72–10,000 J∕m^2	1 log; reduction	Aikaterini et al. [36]
	Mature biofilms (48 and 72 h grown)	20 000 J∕m^2	0.8 _ 0.3 log reductions
Continuous low-intensity UVC irradiation	E. coli	50.5 μW/cm^2 (254 nm)	95%	Torkzadeh et al. [26]
	Catheter biofilm	15 kJ/m^2	99%	Bak et al. [27, 31]
	Wastewater	40 mJ/cm^2

Table 1.

UV doses required for the treatment of biofilms for different microorganisms.

3.3 Ionizing radiation treatment efficiency on biofilms

Ionizing radiation is a non-thermal destruction technique that inactivates pathogens that may contaminate certain food products, by exposing them to irradiation sources such as high-energy X-rays at about 5 MeV, gamma rays at about 2.5 MeV, or electron beams at about 10 MeV [41]. Compared to these conventional high-energy irradiation techniques, low-energy X-rays have a higher linear energy transfer (LET) value, resulting in a greater relative biological effect (RBE)[42]. Some previous studies have shown that low-energy X-rays is effective in destroying certain planktonic germs such as E. coli O157:H7, Salmonella, Listeria monocytogenes, and Shigella flexneri [43, 44, 45]. However, few studies have investigated the effect of low-energy X-rays on more resistant pathogens in mono-microbial or poly-microbial cultured biofilms and on food contact surfaces.

Despite of this, we could not simply conclude that low-energy X-rays destroyed EPS in biofilm. Therefore, we could at least postulate that low-energy X-rays irradiation weakened EPS structure in biofilm. Typical EPS mainly comprises homopolymers like cellulose and dextran and heteropolymers of alginate, emulsan, gellan, and xanthan, which maintain the stability of the biofilm matrix [46]. Ionizing irradiation can break down glycosidic bonds and consequently degrade polysaccharides and destabilize the biofilm [47].

Similarly, some in vitro studies also showed that the direct effect of radiation on oral C. albicans cells leads to a rapid proliferation ability, increase of virulent factors, and resistance to drugs [48]. Moreover, irradiated Klebsiella oxytoca strains of oral origin were more virulent than non-irradiated ones [49]. All of these results indicated that direct exposure of X-rays can affect the virulence of oral bacteria microbes even at therapeutic doses [50].

Concerning gamma irradiation, it is an established technology of well-documented safety and efficacy for the inactivation of pathogenic microorganisms such as Salmonella [51, 52]. Recently, gamma-ray sterilization was proven to be a viable method of sterilization of conducting polymer-based biomaterials for biomedical applications [53].

The study of [54] has demonstrated that in bacterial biofilms attached to stainless steel, gamma irradiation at a dose of 10.0 kGy reduced the counts of S. aureus attached for 1 hr. and overnight by ≥5.1 and 5.0 log CFU/cm², respectively. Gamma irradiation at a dose of 1.0 kGy reduced the counts of P. aeruginosa counts to below the limit of detection (<2logCFU/cm²).

Concerning food sterilization, Salmonella is a problematic bacterium due to its biofilm resistance to chemical sanitizing treatments. Ionizing radiation is known to be used to inactivate Salmonella on a variety of foods and contact surfaces in the food industry. The relative efficacy of the process against biofilm-associated cells versus free-living planktonic cells was tested for three food-borne-illness-associated isolates of Salmonella, by by Niemira and Solomonet [55]. They demonstrated that the dose of radiation required to reduce 90% (D10 values) of Salmonella enterica serovar Anatum was not significantly different between biofilm-forming bacteria (0.645 kGy) and planktonic cells (0.677 kGy). In contrast, biofilm-forming cells of S. enterica serovar Stanley were significantly more sensitive to ionizing radiation, with a D10 of 0.531, than planktonic cells, with a D10 of 0.591 kGy. D10 values of S. enterica serovar Enteritidis were similarly decreased for biofilm-associated cells (0.436 kGy) in comparison to planktonic cells (0.535 kGy). The anti-microbial efficiency of ionizing radiation is therefore guaranteed in the treatment of bacteria associated with a biofilm. Ben Miloud YahiaYahia [52] proposed that the biofilm-forming abilities could be reduced with temperature decrease and increasing gamma radiation doses (Table 2).

Light sources	Microorganisms	Dose	Inactivation	Reference
Gamma irradiation	Biofilm-bound S. aureus attached for 1 h	10.0 kGy	≥5.1 log CFU/cm^2; 5.0 log	[54]
	Biofilm-bound S. aureus attached overnight		5.0 log CFU/cm^2
	Biofilm-bound P. aeruginosa	1.0 kGy	<2 log CFU/cm^2
	Salmonella enterica serovar A	0.645 kGy	1 log	[31]
	S. enterica serovar Stanley	0.531 kGy	1 log
	S. enterica serovar Enteritidis	0.436 kGy	1 log

Table 2.

Gamma irradiation and doses required for the treatment of biofilms for different microorganisms.

4. Conclusion

This study has demonstrated that ionizing and non-ionizing radiation effectively reduces the populations of both planktonic and biofilm-associated bacteria. However, biofilms are confirmed to be more difficult to eradicate and require enhanced doses for their eradication. It was also confirmed that radiation sensitivity is microorganism specific. Likewise, the influence on radiation sensitivity of the cultured state of the organism, between planktonic and biofilm-associated, is also isolate specific, confirmed for gamma-treated Salmonella. But also, the stage of biofilm growth seems to affect the effectiveness of radiations treatment, as confirmed for Pseudomonas and Staphylococcus biofilms. In general, these results show that, in contrast to chemical antimicrobial treatments, the antimicrobial efficacy of radiation is preserved or enhanced when treating biofilm-associated bacteria, compared to planktonic cells.