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BIOFILM RESISTANCE TO HYDROGEN PEROXIDE  

Many chemicals are used in the food industry on a daily basis: cleaning products, disinfectants, antimicrobial soaps, antiseptics, antibiotics, etc. But how does the use of these biocides affect the resistance of bacteria? This concern has grown enormously in recent years because of the increasing number of resistant strains and their ability to survive in the food industry.1 In fact, it has been proven that bacteria can withstand biocides when they live in a biofilm.2

Bacterial resistance

Research into the resistance of bacteria to biocides has been going on since the 1950s.3 Today, the ability of microorganisms to adapt to their environment very quickly by developing resistance to aggressive external factors is well known. Bacteria become increasingly resistant in the presence of biocides that only partially destroy the bacterial flora.4 A recent study 5 has shown that certain biocides are ineffective against bacteria in a biofilm, even when they are effective against the same type of bacteria in planktonic form.

                                                                                                                         1  J.M.,  La  résistance  face  aux  biocides,  in  Microbiologie,  Services  n°0214,  March  2011  2  LEUNG  CY,  CHAN  YC,  SAMARANAYAKE  LP,  SENEVIRATNE  CJ,  Biocide  resistance  of  Candida  and  Escherichia  coli  biofilms  is  associated  with  higher  antioxidative  capacities,  in  J  Hosp  Infect.,  81(2):79-­‐86,  May  2012  3  EUROPEAN  COMMISSION,  Directorate-­‐General  for  Health&Consumers,  Assessment  of  the  Antibiotic  Resistance  Effects  of  Biocides,  Scientific  Committee  on  Emerging  and  Newly  Identified  Health  Risks  SCENIHR,  January  2009  4  BRIANDET  R,  FECHNER  L,  NAITALI  M,  DREANNO  C,  Biofilms,  quand  les  microbes  s’organisent,  Carnets  de  Sciences,  Editions  Quae,  2012  5  SMITH  K,  HUNTER  IS.,Efficacy  of  common  hospital  biocides  with  biofilms  of  multi-­‐drug  resistant  clinical  isolates,  in  J  Med  Microbiol  2008;  57:966-­‐73.  

Over the years, bacteria have been increasing their resistance to biocides by developing highly effective defence mechanisms, such as biofilm. Biofilm,

however, is how most microorganisms live. There are actually 1000 to 10,000 times more bacteria growing in biofilms than there are in planktonic cells. In a biofilm, the bacteria protect themselves with a layer of polysaccharides. Because

of this exopolymer matrix, biofilm can be a thousand times more resistant to biocides thank planktonic cells. Several studies, confirmed by experiment, have shown how ineffective biocides, including hydrogen peroxide, is at completely

eliminating a biofilm. In fact, after a continuous 50 mM dose of hydrogen peroxide for one hour, the integrity of the biofilm remains more or less intact, and almost 80% of the cells survive. Tests also demonstrate the effectiveness of Realco

enzymatic treatment followed by a disinfection phase in eliminating the biofilm completely.

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Bacterial flora have implemented a range of defence mechanisms against biocides:6

1. Reduction of membrane permeability: The bacteria prevent the biocide from entering by altering the permeability of its cytoplasmic membrane. The modification of the permeability limits the quantity of biocides in the cell, which reduces the effective concentration of the biocide. The biocide then becomes ineffective, because the lethal dose is not reached.

2. Biocide efflux: Bacteria use efflux pumps with the ability to expel certain active substances. The efflux pump is a pump located in the cytoplasmic membrane. It prevents the biocide from reaching its target by pumping it out of the bacteria. The biocide can then no longer reach the target in sufficient quantity to destroy the bacteria.

3. Enzymatic transformation of biocides: The bacteria produce new enzymes capable of de-activating the biocide (such as catalase, which degrades the hydrogen peroxide). The biocide then becomes disabled.

4. Mutation of the biocide's target: Each biocide acts by targeting a specific part of the cell: wall, ribosome, etc. The presence of a modification caused by a mutation changes the attachment site and the bond of the biocide. The biocide then becomes useless.

In addition to these four mechanisms, there is another very effective defence mechanism, called a biofilm, which is able to combine all the resistance mechanisms described so far but also has the following characteristic: the bacteria protect themselves with a layer of polysaccharides they secrete, and grow in the form of colonies.

Biofilm

The polysaccharide layer surrounding the bacteria of a biofilm is very difficult, or even impossible, to destroy using traditional methods of disinfection.7 And yet biofilms provide a way of life for most microorganisms.8 There are actually 1000 to 10,000 times more bacteria growing in biofilms than there are in planktonic cells.9 A bacterial biofilm can be a thousand times more resistant to biocides than planktonic cells are.10 This is mainly due to the matrix of exopolymers surrounding the bacteria of the biofilm.11

In view of how difficult it is to eradicate biofilm, very few laboratories perform tests to evaluate the effectiveness of biocides in eliminating it. There are currently no European standards on testing the performance of disinfectants against biofilms in the food industry.12 However, the small number of tests conducted reveal alarming levels of resistance to disinfectants and antibiotics. The biofilm matrix is seen as a barrier to diffusion.13

                                                                                                                         6  EUROPEAN  COMMISSION,  Directorate-­‐General  for  Health&Consumers,  Assessment  of  the  Antibiotic  Resistance  Effects  of  Biocides,  Scientific  Committee  on  Emerging  and  Newly  Identified  Health  Risks  SCENIHR,  January  2009  7  BRIANDET  R,  FECHNER  L,  NAITALI  M,  DREANNO  C,  Biofilms,  quand  les  microbes  s’organisent,  Carnets  de  Sciences,  Editions  Quae,  2012  8  BRIANDET  R,  FECHNER  L,  NAITALI  M,  DREANNO  C,  Biofilms,  quand  les  microbes  s’organisent,  Carnets  de  Sciences,  Editions  Quae,  2012  9  COSTERTON  JW,  GEESEY  GG,  CHENG  Cheng  K-­‐J,  How  bacteria  stick,  in  Sci.Amer,  238:86-­‐95,  1978  10  ROUX  A,  GHIGO  JM,  Les  biofilms  bactériens,  in  Bull.  Acad.  Vét.  France,  Vol.  159,  n°3,  2006  11  BRIANDET  R,  FECHNER  L,  NAITALI  M,  DREANNO  C,  Biofilms,  quand  les  microbes  s’organisent,  Carnets  de  Sciences,  Editions  Quae,  2012  12  EUROPEAN  COMMISSION,  Directorate-­‐General  for  Health&Consumers,  Assessment  of  the  Antibiotic  Resistance  Effects  of  Biocides,  Scientific  Committee  on  Emerging  and  Newly  Identified  Health  Risks  SCENIHR,  January  2009  13  BRIANDET  R,  FECHNER  L,  NAITALI  M,  DREANNO  C,  Biofilms,  quand  les  microbes  s’organisent,  Carnets  de  Sciences,  Editions  Quae,  2012  

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Photo: The five stages of biofilm growth on a hard surface: initial attachment, irreversible attachment, appearance and "Maturation I" of the biofilm, Maturation II, erosion and dispersion/autogenous detachment. The photomicrographs (all on the same scale) show the growth of a Pseudomonas aeruginosa biofilm.

Biofilm resistance to hydrogen peroxide

Several studies (Journal of Bacteriology14, Applied and Environmental Microbiology15 and Journal of Hospital Infection16) have shown that biofilm is resistant to hydrogen peroxide. The study published in Applied and Environmental Microbiology17 analysed the role of two catalases in Pseudomonas aeruginosa bacteria in protecting the cells of a biofilm from hydrogen peroxide. In the presence of hydrogen peroxide, the bacteria release two enzymes: KatA and KatB. Their role is to neutralise this biocide. This study was carried out after one hour of exposure to a continuous 50 mM dose of hydrogen peroxide. It showed that the integrity of the biofilm remains more or less intact, and almost 80% of the cells survive. Even in the absence of catalase activity, biofilms are still relatively resistant to hydrogen peroxide.

FIG. 1: The hollow bars represent the percentage of viability of the remaining cells, and the solid bars represent the percentage of biofilm eliminated.18

Another study, published in the Journal of Hospital Infection19, also showed that after five minutes of exposure to recommended concentrations of hydrogen peroxide (0.017% to 8.75%) during clinical disinfection, microbial cells remain intact and alive.

                                                                                                                         14  KHAKIMOVA  M,  AHLGREN  HG,  HARRISON  JJ,  ENGLISH  AM,  NGUYEN  D,  The  stringent  response  controls  catalases  in  Pseudomonas  aeruginosa  and  is  required  for  hydrogen  peroxide  and  antibiotic  tolerance,  in  J.  Bacteriol.,  195(9):2011,  March  2013  15  ELKINS  JG,  HASSETT  DJ,  STEWART  PS,  SCHWEIZER  HP,  MCDERMOTT  TR,  Protective  role  of  catalase  in  Pseudomonas  aeruginosa  biofilm  resistance  to  hydrogen  peroxide,  in  Appl  Environ  Microbiol.,  65(10):4594-­‐600,  Oct  1999  16  LEUNG  CY,  CHAN  YC,  SAMARANAYAKE  LP,  SENEVIRATNE  CJ,  Biocide  resistance  of  Candida  and  Escherichia  coli  biofilms  is  associated  with  higher  antioxidative  capacities,  in  J  Hosp  Infect.,  81(2):79-­‐86,  May  2012  17  ELKINS  JG,  HASSETT  DJ,  STEWART  PS,  SCHWEIZER  HP,  MCDERMOTT  TR,  Protective  role  of  catalase  in  Pseudomonas  aeruginosa  biofilm  resistance  to  hydrogen  peroxide,  in  Appl  Environ  Microbiol.,  65(10):4594-­‐600,  Oct  1999  18  ELKINS  JG,  HASSETT  DJ,  STEWART  PS,  SCHWEIZER  HP,  MCDERMOTT  TR,  Protective  role  of  catalase  in  Pseudomonas  aeruginosa  biofilm  resistance  to  hydrogen  peroxide,  in  Appl  Environ  Microbiol.,  65(10):4594-­‐600,  Oct  1999  

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FIG. 220: Laser scanning confocal microscopy

Enzyme-based treatment

Because of the ineffectiveness of disinfectants, and particularly hydrogen peroxide, in dealing with biofilm, other methods have been investigated.21 Enzyme-based treatment seems to be an effective solution in eliminating biofilm.22 However, only a complex and very specific cocktail of enzymes will be able to break down the biofilm's protective matrix. A disinfection phase is then applied to destroy the bacteria, which have become planktonic and therefore sensitive to biocides.

Practical validation: the ineffectiveness of biocides (hydrogen peroxide in this case) in eliminating a biofilm23

For this practical case, we use hydrogen peroxide-based Sterilex products to evaluate the effectiveness of a biocide on the elimination of the biofilm.

Protocol

                                                                                                                                                                                                                                                                                                                                                                                           19  LEUNG  CY,  CHAN  YC,  SAMARANAYAKE  LP,    SENEVIRATNE  CJ,  Biocide  resistance  of  Candida  and  Escherichia  coli  biofilms  is  associated  with  higher  antioxidative  capacities,  in  J  Hosp  Infect.,  81(2):79-­‐86,  May  2012  20  LEUNG  CY,  CHAN  YC,  SAMARANAYAKE  LP,  SENEVIRATNE  CJ,  Biocide  resistance  of  Candida  and  Escherichia  coli  biofilms  is  associated  with  higher  antioxidative  capacities,  in  J  Hosp  Infect.,  81(2):79-­‐86,  May  2012  21  LEQUETTE  Y.,  BOELS  G,  CLARISSE  M,  FAILLE  C.  ,  Using  enzymes  to  remove  biofilms  of  bacterial  isolates  sampled  in  the  food-­‐industry,  in  Biofouling,  26(4):421-­‐31.,  2010  May  22  BRIANDET  R,  FECHNER  L,  NAITALI  M,  DREANNO  C,  Biofilms,  quand  les  microbes  s’organisent,  Carnets  de  Sciences,  Editions  Quae,  2012  23  Lab  report  issued  by  Realco,  Biofilm  comparison,  Sterilex  vs  Enzyfoam,  27/05/2014  

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1) Biofilm preparation

The biofilm is formed dynamically in the laboratory on PTFE (polytetrafluoroethylene) tubes at 30°C with Pseudomonas fluorescens. After seven days of circulation, we can consider that the biofilm has formed in the tube. The PTFE tube is then cut into several segments 7 cm in length. For each product, three non-consecutive pieces of tube are chosen. These are then connected to the peristaltic pump for the cleaning in circulation tests.

2) Elimination of biofilm • Water rinse: five minutes at ambient temperature. • Application of Sterilex Ultra Disinfectant Cleaner Solution 1 to 10% + Sterilex

Ultra Activator 10% • Cleaning in circulation time tested at 10, 15, and 20 minutes. • Start temperature of water: 50°C

3) Detection of biofilm • The PTFE tubes are cut longitudinally to obtain a good view inside the tube. The

residual biofilm is then coloured using the Biofilm Detection Kit*, which makes any biofilms that are present visible.

• 5 minutes soaking in REAGENT 1 (a product from the Biofilm Detection Kit) • 5 minutes soaking in REAGENT 2 (a product from the Biofilm Detection Kit)

Results

1) Control

Photo 1. Biofilm coloration before cleaning

2) Rinsing with 50°C water

                                                                                                                         *  The  Biofilm  Detection  Kit  instantly  colours  the  biofilm's  protective  matrix.  It  has  been  approved  by  the  INRA  (French  National  Institute  for  Agronomy  Research)  and    Biotech-­‐Germande  (a  laboratory  specialising  in  the  field  of  hospital  hygiene).    

 

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Photo 2. Coloration of residual biofilm after 50°C water rinse. Left to right: 10, 15 and 20 minutes of circulation

3) Application of Sterilex Ultra Disinfectant Cleaner Solution 1 to 10% + Sterilex Ultra Activator 10%

Photo 3. Coloration of residual biofilm after cleaning with Sterilex products. Left to right: 10, 15 and 20 minutes of circulation

Tubes cleaned with the mixture of Sterilex products still have quite a significant quantity of biofilm (blue colouring) even after 20 minutes of cleaning. There is no major difference between the 10, 15, and 20-minute cleaning times.

Realco's enzymatic cocktail was tested in parallel, according to the same protocol and under the same conditions. The only difference was in the (lower) dosage used: Enzyfoam was used at 1%, and Biorem 20 at 0.2%.

 

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4) Application of Enzyfoam 1% + Biorem 20 0.2%

 Photo 4. Coloration of residual biofilm after cleaning with Realco products. Left to right: 10, 15 and 20 minutes of circulation  

The complex and very specific Realco enzymatic treatment eliminated the EPS matrix in 10 minutes. After a targeted and controlled disinfection phase, the biofilm will be completely eliminated.

Conclusion of the experiment

In circulation, the mixture of enzymatic products is considerably more effective in eliminating a biofilm than the mixture based on hydrogen peroxide.

General Conclusion

  As seen in this article, biofilm accounts for the way of life of most microorganisms. There are 1000 to 10,000 times more bacteria growing in biofilms than there are in planktonic cells. The ability to eliminate biofilm is therefore extremely important, because biofilm can have serious consequences in the food industry, leading to significant financial losses. Several reviews of the literature and a laboratory study, however, have shown that biocides, especially hydrogen peroxide, are ineffective in eliminating biofilm completely. In a biofilm, the bacteria are protected by a layer of polysaccharides, which is an extremely difficult barrier to destroy. Laboratory experiment have shown that Realco's enzymatic treatment was the only one to be able to eliminate this barrier. Once the matrix destroyed, a disinfection phase is necessary in order to eliminate the bacteria that have been released.