assessment of enzymatic cleaning agents and disinfectants ...csps/jpps7(1)/f.atroshi/biofilm.pdf ·...

J Pharm Pharmaceut Sci (www.ualberta.ca/~csps) 7(1):55-64, 2004

Corresponding Author: Faik Atroshi, Department of Clinical Medi-cine, Pharmacology & Toxicology, Faculty of Veterinary Medicine, P.O.Box 57, FIN-00014 Helsinki University, Finland [email protected]

Assessment of enzymatic cleaning agents and disinfectants against bacterial biofilms.

Mona Augustin, Terhi Ali-VehmasDepartment of Basic Veterinary Sciences, Section of Veterinary Microbiology, Faculty of Veterinary Medicine, University of Helsinki, Finland

Faik AtroshiDepartment of Clinical Medicine, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Helsinki, Finland

Received 22 November 2003, Revised 13 January 2004, Accepted 10 February 2004, Published 18 February 2004

Abstract: Purpose: Microbial biofilm has become dif-ficult to control by antibiotic and biocide regimes thatare effective against suspended bacteria. Their coloniza-tion of surfaces can be a problem and is generally con-trolled through cleaning and disinfection. This studywas undertaken to examine the efficacy of the disinfec-tants including Bio-Ow, Econase CE, Gamanase GC140, IndiAge 44L, Mannanase AMB, Multifect P-3000,Neutrase, Pandion, Paradigm, Pectinex Ultra SP-L,Promozyme, Resinase A2X, Spezyme AA300,Spezyme GA300 and Vinozym EC, and the proteinaseagainst bacterial biofilms. Methods: The effectivenessof 20 commercial disinfectants against Pseudomonasaeruginosa (P. aeruginosa) biofilms using a fluorometrictechnique was examined. Additionally the disinfectantswere also tested against Lactobacillus bulgaricus (L. bul-garicus), Lactobacillus lactis (L. lactis) and Streptococcusthermophilus (S. thermophilus) isolates using microtitra-tion tray based turbidimetric techniques. Escherichiacoli (E. coli) was used as the test bacteria in the fluoro-metric control method. Results: Among the firstgroup of the enzymatic cleaning agents tested, four dis-infectants (Pandion, Resinase A2X, Spezyme GA300and Paradigm) were the most potent against bacterialbiofilms after 30 min incubation time (residual bacte-rial count less than 103 CFU (colony forming units)/ml). However, only Resinase A2X and Paradigmshowed a good effect on bacterial biofilms after 15 minincubation time. Proteinase disinfectants (alkalase, chy-motrypsin, cryotin and krilltrypsin) from the secondgroup of the disinfectants showed a good effect againstP. aeruginosa biofilm when tested in the absence ofmilk. The performance of the disinfectants was

reduced in the presence of milk. The minimum inhibi-tory concentration (MIC) of the cleaning agents wasdetermined as the lowest concentration inhibiting bac-terial growth. The MIC was tested on Lactobacillus bul-garicus (L. bulgaricus), Lactobacillus lactis (L. lactis) andStreptococcus thermophilus (S. thermophilus) isolates.The minimum inhibitory concentrations (MIC) forParadigm against S. thermophilus and L. Lactis werelower than L. Bulgaricus. Whereas, the MIC of Pandionagainst L. bulgaricus was lower than MIC against L. lac-tis. Resinase A2X had no inhibitory effect on bacterialgrowth when the concentration was less than or equalto 2.4 mg/ml and Spezyme GA 300 concentration lessthan or equal to 7.3 mg/ml. Minimum inhibitory con-centration of Pandion against L. bulgaricus was 2.7 µg/ml and against L. lactis 5.3 µg/ml. Growth of S. thermo-philus was inhibited in all concentration of Pandiontested. Conclusions: The choice of disinfectant orcleaning agent along with the optimum concentrationand the time of action is very important when destroy-ing microbes. It is also important that the resistances ofmicrobes to different disinfectants and cleaning agentsbe taken into account when planning the cleaning pro-cess.

INTRODUCTION

The development of pharmaceuticals for the antimi-crobial agents including disinfectants is well known.However, increasing problems associated with the useof these agents’ calls for the development of ideal disin-fectants, which would act quickly and not produceunknown byproduct remains the primary challenge toapproach. Disinfection is a process where pathogenicmicroorganisms are removed or inactivated by chemi-cal (e.g., chlorination) or physical (e.g., filtration, UVradiation) means such that they represent no signifi-

55


cant risk of infection (1). An ideal disinfectant shouldhave a broad spectrum of antimicrobial activity, but itshould be nontoxic and nonirritating (1). It should alsobe compatible with the surfaces to be disinfected, easyto prepare and use, stable, affordable, and readily avail-able, and it should lack any unpleasant odor. However,no antiseptic or disinfectant is ideal, each falling shortof meeting the aforementioned criteria. Mandell et al(2) defined three different categories of disinfectants:(a)high-level disinfectants that are effective against viableendospores if the exposure is long enough; (b) interme-diate-level disinfectants that are tuberculocidal but notsporicidal; (c) low-level disinfectants that do not reli-ably destroy bacterial endospores, tuberculous bacilli,or small, non lipid RNA viruses (e.g. enterovirus).Consequently, efforts are continuously under way toimprove the performance of existing disinfectants ordevelop new ones. The efficacy of antimicrobial activeingredients may be greatly affected by the formulationof the product, such that products with the same levelsof antimicrobial may demonstrate varying levels ofeffectiveness. Efficacy may be affected by pH, deter-gent base, emollients and humectants, ionic nature ofthe formulation, and type of surfactants.

Bacteria are amongst the most successful living organ-isms and their ubiquity ensures that humans areobliged to live in constant and intimate contact with awide variety of species. Although the number of spe-cies capable of causing disease is relatively few, manyothers have the ability to do so given the right condi-tions (3). During the last 10–15 years, antibiotic resis-tance has increased (4). Over use of antibiotics inhumans has led to the rapid evolution of bacteria thatare resistant to multiple drugs. Apart from the discov-ery and exploitation of the natural peptide antimicro-bial agents that form part of the innate immunesystems of plants and animals, there have been fewnew antibiotics developed in recent years (5). In addi-tion, some bacteria have resistance to biocides such asantiseptics, disinfectants and preservatives, which posesa potential challenge for future bacteria control (6).With this in mind, there will be a continued require-ment for new and potent antimicrobial agents,together with techniques suitable for the control anddestruction of bacteria.

Biofilms are composed of populations of microorgan-isms in an extra cellular matrix adhering to surfaces in

which sufficient moisture is available. They are presentin living organisms in addition to the surfaces of inani-mate objects (7, 8, 9, 10, 11, and 12). Biofilm formationcauses problems in many areas, e.g. in industrial watersystems, in medicine and in food processing industry(13, 14, 15,16,17,18). There are serious health and med-ically related problems caused because of microbialbiofilm growth. Bacteria growing as surface associatedbiofilms are much harder to treat with anti-microbialsthus potentially leading to the build up dangerousinfections. The growth patterns, coverage and adher-ence of the biofilms depend critically on the substrateroughness, composition, type of microorganisms andother factors (17, 18, 19, 20, 21, and 22).

Several reasons can account for the reduced sensitivityof bacteria within a biofilm. There may be (i) reducedaccess of a disinfectant to the cells within the biofilm,(ii) chemical interaction between the disinfectant andthe biofilm itself, (iii) modulation of the microenviron-ment, (iv) production of degradative enzymes (andneutralizing chemicals), or (v) genetic exchangebetween cells in a biofilm. However, bacteria removedfrom a biofilm and recultured in liquid culture mediaare generally no more resistant than the “ordinary”planktonic cells of that species (25). Milk is a goodgrowth medium for many microorganisms because ofits high water content, neutral pH, and variety of avail-able nutrients. Dairy products provide substantiallydifferent growth environments than fluid milk becausethese products have nutrients removed or concentratedor have lower pH water activity (26).

Enzymes can be used for degradation of biofilm butdue to the heterogeneity of the extracellular polysac-charides in the biofilm, a mixture of enzyme activitiesmay be necessary for a sufficient degradation of a bac-terial biofilm. The application of enzymes for controlof protein biofilm on surfaces and in closed pipelines iswell known. In particular, proteases are used in pipe-lines and for removal of protein from contact lenses.The use of enzymes for removal of bacterial biofilm isstill limited, partly due to the very low prices of chem-icals in use. In addition, the lack of techniques forquantitative evaluation of the effect of enzymes, as wellas the commercial accessibility of different enzymeactivities, limits their usage. It is known that mono-component enzymes can be used for biofilm removal.However, the heterogeneity of the biofilm matrix lim-

56


its the potential of monocompound enzymes (27).

The activity of disinfectants in vitro are currentlytested either on microorganisms in suspension or ongerm carriers. The mix microorganisms in suspensionwith a solution of the compound under test, in thepresence or absence of interfering substances selectedas a function of the field of application of the agent andthe mode of use seems the most common method (28,29,30,31,32,33). However, marked discrepanciesbetween these methods are not uncommon. Therefore,it is necessary to perform and develop a rapid and pre-dictive tool for in vitro antimicrobial studies of the effi-cacy of the disinfectants. No information is availableregarding the efficacy of the disinfectants studiedincluding Bio-Ow, Econase CE, Gamanase GC 140,IndiAge 44L, Mannanase AMB, Multifect P-3000, Neu-trase, Pandion, Paradigm, Pectinex Ultra SP-L, Pro-mozyme, Resinase A2X, Spezyme AA300, SpezymeGA300 and Vinozym EC, and the proteinase disinfec-tants against bacterial biofilms. Furthermore, we stud-ied the sensitivity of microtitration tray basedfluorometric assay in analyzing enzymatic activity ofP. aeruginosa and E. coli and finally specified the effectof disinfectants against biofilms.

MATERIALS AND METHODS

Disinfectants. The disinfectants (enzymatic cleaningagents) used in this study were tested. Enzymes wereoriginated from two sources. First group were origi-nated from VTT Biotechnology and Food ResearchInstitute (Espoo, Finland). They include Bio-Ow, Eco-nase CE, Gamanase GC 140, IndiAge 44L, MannanaseAMB, Multifect P-3000, Neutrase, Pandion, Paradigm,Pectinex Ultra SP-L, Promozyme, Resinase A2X,Spezyme AA300, Spezyme GA300 and Vinozym EC.The second group was proteinase agents, they wereoriginated from University of Iceland: Cryotin andChymotrypsin were from cod, alkalase was fromNovo, Krilltrypsin was from Antarctic krill. The pro-teinase samples were isolated and prepared in Iceland.

Microorganisms. Escherichia coli (PT 238), Pseudomo-nas aeruginosa (ATCC-215442), Lactobacillus lactis(VTT E-85 222), Streptococcus thermophilus (VTT E-96665) and Lactobacillus bulgaricus (VTT E-96662) wereused as test organisms.

Media. The broth media used in the study were ISB(Iso-Sensitest Broth, Unipath ltd., Basingstoke, Hamp-shire, England) and M.R.S (M.R.S Broth,Unipath ltd.,Basingstoke, Hampshire, England). The media wereprepared according to the manufacturer's instructions.

Fluorogenic substrates. The fluorogenic substrates usedwere 4-methylumbelliferyl- β -D-glucuronide (4-MUG) (Sigma Inc., St Louis, MO, USA), 4-methylum-belliferyl phosphate-Li2 (4-MUP), (Sigma Inc., St Louis,MO, USA) and resazurin (Sigma, R-217, St Louis, MO,USA).

Major equipment. Fluorometric studies of bacterialenzymatic activity were carried out using microtitra-tion-tray reading fluorometer Fluoroskan 2 (Lab-systems, Helsinki, Finland). For washing procedures,the Labsystem's Wellwash 4 microplate ELISA washer(Labsystems, Helsinki, Finland) was used. Water bathwas used for incubation. Turbidimetric studies of bac-terial growth were carried out using the Bioscreen®analyzer (Labsystems, Helsinki, Finland), a fully auto-mated analyzing system for measuring bacterialgrowth with a vertical light path, 200 individual testscould be run simultaneously.

Preparation of biofilms. 50 µl of milk was allowed todry three days on surfaces of the test wells of microti-tration trays by evaporation in a laminar. After drying,the plates were covered with plastic coat and stored incold (4° C). P. aeruginosa were allowed to grow anddevelop biofilm for 1 day (+37° C) in wells using ISBas the growth medium. This method produced biofilmwith about 107 CFU/ml.

Washing procedure and tests on cleaning agents. Afterincubation of P. aeruginosa, the wells were rinsed twicewith sterile water and then washed once with ELISAwasher before enzyme treatment. A dilution series ofenzymes (100%-1.5% enzyme product) was prepared insterile water. The enzyme dilutions were added towells of a microtitration tray (200 µl to each well, 8repeats). The tray containing the enzyme dilutions wasincubated 30 min and 15 min at optimal conditions foreach enzyme (pH and temperature). After incubation,the enzyme product was removed from the wells andwashed three times with sterile water. Medium and theflurogenic substrate were added to each well before themeasurement. With proteinase samples, two different

57


concentrations were used in final assay system. Sampledilutions were buffered at pH 8 and temperature usedwas 5° C. Duration of incubation was 2 h and 24 h.

Fluorometric analysis of bacterial phosphatase activ-ity. The bacteria used was P. aeruginosa (ATCC - 215442). The bacteria were harvested from blood-agarplates, inoculated into ISB-broth and allowed to growovernight at 37° C. A tenfold dilution series was pre-pared in the 0.9% NaCl. The initial bacterial numbersin the wells of the microtitration trays corresponded to103-107 CFU/ml. A stock solution of 4-MUP (4-Meth-ylumbelliferyl phosphate-Li2) was prepared freshly bydissolving 4-MUP (1mg/ml) in saline and filter-steril-ized (34). The final concentration of 4-MUP in samplemixtures was 0.2 mM. The bacterial suspension (50µl, initial concentration 103-107 CFU/ml in wells, 8replicates) was dispensed into microtitration tray wellscontaining 50 µl dry milk, 30 µl 4-MUP and 220 µl ISB.The release of 4-MU over time by bacterial phos-phatase was automatically monitored at 37° C for 12hour (excitation 355 nm and emission 460 nm).

Fluorometric analysis of bacterial β-glucuronidaseactivity. The bacteria strain used was E.coli PT-238.The bacteria were harvested from blood-agar plates,inoculated into ISB-broth and allowed to grow over-night at 37° C. A tenfold dilution series was preparedin the 0.9% NaCl. Therefore, the initial bacterial num-bers in the wells of the microtitration trays corre-sponded to 103-107 CFU/ml. A stock solution of 2 mM[4-MUG (4- Methylumbelliferyl-β-D-glucuronide)]was prepared freshly by dissolving 7 mg 4-MUG in ISBand filter-sterilized (35). A final concentration of 4-MUG in sample mixtures was 0.2 mM. The bacterialsuspension (50 µl, initial concentration 103-107 CFU/ml in wells, 8 replicates) was dispensed into microtitra-tion tray wells containing 50 µl dry milk, 30 µlMUG-solution and 220 µl ISB. The release of 4-MU(4- Methylumbelliferone) over time by bacterial β-glu-curonidase was automatically monitored at 37° C for12 hour (excitation 355 nm and emission 460 nm).

Bacterial growth studies using resazurin as a fluorogenicsubstrate. The bacteria used was P. aeruginosa (ATCC -2 15442). Bacteria were harvested from blood-agarplates, inoculated into ISB-broth and allowed to growovernight at 37° C. A ten fold dilution series was pre-pared in the 0.9% NaCl. The initial bacterial numbers

in the wells of the microtitration trays corresponded to103-107 CFU/ml. A stock solution of resazurin (0.05mg/ml) was prepared by dissolving resazurin in sterilewater. The final concentration of resazurin in samplemixtures was 0.006 mg/ml. The bacterial suspension(50 µl, initial concentration 107 CFU/ml in wells, 8replicates) was dispensed into microtitration tray wellscontaining 50 µl dry milk, 20 µl resazurin and 230 µlISB. P. aeruginosa were allowed to grow for one day.The fluorogenic resazurin reaction for bacterial activi-ties was monitored 12 hour at 37° C (excitation 544nm and emission 584 nm).

Analysis of the minimum inhibitory concentration(MIC). Bacteria were harvested from MRS-plates andallowed to grow overnight at 37° C. To specify theminimum inhibitory concentration (MIC), turbido-metric bacterial growth studies (36) were carried out,using the Bioscreen analyzer (Labsystems). L. bulgari-cus, L. lactis and S. thermophilus were used as teststrains. Bacterial suspension (30 µl initial concentra-tion 106-107 CFU/ml in wells) was dispensed into hon-eycomb wells containing 240 µl MRS-broth and 30 µlenzyme dilution. The honeycomb plates were coveredand pre-incubated for 10 min. The change in turbiditywas monitored automatically every 20 minutes for 24hour at 37° C using wide-band filter (wide band,absorbance at 420, 450, 492, 540, and 580 nm). Theminimum inhibitory concentrations of the cleaningagents were determined as the lowest concentrationinhibiting bacterial growth.

Statistical analysis. Data was expressed as the mean ±standard error of the mean (SEM). Statistical proce-dures were performed using linear regression analysis.Statistical significance was defined as P< 0.05.

RESULTS

Determination of the disinfectants effect on bacterialcells. The resazurin-based fluorometric method foundto be more promising to evaluate the functional statusof disinfectants than 4-MUP (P. aeruginosa) and 4-MUG (E. coli)-based methods giving a combination ofgreater fluorescence over a broad pH range andreduced growth inhibition with P. aeruginosa biofilms.The resazurin-based fluoromety method generated amore rapid fluorescence than any other tested sub-strate.

58


Even with the smallest E. coli inoculum in milk, CFUmore than 103 with 4-MUG (Figure 1) and 106 with P.aeruginosa 4-MUP (Figure 2) could be quantifiedwithin 8 hours of incubation.

Figure 1: Time-fluorescence curves of E. coli 4-MUG as afluorogenic substrate.

Figure 2: Time-fluorescence curves of P. aeruginosa 4-MUP as a fluorogenic substrate.

The fluorescence of 4-MU did not reach the maximumvalue due to the decreasing pH caused by bacteria.However, 4-MU is more fluorigenic at alkalic condi-tions. The timing of the appearance of maximum fluo-rescence of resazurin was directly related to thelogarithm of the number of colony-forming units (Fig-ures 3 and 4).

Cleaning agents and proteinases. Four of the cleaningagents (Pandion, Resinase A2X, Spezyme GA300 andParadigm) had a significant effect against bacterial bio-films after 30 min incubation time using the resazurinmethod (Table 1).

Figure 3: Time-fluorescence curves of P. aeruginosaResazurin as a fluorogenic substrate.

Figure 4: Time-fluorescence curves of P. aeruginosaResazurin as a fluorogenic substrate (in ISB).

In Pandion and Spezyme GA300, the dropped in theresidual bacterial counts were from 107 to 103 CFU/ml(Figure 5).

Whereas, Resinase A2X and Paradigm showed a reduc-tion effect from 106 to 103 CFU/ml against bacterialbiofilms after 15 min exposure (Table 1 and Figure 6).

When proteinase agents were considered, a significanteffect against Pseudomonas biofilm was seen when milkwas not included in the suspension. However, the pro-teinase agents had no effect on biofilm in the present ofmilk (Table 2).

Minimum inhibitory concentration (MIC). The MICof the enzymatic cleaning agents is given in Table 3.

59


Table 1: Effect of enzymes treatment on P. aeruginosabiofilm. Bacterial concentrations were interpolated fromthe time-fluoresence response curves of P. aeruginosa(CFU/ml). Bacterial biofilms on microtitration trays wereincubated over 15 and 30 minuets at optimaltemperatures and pH before measurement. Enzymesconcentrations are indicated in brackets.

The MIC of the cleaning agents was determined as thelowest concentration inhibiting bacterial growth.Resinase A2X and Spezyme GA 300 had no inhibitoryeffect on bacterial growth when Resinase A2X andSpezyme concentrations were less than or equal to 2.4mg/ml and 7.3 mg/ml simultaneously. Minimuminhibitory concentration of Pandion against Lactobacil-lus bulgaricus and Lactobacillus lactis were 2.7 µg/mland 5.3 µg/ml simultaneously. Streptococcus thermo-philus growth was inhibited by all concentrations ofPandion tested. Minimum inhibitory effect of Para-digm against Lactobacillus bulgaricus was 14 µg/ml,against Lactobacillus lactis was 7.3 µg/ml and againstStreptococcus thermophilus was 0.5 µg/ml (Table 3 andFigure 7).

Figure 5: The effect of Pandion on P. aeruginosa biofilmafter 30 min incubation.

Figure 6: The effect of Paradigm on P. aeruginosa biofilmafter 15 min incubation.

Table 2: Effect of proteinase agents on P. aeruginosabiofilms.

60


Table 3: The minimum inhibitory concentration (MIC) forthe most effective cleaning agents on Lactobacillusbulgaricus, Lactobacillus lactis and Streptococcusthermophilus.

Figure 7: The effect of Pandion on Streptococcusthermophilus E-96 665.

DISCUSSION

In this study, we have demonstrated the rapid microbi-cidal action of the disinfectants Pandion, ResinaseA2X, Spezyme GA300 and Paradigm on P. aeruginosabiofilms. Although Pandion and Spezyme GA300demonstrated, potent activity against strains tested atinitial cell densities as high as 107 CFU/ml, the micro-bicidal effect decreased with increasing dilution of thedisinfectants. However, when the initial cell densitywas reduced to 103 CFU/ml, 100% killing of the teststrain was achieved with only 10% of these enzymes.

In contrast to many other chemical disinfectants,which may require high concentrations and severalminutes of exposure (37), the lethal microbicidal effectsof the cleaning enzymes of the present study occurredwithin the first minute of exposure. The absence of sig-nificant microbicidal activity after 1 minute of expo-sure suggests that enzymes used might be veryunstable. However, preliminary data (not shown) dem-onstrate that Pandion remains active in both undilutedand diluted forms for at least 24 hours after prepara-

tion. These results suggest that the loss of microbicidalactivity after 1 minute of exposure is associated withthe presence of the microorganisms rather than theinnate instability of Pandion. There are two possibleexplanations for this mechanism; the first one is thepossibility of slow or incomplete penetration of thedisinfectants into the biofilm. For example, measure-ments of antibiotic penetration into biofilms in vitrohave shown that some antibiotics readily permeate bac-terial biofilms (38). The second explanation depends onan altered chemical microenvironment within the bio-film. Microscale gradients in nutrient concentrationsare a well known feature of biofilms. Findings fromstudies with miniature electrodes have shown that oxy-gen can be completely consumed in the surface layersof a biofilm, leading to anaerobic niches in the deeplayers of the biofilm (39). Concentration gradients inmetabolic products mirror those of the substrates.Local accumulation of acidic waste products might leadto pH differences greater than 1 between the bulk fluidand the biofilm interior (40) which could directlyantagonise the action of an antibiotic.

The results of the present study show that individualproducts may show variations in efficacy againstmicrobial biofilms. Paradigm and Resinase A2X werethe most potent disinfectants at low concentrationsand short test period against P. aeruginosa biofilm(residual bacterial count less than 103 CFU/ml). Para-digm and Resinase A2X may be useful for inactivatingof biofilms produced in milk and in other lipid foodresidues that may remain after poor cleaning in manyindustrial types of equipment.

When proteinase disinfectants were considered, theresults of this study show that these disinfectants wereeffective against P. aeruginosa biofilms when milk wasnot added directly to the proteinase solution. The elim-ination of biofilm is a very difficult and demandingtask because many different factors affect the detach-ment. It is known that monocomponent enzymes canbe used for biofilm removal. However, the heterogene-ity of the biofilm matrix limits the potential of theseenzymes. Our findings suggest that numerous factorsplay a role in the resistance of biofilm-embedded bacte-ria. Any of the following alone or in combination,may contribute to compromised antibacterial activity:interaction between constituents of the exopolymermatrices and the antimicrobial agent’s suppression of

61


growth rate due to a modified nutrient environment,and dense bacterial inocula. Hostacka (41) studied theeffect of commercially manufactured disinfectantsagainst P. aeruginosa and found that the disinfectantstested depressed Pseudomonas aeruginosa virulence fac-tors (elastase and proteinase). The degree of inhibitionwas directly proportional to the disinfectant concen-tration.

Since the duration of treatment is one of the importantfactors affecting the antimicrobial activity of disinfec-tants, it is desirable to use a sufficiently long cleaningprocedure in the food industry. The results after 15min exposure times showed high activity against P.aeruginosa. According to the provisional European andnational surface test methods, 60 min disinfection maybe suitable as a disinfection time (42, 29). However, inthe food industry there is a trend towards longer pro-duction runs with shorter intervals for disinfection andcleaning (43). On the other hand, not only the contacttime but also the concentration and the composition ofthe agent affect the antimicrobial activity. In this studyfour of the cleaning agents tested (Pandion, ResinaseA2X, Spezyme GA300 and Paradigm) had a significanteffect (residual bacterial count less than 103 CFU/ml)against bacterial biofilms in 30 min. Accordingly it isimportant to use higher concentrations of the cleaningand disinfection agents, especially in the presence oforganic materials. Considerable variation in resistancehas been observed between the different species ofmicrobes. It has been demonstrated that gram-negativebacteria biofilms are more resistance than other spe-cies. On the other hand, spores have several strong lay-ers, which make it difficult for disinfectants topenetrate them (44, 45). Therefore, it is necessary touse a longer disinfection time and a stronger concentra-tion of disinfectants to destroy biofilms. These findingsshow the importance of correct application of disinfec-tants for both concentration and exposure time in thedairies.

Another interesting point in this study was the meth-ods used to study the effect of the disinfectants againstbacterial biofilms. We have shown that the susceptibil-ity of bacterial biofilms to the disinfectants tested canbe assessed by resazurin based fluorometry. The resa-zurin based fluorometric method used in this studyseems to be more sensitive and reliable than 4-MUP or4-MUG based fluorometry. 4-MU was more fluores-

cent at alakaline pH. Therefore, with resazurin basedfluorometric method it was possible to define the mosteffective disinfectants.

CONCLUSION

Bacteria that adhere to implanted food plants, medicaldevices or damaged tissue can become the cause of per-sistent infections (39, 41, and 42). These bacteria encasethemselves in a hydrated matrix of polysaccharide andprotein, forming a slimy layer known as a biofilm. Themechanisms of resistance to disinfectants in bacterialbiofilms can be explained by an altered chemicalmicroenvironment. Microscale gradients in nutrientconcentrations are a well known feature of biofilms.Earlier studies with miniature electrodes have shownthat oxygen can be completely consumed in the surfacelayers of a biofilm, leading to anaerobic niches in thedeep layers of the biofilm (44). The other possiblemechanism is that a subpopulation of micro-organismsin a biofilm forms a unique, and highly protected, phe-notypic state––a cell differentiation similar to sporeformation. This explanation is lent support by findingsfrom studies that show resistance in newly formed bio-films, even though they are too thin to pose a barrierto the penetration of either an antimicrobial agent ormetabolic substrates (45). Nevertheless, the hypothesisof a spore-like state entered into by some of the bacte-ria in a biofilm provides a powerful, and generic, expla-nation for the reduced susceptibility of biofilms toantibiotics and disinfectants of widely different chemis-tries (46). Our results demonstrate that the choice ofdisinfectant or cleaning agent along with the optimumconcentration and the time of action is very importantwhen destroying microbes. It is also important that theresistance of microbes to different disinfectants andcleaning agents be taken into account when planningthe cleaning process. Another out come of this study isthat the microtitration tray based fluorometric assayprovided high throughput capacity system of analyz-ing various enzymatic cleaning agents for biofilmsformed by bactaria.

REFERENCES

[1] McDonnell G, and Russell D. Antiseptic and Disin-fectants: Activity, action, and resistance. Clin Micro-biol Rev 1999; 12:147-179.

[2] Mandell GL, Bennet JE y, Dolin R., Principles andPractice of Infectious Diseases. New York, ChurchillLivingstone, pp.199-212, 1995.

62


[3] Schorer M, Eisele M. Accumulation of inorganic andorganic pollutants by biofilms in the aquatic environ-ment. Water, Air, Soil Pollut 1997; 99:651–659.

[4] Percival SL, Knapp JS, Edyvean R, Wales DS. Biofilmdevelopment on stainless in mains water. Water Res1998; 32:243–253.

[5] Nielsen PH, Jahn A, Palmgren R. Conceptual modelfor production and composition of exopolymers inbiofilms. Water Sci Technol 1997;36:11–19.

[6] Fuchs S, Haritopoulou T, Wilhelmi M. Biofilms infreshwater ecosystems and their use as a pollutantmonitor. Water Sci Technol 1996; 34:137–140.

[7] Drexler KE. Molecular nanomachines: physical prin-ciples and implementation strategies. Ann Rev Bio-phys & Biomol Structure 1994; 23:377-405.

[8] Stoffler D, Steinmetz MO, Aebi U. Imaging biologi-cal matter across dimensions: from cells to moleculesand atoms. FASEB J 1999; 13 :S195-S200.

[9] Mattila K, Weber A, Salkinoja-Salonen MS. Struc-ture and on-site formation of biofilms in papermachine water flow. J Indust Microbiol & Biotech2002; 28:268-279.

[10] O’Toole H, Kaplan B, Kolter R. Biofilm formationas microbial development. Ann Rev Microbiol 2000;54:49–79.

[11] Gilbert P, Allison DG, McBain AJ. Biofilms in vitroand in vivo: do singular mechanisms imply cross-resistance? J Appl Microbiol 2002; 92:S98-S110.

[12] Carpentier B, and Cerf O. Biofilms and their conse-quences, with particular

[13] references to hygiene in the food industry: a reviewof the literature. J Appl Bacteriol 1993; 75, 499-511.

[14] Grobe S, Wingender J, Flemming HC. Capability ofmucoid Pseudomonas aeruginosa to survive in chlori-nated water. Inter J Hygien & Environ Health 2000;204:139-142.

[15] Wirtanen G. Biofilm formation and its eliminationfrom food processing equipment. Helsinki Univer-sity of Tecnology. PhD thesis, Espoo, Finland,1995.

[16] Gibson H, Taylor JH, Hall KE, Holah JT. Effective-ness of cleaning techniques used in the food industryin terms of the removal of bacterial biofilms. J ApplMicrobiol 1999; 87:41-48.

[17] Moller S, Pedersen AR, Poulsen LK, Arvin E, MolinS. Activity and three-dimensional distribution of tol-uene-degrading Pseudomonas putida in a multispeciesbiofilm assessed by quantitative in situ hybridizationand scanning confocal laser microscopy. Appl &Environ Microbiol 1996; 62:4632-4640.

[18] Schwartz T. Hoffmann S. Obst, U. Formation ofnatural biofilms during chlorine dioxide and U.V.

disinfection in a public drinking water distributionsystem. J Appl Microbiol. 2003; 95:591-601.

[19] Codony F, Domenico P, Mas J. Assessment ofbismuth thiols and conventional disinfectants ondrinking water biofilms. J Appl Microbiol 2003;95:288-293.

[20] Jayaraman A, Sun AK, Wood TK. Characteriz ationof axenic Pseudomonas fragi and E. coli biofilms thatinhibit corrosion of SAE 1018 steel. J Appl Microbiol1998; 84:485–492.

[21] Ben-Jacob E, Schochet O, Tenenbaum A, Cohen I,Czirok A, Vicsek T. Generic model of co-operativegrowth patterns in bacterial colonies. Nature 1994;368:46–49.

[22] Moller S, Korber DR, Wolfhaardt GM, Molin S,Caldwell DE. Impact of nutrient composition on adegradative biofilm community. Appl EnvironMicrobiol 1997; 63:2432–2438.

[23] Stephens C. Microbiology: breaking down biofilms.Current Biol 2002; 12:R132-R134.

[24] Richter A, Smith R, Ries R, Wildenauer F. Featuresof biofilm growth on solid surfaces analysed byMonte Carlo simulations. Phys Stat Sol A 1999;176:953–967.

[25] Hentzer M, Eberl L, Nielsen J. Givskov, M. Quo-rum Sensing: A Novel Target for the Treatment ofBiofilm Infections. Biodrugs. 2003; 17:241-250.

[26] Brown MRW and Gilbert P. Sensitivity of biofilmsto antimicro-bialagents. J Appl Bacterio 1993;74:S87–S97.

[27] Frank, JF., Milk and dairy products, in Food Micro-biology, Fundamentals and Frontiers: Doyle MP,Beuchat LR, Montville TJ (eds), ASM Press, Wash-ington. pp 101, 1997.

[28] Johansen C, Falholt P, and Gram L. Enzymaticremoval and disinfection of bacterial biofilms ApplEnvironment Mikrobiol 1997; 63: 3724-3728.

[29] Kelsey JC. Disinfectants and methods of testing. JMed Lab Tech 1969; 26:79-89.

[30] Van Klingeren B. Disinfectants testing on surfaces. JHosp Infect 1995; 30: S397-S408.

[31] Bloomfield SF, Scott E. Cross-contamination a,dinfection in the domestic environment and the role ofchemical disinfectants. J Appl Microbiol 1997; 83:1-9.

[32] Ntsama-Essomba C, Bouttier S, Ramaldes M,Dubois-Brissonnet F, Fourniat J. Resistance ofEscherichia coli growing as biofilms to disinfectants.Vet Res 1997; 28:352-363.

[33] Langsrud S, Sundheim G, Borgmann-Strahsen R.Intrinsic and acquired resistance to quaternary

63


ammonium compounds in food-related Pseudomonasspp.. J Appl Microbiol 2003; 95:874-882.

[34] Cole EC, Addison RM, Rubino JR, Leese KE,Dulaney PD, Newell MS, Wilkins J, GaberDJ, Wineinger T, Criger DA. Investigation of anti-biotic and antibacterial agent cross-resistance in targetbacteria from homes of antibacterial product usersand nonusers. J Appl Microbiol 2003; 95:664-676.

[35] Ali-Vehmas T, Rizzo A, Westermarck T, Atroshi F.Measurement of antibacterial activities of T-2 Toxin,deoxynivalenol, ochratoxin A, aflatoxin B1 andfumonisin B1 using microtitration tray-based turbidi-metric techniques. J Vet Med A 1998; 45:453-458.

[36] Atroshi F, Ali-Vehmas T, Westermarck T, Rizzo A,Selga G, Baltais A, Linars J, Saulite V, Daukste A.Evaluation of the antibacterial and hemolytic activi-ties of Latvian herbal preparation. Vet & HumanToxicol 2000; 42:341-344.

[37] Ali-Vehmas T, Westphalen P, Myllys V, SandholmM. Binding of Staphylococcus aureus to milk fatglobules increases resistance to penicillin-G. J DairyRes 1997 64:253-260.

[38] Olmsted, RN.; APIC Infection Control and AppliedEpidemiology: Principles and Practice. St Louis. MO:Mosby-Year Book, Inc; pp 34-39, 1996.

[39] Stewart PS. Theoretical aspects of antibiotic diffusioninto microbial biofilms. Antimicrob AgentsChemother 1996; 40: 2517–2522.

[40] de Beer D, Stoodley P, Roe F, Lewandowski Z.Effects of biofilm structure on oxygen distributionand mass transport. Biotechnol Bioeng 1994; 43:1131–1138.

[41] Zhang TC, Bishop PL. Evaluation of substrate andpH effects in a nitrifying biofilm. Wat Environ Res1996; 68: 1107–1115.

[42] Hostacka A. Influencing virulence factors ofPseudomonas aeruginosa by commercially manufac-tured disinfectants. Pharmazie 1997; 52:317-319.

[43] Bloomfield SF, Arthur M, Van Klingeren B, PullenW, Holah JT, Elton R. An evaluation of the repeat-ability and reproducibility of a surface test for theactivity of disinfectants. J Appl Bacteriol 1994; 76:86-94.

[44] Rutala WA. APIC guideline for selection and use ofdisinfectants. Amer J Infec Control 1996; 24:313-342.

[45] Wirtanen G, Salo S, Allison DG, Mattila-SandholmT, Gilbert P. Performance evaluation of disinfectantformulations using poloxamer-hydrogel biofilm-con-structs. J Appl Microbiol 1998; 85:965-971.

[46] Gilbert P, Allison DG, McBain AJ . Biofilms in vitroand in vivo: do singular mechanisms imply cross-resistance? J Appl Microbiol 2002; 92: 98S-110S.

64

assessment of enzymatic cleaning agents and disinfectants ...csps/jpps7(1)/f.atroshi/biofilm.pdf ·...

Documents