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 Table of Contents  
Year : 2016  |  Volume : 5  |  Issue : 3  |  Page : 154-157

Rethink on recommended concentrations of disinfectants in the light of biofilm, based on in vitro study

1 Department of Microbiology, Burdwan Medical College, Burdwan, West Bengal, India
2 Department of Microbiology, Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India
3 Department of Microbiology, Nil Ratan Sircar Medical College and Hospital, Kolkata, West Bengal, India
4 Department of Microbiology, College of Medicine and Sagar Dutta Hospital, Kolkata, West Bengal, India

Date of Web Publication5-Aug-2016

Correspondence Address:
Dr. Bipasa Chakraborty
lat-1B, Anandam Apartment, 107, Garfa Main Road, Kolkata - 700 075, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2278-344X.187803

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Background: Some medical devices are reused after treatment with disinfectants at manufacturer's recommended concentrations (RCs), based on experimental evaluation studies of their bactericidal, sporicidal, and mycobactericidal concentrations. However, this may not be sufficient to eliminate all colonized highly drug-resistant biofilms on medical devices, resulting possibility in iatrogenic infections by recolonization of highly resistant persisters. The objective of this study is to evaluate the antibacterial efficacy of different concentrations of novel and conventional disinfectants on in vitro grown biofilm of multidrug-resistant nosocomial bacterial isolates and a reference strain. Materials and Methods: Multidrug-resistant and strong biofilm producers Pseudomonas aeruginosa (n = 4) and Escherichia coli (n = 5) nosocomial isolates and one reference strain, P. aeruginosa PAO-1, were selected after testing their biofilm status by modified Christensen's method and Stepanovic's interpretative criteria. The activity of three different groups of new disinfectants, Novacide, Virkon, Silvicide and two conventional disinfectants, phenol and glutaraldehyde, were assessed on their in vitro grown biofilms as percentage of total surviving bacteria within biofilm matrix after challenge at different concentrations in terms of multiples of RC and different contact time. Results: Modified RCs for Virkon, phenol, and Silvicide, as pointed out by in vitro study, were 4, 8, and 10 times higher than the one suggested by the manufacturer. Novacide was least effective, whereas glutaraldehyde at its available concentration was also not effective in removal of biofilm bioburden. Conclusions: RC was not sufficient to eliminate the in vitro biofilms tested in this study. Further studies using medical devices materials should be performed for clarification. However, this study points out possible relevant limitations of currently used hospital disinfectants and concentrations against biofilms. Additional measures to prevent reinfections related to their use of medical devices are mandatory, and this study suggests that optimization of disinfectants concentrations may be highly relevant.

Keywords: Biofilm, disinfectants, hospital acquired infections, recommended concentration

How to cite this article:
Chakraborty B, Chatterjee S, Ray R, Pal NK, Patra SK, Maiti PK. Rethink on recommended concentrations of disinfectants in the light of biofilm, based on in vitro study. Int J Health Allied Sci 2016;5:154-7

How to cite this URL:
Chakraborty B, Chatterjee S, Ray R, Pal NK, Patra SK, Maiti PK. Rethink on recommended concentrations of disinfectants in the light of biofilm, based on in vitro study. Int J Health Allied Sci [serial online] 2016 [cited 2022 Aug 11];5:154-7. Available from: https://www.ijhas.in/text.asp?2016/5/3/154/187803

  Introduction Top

A subpopulation of bacteria within biofilms formation (BF) show persistent resistance to almost all regular disinfectants at recommended concentrations (RCs) and time exposure. [1] Biofilms can occur on almost any biological or abiotic surface and generally have 100-1000 fold increased resistance toward antimicrobials than equivalent populations of planktonic bacteria, [2],[3],[4] mostly due to restricted drug permeability across matrix and dormant state of bacteria. [5] Except genetic mechanism of drug-resistance, these factors are reversible on planktonic conversion. Manufacturer's RCs of disinfectants are based on experimental evaluation studies of their bactericidal, sporicidal, and mycobactericidal concentrations. However, this may not be sufficient to eliminate any colonized high resistant BF on medical devices, resulting possibility in iatrogenic infection by recolonization of highly resistant BF persisters. Prevention of device associated hospital-acquired infections (HAIs) has become a serious challenge. Most nosocomial infections from reusable medical devices such as endoscopes, cystoscopes, bronchoscopes, laryngoscopes, and implants are due to multidrug-resistant persisters within biofilm. [6] Hence, RCs, as set by the manufacturer for disinfectants, should be reviewed in light of BF.

In the backdrop of the above scenario, the following study was planned with the objective of evaluating the efficacy of three novel hospital disinfectants against biofilm organisms in their sessile form - (a) Virkon: Triple salt of potassium monopersulfate, potassium sulfate and potassium hydrogen sulfate (strong oxidising agents), RC of 1% solution; (b) Novacide: 3% w/v polyhexamethylene biguanide and 10% w/v didecyldimethylammonium chloride (fourth generation quarternary ammonium disinfectant with surfactant properties), RC of 2.5% solution; (c) Silvicide: 0.01% silver nitrate and 10% hydrogen peroxide (strong oxidising agent with stabilizer incorporated), RC of 5% solution. Two conventional disinfectants were also tested - (d) phenol (80%) (protoplasmic poison and enzyme inactivation agent), RC of 5% solution; and (e) glutaraldehyde (2.5%) (alkylating agent), RC of 2% solution.

  Materials and Methods Top

This study was carried out from March 2013 to October 2013, after clearance from Institute Ethical Committee. From endotracheal tubes, central venous lines, urinary catheters, and stents of ventriculoperitoneal shunts, forty multidrug-resistant bacteria were obtained and identified by standard methods of identification. Their in vitro BF production was checked by modified Christensen microtiter plate assay, according to literature. [7],[8],[9] Optical density (OD) were measured by ELISA Reader (Bio-Rad 680) at 570 nm and according to interpretative criteria of Stepanovic et al. [7],[10] Only strong biofilm producers were selected and included for this study. Four Pseudomonas aeruginosa and five  Escherichia More Details coli isolates, along with one reference strain of a strong biofilm producing P. aeruginosa PAO-1 were used, in a total of ten strong biofilm producing strains.

Selected biofilm producing bacteria were subcultured in solid agar plate and isolated colonies were inoculated in brain-heart infusion (BHI) broth. After overnight incubation at 37°C, OD at 600 nm was adjusted to 0.2 by diluting with broth and further diluted to 1:100 to be used as the inoculum for the in vitro experiments. Microtiter plate with preformed biofilms was first prepared. For each strain, 0.2 ml of diluted inoculum was added in each well. Plates were incubated for 48 h, 100 rpm, 37°C, allowing for BF formation. Plates washed with phosphate buffered saline (PBS) three times for removal of planktonic form of bacteria, leaving only the BF sessile organisms. Plates were air dried and made ready for disinfectant challenge test on the first six wells leaving the seventh well as a positive control. Eighth row, containing only BHI broth, was the negative control. All experiments were performed in triplicate.

The used conventional disinfectants were manufactured by Indian Drug House, India (phenol) and BioShields, Tulip Group, India (glutaraldehyde). Regarding the three new ones, Virkon was produced by Antec International Limited, India; and Novacide and Silvicide by BioShields, India. All the liquid disinfectants and Virkon stock solution (4%) were serially diluted by double dilution method using BHI as diluent, starting at crude concentration. A volume of 0.2 ml of each dilution was added in each well with preformed biofilm. Different contact times, 2, 3, 4, 5, and 6 h, were also tested. At the end of each experiment, microtiter plates were washed three times separately with PBS, 0.2 ml BHI was added and mixed thoroughly by vigorous pipetting, kept for 15 min and surviving bacteria was detected by measuring OD at 630 nm. [11]

The percentage of survival bacteria in biofilm was calculated using the following formula, as given by Wasfi et al. [11]

Results were statistically analyzed by Graph Pad Prism version 4.03 (Graph Pad Software, San Diego, CA, USA).

  Results Top

In this study, P. aeruginosa was the strongest biofilm producer and showed higher resistance for all disinfectants [Figure 1]. Comparative efficacy of all five disinfectants against biofilms at 2, 3, 4, 5, and 6 h contact time at their highest concentrations was analyzed by two-way ANOVA and was statistically significant (P < 0.0001). 4% Virkon (4 × RC) reduced biofilm maximally to 0.38% of surviving bacteria levels. About 40% phenol (8 × RC) also reduced surviving bacteria levels to 1.93% and 50% Silvicide (10 × RC) to 5.20% surviving bacteria levels. For 2.5% glutaraldehyde (1.25 × RC) percentage of surviving bacteria were more than 18.12% approximately, whereas 40% Novacide (16 × RC) has shown the worst result, with 53.91% bacteria surviving even after 6 h of contact time with this disinfectant [Figure 1]. At lesser contact time, percentages of surviving bacteria were even more.
Figure 1: Comparative efficacy of all five disinfectants against biofilms formed by Escherichia coli and Pseudomonas aeruginosa at 6 h contact time and their highest test concentrations used

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Biofilms could not be completely removed by any of the tested disinfectants used at a much higher concentration than their RC and with a much greater contact time (6 h exposure). Still, good amount of BF reduction (≤10% survival of bacteria within biofilm) was possible to obtain with Virkon, phenol, and Silvicide, which may be considered as good disinfectants against biofilms, only after 6 h treatment.

  Discussion Top

Resistance due to BF is conferred by both biotic and abiotic components. The restricted permeability generated by the BF exopolysaccharide matrix and the dormant state of BF cells potentiates an increased but reversible resistance to antimicrobials and disinfectants, whereas genetically determined resistance is irreversible. The selection pressures for planktonic and sessile forms are not equally applicable. Hence, the sessile forms, if reverted to planktonic forms may not produce similar highly resistant planktonic form. But for the highly resistant sessile BF organisms, the use of disinfectants RC, determined on planktonic experimental tests, may not be sufficient to eliminate biofilm colonizers on reusable medical instruments and implants. Revised disinfectants RC may be required, considering biofilm biocidal experiments, as suggested by current study.

Biofilms are present in reusable medical devices and implants. It has been observed that potential biofilm usually colonizes on fluid flow line like urinary catheters. Unnoticed biofilm colonizers are present in ventilator connectors and humidifiers, which may not be removed by routine methods. Based on this observation, thus, a fair possibility of biofilm colonization of new connectors by previous contaminated ones exists and may be responsible for the spread of nosocomial infections. This is a presumptive comment which we tried to substantiate with this study. Further studies on biofilm persisters are required to explore their contribution in the still not fully understood current high infection rates due to the re-use of medical devices.

For practical purposes ≤10% survival of bacteria within biofilms is accepted as a good result because a few high resistant sub-population of persisters (0.1-10%) always remain within biofilm. [12] In fact, in this study, none of the tested disinfectants could completely eradicate biofilm organisms. Virkon used at 4 times RC, during 6 h, showed the highest bactericidal activity, followed by phenol at 8 times the RC. However, phenol is toxic, carcinogenic and bio-incompatible to medical devices, thus not applicable for our goals of medical devices disinfection in the practice. Regarding Silvicide, we concluded that 10 times RC returns a good result for BF control [Figure 2]. Glutaraldehyde was effective only in partial removal of biofilms at its highest available concentration and Novacide had the worst action tested.
Figure 2: Comparison between RC and revised concentration of the disinfectants that reduced ≤10% surviving bacteria within biofilm matrix following disinfectant exposure. RC: Recommended concentration, TC: Test concentration

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Based on our recent published paper, Chakraborty et al., [13] as part of this study with newer and conventional disinfectants, we showed that planktonic form of bacteria and spores were susceptible at RC or sometimes even lower than RC. [13] However, when we performed efficacy testing in BF organisms in sessile form, much higher concentrations was required, emphasizing the need of conducting tests with BF and not only with planktonic forms for future RC determinations by disinfectants manufacturers. Indeed, Novacide at 16 times RC were only able to destroy <50% BF bacteria while presenting an excellent bactericidal efficacy against planktonic vegetative bacteria and spores based on our recent published work. [13] The importance of the use of disinfectants in reusable medical devices is mainly associated to those that are heat sensitive. Proper cleaning of instruments followed by mechanical removal of biofilm before application of disinfectant is the most important prerequisite, which helps in partial dislodgement of adherent bacteria and still remains a priority for reduction of biofilm bioburden.

  Conclusions Top

Application of appropriate disinfectant at much higher concentrations than current RC and at much greater exposure times appears to be mandatory to control biofilm hazards. This study reveals limitation of common hospital disinfectants against biofilms. From this study, we obtained a comparative efficacy of disinfectants against in vitro generated biofilms, but ideal concentration of disinfectants may vary in practical purpose, depending on the degree of BF on medical devices, material compatibility, and environmental conditions. This should be further substantiated by disinfection efficacy studies performed directly on medical devices with biofilms.

This study is a part of HAI control measure, generated relevant data regarding the activity of currently used hospital disinfectants against multidrug-resistant bacterial biofilms, and showed that RC may be insufficient for biofilm removal. Altogether, the obtained information can be used to update hospital infection control policies and increase the awareness of biofilm colonization in medical devices and the adequacy of control measures related to them. We believe it is time to rethink the use of common hospital disinfectants at its RCs, to help achieve the necessary changes to prevent HAIs.


We express our sincere thanks to Dr. Pramit Ghosh, Department of Community Medicine, Calcutta Medical College, Kolkata, West Bengal, India for his valuable help in statistical analysis of this work.

We heartily acknowledge Dr. Manju Banerjee: Director of Institute of Post Graduate Medical Education and Research, 244 AJC Bose Road, Kolkata - 700 020, West Bengal, India and Dr. Pradip K Mitra: Ex-Director of Institute of Post Graduate Medical Education and Research, 244 AJC Bose Road, Kolkata - 700 020, West Bengal, India for their kind support and permission to carry out this work.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Epstein AK, Pokroy B, Seminara A, Aizenberg J. Bacterial biofilm shows persistent resistance to liquid wetting and gas penetration. Proc Natl Acad Sci U S A 2011;108:995-1000.  Back to cited text no. 1
Gander S. Bacterial biofilms: Resistance to antimicrobial agents. J Antimicrob Chemother 1996;37:1047-50.  Back to cited text no. 2
Gilbert P, McBain AJ. Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance. Clin Microbiol Rev 2003;16:189-208.  Back to cited text no. 3
Buffet-Bataillon S, Branger B, Cormier M, Bonnaure-Mallet M, Jolivet-Gougeon A. Effect of higher minimum inhibitory concentrations of quaternary ammonium compounds in clinical E. coli isolates on antibiotic susceptibilities and clinical outcomes. J Hosp Infect 2011;79:141-6.  Back to cited text no. 4
Mah TF, O′Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001;9:34-9.  Back to cited text no. 5
Vickery K, Pajkos A, Cossart Y. Removal of biofilm from endoscopes: Evaluation of detergent efficiency. Am J Infect Control 2004;32:170-6.  Back to cited text no. 6
Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 2000;40:175-9.  Back to cited text no. 7
Mathur T, Singhal S, Khan S, Upadhyay DJ, Fatma T, Rattan A. Detection of biofilm formation among the clinical isolates of staphylococci: An evaluation of three different screening methods. Indian J Med Microbiol 2006;24:25-9.  Back to cited text no. 8
[PUBMED]  Medknow Journal  
Oliveira A, Cunha Mde L. Comparison of methods for the detection of biofilm production in coagulase-negative staphylococci. BMC Res Notes 2010;3:260.  Back to cited text no. 9
Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis 2011;15:305-11.  Back to cited text no. 10
Wasfi R, Abd El-Rahman OA, Mansour LE, Hanora AS, Hashem AM, Ashour MS. Antimicrobial activities against biofilm formed by Proteus mirabilis isolates from wound and urinary tract infections. Indian J Med Microbiol 2012;30:76-80.  Back to cited text no. 11
[PUBMED]  Medknow Journal  
Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 2004;230:13-8.  Back to cited text no. 12
Chakraborty B, Pal NK, Maiti PK, Patra SK, Ray R. Action of newer disinfectants on multidrug resistant bacteria. J Evol Med Dent Sci 2014;3:2797-813.  Back to cited text no. 13


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