Are Antiseptics and Disinfectants Commonly Used Against Methicillin Resistant Staphylococcus Aureus Effective? a Study in a Tertiary Care Hospital

Indian Journal of Basic and Applied Medical Research; September 2016: Vol.-5, Issue- 4, P. 640-648

Original article Are antiseptics and disinfectants commonly used against methicillin resistant staphylococcus aureus effective? A study in a tertiary care hospital

Srijita Ghosh 1, Nikhil Kumar Tudu 2, Tapashi Ghosh 3, Aditya Prasad Sarkar 4, Jayanta Bikash Dey 5 , Tapan Kumar Chatterjee 6

1- 3rd Year Post Graduate Trainee, Department of Microbiology, Bankura Sammilani Medical College, Bankura, West Bengal 2- Associate Professor, Department of Microbiology, Bankura Sammilani Medical College, Bankura, West Bengal 3- Associate Professor, Department of Microbiology, Bankura Sammilani Medical College, Bankura, West Bengal 4- Associate Professor, Department of Community Medicine, Bankura Sammilani Medical College, Bankura, West Bengal 5- Professor and HOD, Department of Microbiology, Bankura Sammilani Medical College, Bankura, West Bengal 6- Professor, Department of Microbiology, Bankura Sammilani Medical College, Bankura, West Bengal Corresponding author: Dr. Aditya Prasad Sarkar

Abstract: Introduction : Strains sensitive to beta-lactam antibiotics are called methicillin sensitive Staphylococcus aureus (MSSA) and strains resistant to beta-lactams are defined as methicillin resistant Staphylococcus aureus(MRSA). The acquisition of extra gene has rendered them to be named asone of the serious emergent pathogens in the hospital environment. The aim of this study was to assess the efficacy of eight antiseptics and disinfectants used commonly against MRSA and MSSA in a tertiary care hospital. Methods: Few of the commonly used disinfectants were tested against isolated strains of MRSA and MSSA from clinical samples at the stock strength and upto its 16 th fold dilution according to the methods recommended in CLSI guidelines. The antiseptics and disinfectants used were 10%povidone iodine, 4% chlorhexidine gluconate solution, 4.8% chloroxylenol dettol,2% lysol, 2% glutaraldehyde, 1% sodium hypochlorite solution, 2% liquid phenol, chlorophenol , each in different dilutions for both MRSA ans MSSA strains. Observations and Results: Chloroxylenol and chlorhexidine gluconate were most effective disinfectants on MRSA and MSSA strains, followed by povidone iodine, lysol, phenyl chlorophenol. While interestingly 1% sodium hypochlorite and 2% glutaraldehyde showed full resistance against both the strains. The most important finding being resistance of all the MRSA isolates against 2% liquid phenol but uniquely this is effective on MSSA isolates. Conclusion : The wide spread use of these products in the hospital environment has prompted some speculation that these they can induce resistance gene in the microbes to be propagated to the hospital environment. Keywords : Antiseptics, Disinfectant, MRSA, MSSA, Hypochlorite, Glutaraldehyde, Phenol

Introduction: of pathological flora.Chemicals used to destroy all Sterilization is a process by which all forms of forms of microbilogical life, can be called chemical microbial lifes are destroyed.It is an absolute term sterilants. They can be used for shorter exposure whereas disinfection refers only to the destruction periods as part of the disinfection process 640 www.ijbamr.com P ISSN: 2250-284X , E ISSN : 2250-2858

Indian Journal of Basic and Applied Medical Research; September 2016: Vol.-5, Issue- 4, P. 640-648

[1] .Antiseptics and disinfectants are part and parcel concentration for spillage and cleaning in general of hospital infection control management policy. bacteriology in presence of organic and tuberculous These are extensively used in hospital and other materials [3] whereas lysol(cresol 50% v/v + soap health care setups for a variety of topical and 50% v/v) acts as a surfactant disrupting the surface application. [2] A wide variety of active cytoplasmic membrane. Povidone iodine acts as chemical agent [biocides] are found in these bacteriocidal, fungicidal, virucidal, sporicidal with products. Many of which have been used for the help of its activated iodine.Other phenol hundred of years including alcohol, phenol, iodine, derivatives like chlorhexidine gluconate and chlorine. Though most of these active agents chloroxylenoldamages cell membrane demonstrate broad spectrum antimicrobial activity, irreversibly [3] .Among the two high level the mode of their action is still evolving in disinfectants1% sodium hypochlorite acts as comparision to antibiotics. The wide spread use of bacteriocidal and sporicidal interfering in the these products has shortly induced the cytoplasmic membrane integrity with an developement of microbial resistance towards irreversible enzymatic inhibitionand 2% them. ‘Biocide’ can act as ‘cidal’ and ‘static’ form glutaraldehyde alters RNA, DNA, and protein for microbes.Antiseptics are biocides that destroy synthesis. Chlorophenol 5% V/V (blackfluid)which or inhibit growth of microorganisms on living is a low level disinfectant inhibits the membrane tissue, mainly used for hand washes and surgical bound part of the electron transport chain, and also scrubs [3] .Alcohol is used for sterilizing skin induces leakage, causes protoplast lysis, and whereas povidone iodine is applied on skin before inhibits respiration. any operating manuevers [3] . Aims and objectives: To assess the efficacy of The different disinfectants and antiseptics used in eight antiseptics and disinfectants used commonly this study were10% w/v povidone iodine, 4% against MRSA and MSSAin a tertiary care hospital. chlorhexidine gluconate solution, 4.8% w/v Materials and methods: chloroxylenol(dettol),2% lysol(cresol 50% v/v + It is a laboratory based interventional study soap 50% v/v) , 2% glutaraldehyde, 1% sodium conducted during one year period. This study hypochlorite solution, 2% liquid phenol(carbolic focuses on Staphylococcus aureus with its MRSA acid), chlorophenol 5% v/v (phenyl liquid/black and MSSA variants. Pure cultures of MRSA and disinfectant fluid/per-fenol). Phenolics (carbolic MSSA were isolated from various clinical samples acid) which are intermediate level disinfectants act received in bacteriology laboratory in the as bacteriocidal, fungicidal, virucidal, Department of Microbology in B.S.M.C.H. The test tuberculocidal and clean floor, walls, hard surfaces organisms were identified to the species level based and equipements that doesnot touch mucous on standard microbiological methods such as ; membrane. Phenol leaves residual film on culture characteristics [4], cellular characteristics environmental surfaces. In some cases, they have (microscopic examination) [4] and biochemical tests been used for handwashing, cleaning in low [4]and susceptibility patterns wereidentified as per dose.Phenol acts specifically on the cell membrane CLSI guidelines 2015 [3] .Strains were differentiated and inactivates intracytoplasmic enzymes by into individual isolates according to their different forming unstable complexes [3] .Among the resistogram patterns. The antiseptic and phenolics 2% liquid phenolis the effective disinfectant solutions were procured from different

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Indian Journal of Basic and Applied Medical Research; September 2016: Vol.-5, Issue- 4, P. 640-648 manufacturing companies and they were purchased guidelines 2012 depending on the turbidity of the as hospital supplied .Phenol / carbolic acid(80% bacterial growth in overnight culture suspensions of w/v)was from Indian Drug House, Sonarpur,24Pgs, individual bacterial isolate as measured by West Bengal whereas hypochlorite solution (4%) colorimeter in terms of optical value [4][7][8].The was from Stan Bio Reagent Pvt. Ltd,Kolkata. MIC was recorded as the lowest concentration of Both lysol and Chlorophenol(5% V/V ) were the tested disinfectant/antiseptic that will inhibit the manufactured by P. H. Laboratory, Madhyamgram. visible growth of the organism or that prevents 2% glutaraldehyde and chloroxylenol (dettol) were bacteria under test to grow after overnight manufactured from D.P.Enterprise,Kolkataand incubation. from Hootagali Ind area, Mysore respectively. The Observations and Results: In order to assess the source of povidone iodine was Win-Medicare Pvt. efficiency of commonly used antiseptics and Ltd., New Delhi. Lastly the manufacturer of the disinfectants in various fields of healthcare setup, chlorhexidine gluconate was 3M India Limited, eight of them were tested towards the two emerging Ranjangaon, Pune. All these disinfectants and strains of MRSA and MSSA. Results are depicted antiseptics had been used according to the in table (1) and table (2). procedure as per recommended by the Results of disc diffusion method: If thezone of manufacturer. The antimicrobial activities of these inhibitions were compared between MRSA and tested agents were measured against all the isolates MSSA for the eight disinfectants and antiseptics of MRSA and MSSA by disc diffusion method and they were (8-14)mm and (9-19)mm; (9- 25)mm and minimum inhibitory concentration method. (10-19)mm ; (11-26)mm and (14-26)mm ; (7- Susceptibility testing by disc diffusion method: 23)mm and (10-25)mm; (7-18)mm and (7-28)mm For each antiseptic and disinfectant 6mm discs for povidone-iodine [Figure 1], black disinfectant were prepared following standard protocol, where fluid [Figure 2], chlorhexidine gluconate [Figure 3] Whatman no 1 filter paper had been used to absorb , lysol [Figure 4], chloroxylenol [Figure 5] approximately the whole volume of one drop respectively.Secondly, as found in the disc (0.01ml) from micropipette [5].After impregnation, diffusion method the doses of resistance for the discs were dried by leaving them in an incubator tested disinfectants and antiseptics for MRSA and for 2hrs. For each of the eight different types of MSSA were 12.5mg/ml and 6.5mg/ml ; 8mg/ml disinfectants and antiseptics discs were prepared and 16mg/ml; 130mg/ml and 65mg/ml; 6mg/ml upto 16 th fold of dilution [6].Disc diffusion methods and 1.5mg/ml for povidone-iodine, blackfluid, lysol of susceptbility testing was performed following and chloxylenol respectively. Chlorhexidne standard method of Kirby-Bauer disc diffusion gluconate showed full susceptibility against both method as per CLSI guideline 2015 [2][3]where MRSA and MSSA strains till its 16 th fold dilutions standardisation of the bacterial suspension was with a variable zone of inhibition. 2% liquid phenol done by adjusting the turbidity with 0.5Mc Farland was established as totally resistant against MRSA standard (i.e., 1.5 × 10 8 CFU/mL) [4]. whereas for MSSA only the stock strength ie. Susceptibility testing by minimum inhibitory 20mg/ml showed susceptibility and rest all the concentration (MIC) method: This MIC test was dilutions came to be resistant [Figure 6]. 1% determined by broth microdilution technique hypochlorite and 2% glutaraldehyde expressed according to the method recommended in CLSI

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Indian Journal of Basic and Applied Medical Research; September 2016: Vol.-5, Issue- 4, P. 640-648 their full resistant for both MRSA and MSSA resistant to MRSA at a lower concentration (higher strains even at their stock strengths interestingly. dilution) in comparision to MSSA for each of the Results of minmum inhibitory concentration strengths.Only the blackfluid/ phenyl chlorophenol method: Comparision of the MIC values between showed the alteration. Among the eight disinfect- MRSA and MSSA for the eight tested disinfectants ants comparative results of the MIC values showed showed values were 2.5mg/ml,12.5mg/ml, MSSA strains having lesser MIC values than that 33mg/ml in case of chlorhexidine gluconate, of MRSA in case of blackfluid and 2% liquid phe- povidone-iodine and lysol respectively. Whereas nol. Rest all the disinfectants were having the same MIC values of the phenyl chlorophenol liquid for MIC values for both MRSA and MSSA strains. MRSA and MSSA it was 16mg/ml and 8mg/ml Table no. 1 shows variability of sizes of zone (mm) respectively. Chloroxylenol (dettol) showed full of inhibition in 8 different disinfectants and susceptibility even at its 16 th dilution. In MIC antiseptics (average values of 10 MRSA and 5 method 2% liquid phenol revealed the complete MSSA strains were calculated) concluded complete resistance against MRSA but showed MIC value of resistance in both MRSA and MSSA against 2% 20mg/ml for MSSA strains. In case of 1% glutaraldehyde and 1% hypochlorite while both the hypochlorite and 2% glutaraldehyde both MRSA strains were resistant against 2% liquid phenol and MSSA showed complete resistance even at except MSSA is susceptible to the stock strength of their original stock strengths as depicted in MIC 2% liquid phenol only. Among rest of the 5 disinf- procdure. ectants MSSA showed a higher range of zone of In this short study the tested isolates of MRSA and inhibition in comparison to MRSA.Considering 10 MSSA were selected depending on their significant MRSA strains and 5 MSSA strains positive control recurrence in nosocomial infection (nosocomial for MRSA and MSSA were 1.82-1.84 and 1.87-1.9 infection with MRSA is of 16%) Comparision of respectively. Negative control for povidone iodine, the results of the disc diffusion method itself, show chlorhexidine gluconate, dettol, blackfluid, 1% ed hospital supplied commercial povidone iodine hypochlorite, 2% glutaraldehyde, lysol and 2% (10% w/v), lysol (cresol + soap solution), chlorhe phenol were 0.3, 0.12, 0.24, 1.11, 1.2, 1.0, 0.33, 1.0 xidine gluconate,chloroxylenol (dettol), blackfluid respectively. Table no 2 showing variability of the (chlorophenyl) were variably susceptible against optical values for 8 different disinfectants and both MRSA and MSSA strains. It varied not only antiseptics against MRSA and MSSA strains with the type of disinfectants but also with the (average of 10 different MRSA and 5 different different concentrations of each disinfectant. For an MSSA taken) determined the MIC values of lysol, individual isolate and for a particular type of povide iodine , chlorhexidine gluconate as disinfectant’s strength MSSA showed larger zone 33mg/ml,12.5mg/ml, 2.5mg/ml. Dettol showing size in comparision to MRSA in case of povidone- susceptibility in all strengths. Blackfluid having iodine, lysol, dettol, chlorhexidine gluconate. Only MIC at 16mg/ml for MRSA and 8mg/ml for in phenyl blackfluid zone size of MSSA remains MSSA.1% hypochlorite and 2%glutaraldehyde unchanged in comparision to MRSA. Disc diffu both were showing full resistance for MRSA and sion also showed that for all the disinfectants inclu MSSA where 2% liquid phenol showed the only ding povidone-iodne, lysol, chlorhexine gluconate, sensitivity at the stock strength for MSSA. chloroxylenol(dettol) , 2% liquid phenol showed

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Indian Journal of Basic and Applied Medical Research; September 2016: Vol.-5, Issue- 4, P. 640-648

Discussion: chlorhexidine and 1% hypochlorite solution Disinfectants are usually tested against Salmonella whereas 15% MSSA with 29% of MRSA were typhi in Rideal-Walker coefficient test but with an resistant to betadine and 68% MSSA with (46- emergence of resistant strains of different types of 55)% MRSA had growth in presence of bacterial isolates it requires that a continuous phenol [11] .Limitation of this study was that the evaluation should be done for emerging and susceptibility pattern of the disinfectants were emergent organisms like ESBL, MRSA etc.The tested against one bacterial agent named present study shows the emergence of resistance of Staphylococcus aureus only. MRSA to commonly used disinfectants specially in Conclusion: hospital environment being invaded by 16% of Increasing prevalence (>16%) of MRSA infection MRSA. So this is required that antiseptics and in the hospital care environment has become an disinfectants should be evaluated in this light.This emerging problem worldwide. The antiseptics and study results show chloroxynelol (dettol) and disinfectants are part of cleaning maneuvers in chlorhexidine gluconate as the two maximally different high risk areas like emergency ward, effective disinfectants against Staphylococcus operation theatre, ICCU, ITU etc.In the system of aureus. Least susceptibility was found toward 1% hospital infection control it is essential to make the hypochlorite and 2% glutaraldehyde solution. 2% hospital atmosphere absolutely free from dangerous hospital supplied liquid phenol(carbolic acid) is organism like MRSA. In this study certain better suited for MSSA than MRSA.One of the commonly used antiseptics and disinfectants were previous studies where both disc diffusion and selected for hospital infection control purposes to broth dilution method were used for study the see their affect on MSSA and MRSA. efficacy of disinfectants and antiseptics results of Therefore inability to kill multidrug resistant MIC method showed chlorhexidine/hibitane as the organisms like MRSA, MSSA even by two such most effective disinfectant on MRSA followed by high level disinfectants(1% hypochlorite and 2% chloxylenol,hydrogen peroxide, sodium glutaraldehyde) and also by another intermediate hypochlorite , formaldehyde, sodium dichloro- level disifectant 2% liquid phenol carries an isocyanurate and povidone-iodine, while increased risk of occurance of uncontrolled severe chlorhexidine cetramide (savlon) showed no infections and its transmission in health care set up. efficacy against MRSA[9]Some other study result Regular follow up of the patients with their established povidone-iodine antisepticsolution as infection profile may uncover this fact. the most rapid bacteriocidal and having maximum Disinfectants are often misused and rationalization potency till date where both chlorhexidine and of their use in hospitals is desirable for control of hexachlorophene have persistent antibacterial infections. Therefore, in health care settings with effects even at low concentration [10].Another study high prevalence of MRSA4.8% chlorxylenol from WestBengal, that was done on testing the (dettol)& 4% chlorhexidine gluconate solution are sensitivity of MRSA and MSSA to different the most effective disinfectants to avoid cross antiseptics and disinfectants concluded that all the contamination in the hospital environment. MRSA and MSSA were sensitive to dettol,lysol,

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Table no.1: Disc diffusion method for selected disinfectants and antiseptics against MRSAand MSSA isolates

Lysol Povidone Chlorhex chloroxyle Blackflui 1% 2% 2% iodine idine nol d hypochl glutarylde liquid gluconate (Dettol) orite hide phenol D C Zone C Zone C Zone C Zone C Zone C Zone C Zone C Zone il o of o of o of o of o of o of o of o of u nc inhibi n inhibit n inhibi n inhibit n inhibi nc inhibi nc inhibi nc inhibi ti m tion c ion c tion c ion c tion m tion m tion m tion o g/ M M m M M m M M m M M m M M g/ M M g/ M M g/ M M n m R S g R S g R S g R S g R S m R S m R S m R S s l S S / S S / S S / S S / S S l S S l S S l S S A A m A A m A A m A A m A A A A A A A A m m l m l l m m l m m m m m m m m m m m m m m m m m m m m m m m m m m m

D 5 1 2 1 12 1 4 1 2 4 1 2 6 R 7 1 R R 2 R R 2 R 1 2 3 1 0 6 0 8 2 8 9 3 3 0 0 0 0 0 0 D 2 6 1 5 10 1 2 1 1 2 1 1 3 R 5 5 R R 1 R R 1 R R / 6 . 0 0 3 0 7 7 4 8 8 2 0 0 2 0 0 D 1 R R 2 8 9 1 1 1 1 1 1 1 R R 2. R R 5 R R 5 R R / 3 5 . 0 6 6 2 3. 0 6 5 4 0 6 2 D 6 R R 1 R 9 5 1 1 6 R 7. 8 R R 1. R R 2. R R 3 R R / 5 2 4 5 6 2 5 8 . 5 5 D 3 R R 6 R R 2 1 1 3 R 7 4 R R 0. R R 1. R R 2 R R / 3 . . 4 5 6 2 1 2 5 2 5 6 5 D 1 R R 3 R R 1 1 1 1 R R 2 R R 0. R R 0. R R 1 R R / 7 . . 3 4 . 3 6 3 1 2 5 1 3 2 2 5

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Table no. 2: Minimum inhibitory concentration for selected disinfectants and antiseptics against MRSA and MSSAisolates

Lysol Povidone Chlorhexid Chloroxyle blackfluid 1% 2% 2% liquid iodine ine nol (dettol) hypochlo glutarylde phenol gluconate rite hide D C Optic C Optical C Optical C Optical C Optical C Optical C Optica C Optica il o al o density o density o density o density o density o l o l u nc densit n value n value n value n value n value nc densit n density ti m y c c c c c m y c value o g/ value m m m m m g/ value m n m M M g M M g M M g M M g M M g M M m M M g M M l R S / R S / R S / R S / R S / R S l R S / R S S S m S S m S S m S S m S S m S S S S m S S A A l A A l A A l A A l A A l A A A A l A A

D 5 0 0 1 0. 0. 4 0. 0. 4 0. 0. 6 1. 1. 1 1. 1. 2 1 1. 2 1. 1. 2 . . 0 3 3 0 1 1 8 2 2 3 1 1 0 5 3 0 . 4 0 2 O 0 3 3 0 0 0 2 2 4 4 1 1 1 3 3 3 D 2 0 0 5 0. 0. 2 0. 0. 2 0. 0. 3 1. 1. 5 1. 1. 1 1 1. 1 1. 1. / 6 . . 0 3 3 0 1 1 4 2 2 2 1 1 5 5 0 . 5 0 3 3 2 0 3 3 0 0 2 2 4 4 1 1 7 5 2 3 3 7 D 1 0 0 2 0. 0. 1 0. 0. 1 0. 0. 1 1. 1. 2 1. 1. 5 1 1. 5 1. 1. / 3 . . 5 3 3 0 1 1 2 2 2 6 1 1 . 6 6 . 5 5 5 4 0 3 3 0 0 2 2 4 4 1 1 5 8 6 4 7 3 3 8 D 6 0 0 1 0. 0. 5 0. 0. 6 0. 0. 8 1. 1. 1 1. 1. 2. 1 1. 3 1. 1. / 5 . . 2 3 3 1 1 2 2 4 1 . 7 7 5 . 6 5 7 8 3 3 . 0 0 2 2 4 4 0 1 2 1 2 7 2 6 3 3 5 5 1 D 3 0 0 6 1. 0. 2 0. 0. 3 0. 0. 4 1. 1. 0 1. 1. 1. 1 1. 2 1. 1. / 3 . . . 1 6 . 1 1 2 2 5 6 . 8 7 2 . 7 7 7 1 3 3 2 1 7 5 2 2 4 4 2 0 6 9 5 7 2 8 6 3 3 5 3 8 D 1 1 0 3 1. 1. 1 1. 0. 1 0. 0. 2 1. 1. 0 1. 1. 0. 1 1. 1. 1. / 7 . . . 7 6 . 7 7 . 2 2 7 7 . 8 7 6 . 7 1 8 7 3 7 5 1 0 3 2 0 9 5 4 4 7 9 3 9 3 8 4 0 9 2 8 4 2 5 1

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Figures: Bar graphs showing the zone of inhibition(in mm)of the selected antiseptics and disinfectants towardMRSA and MSSA with their stock strengths (D) and 16 th fold serial dilutions (D/2-D/32) .

Zone of inhibition at different Zone of inhibition at different dilutions of Chlorhexidine dilutions of Povidone Iodine 25 gluconate 20 20 15 15 MRSA

10 MRSA mm MSSA

mm 10 5 MSSA 5

0 0 D D/2 D/4 D/8 D D/2 D/4 D/8 D/16D/32

Fig.1 Fig.2

Zone of inhibition at different Zone of inhibition at different dilutions of Black fluid dilutions of Lysol 18 25 16 14 20 12 MRSA 10 15 mm MRSA MSSA 8

mm MSSA 10 6 4 5 2 0 0 D D/2 D/4 D D/2 D/4

Fig.3 Fig.4

Zone of inhibition at different Zone of inhibition at different dilutions of Dettol dilutions of 25 2% phenol 20 MRSA 30 15 20 mm 10 MSSA 5 mm 10 MSSA 0 0 D D/2

Fig. 5 Fig.6

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