Evaluation of the Anti-enterococcal Activity of Disinfectants and Medicated Soaps on Vancomycin-resistant Enterococcus faecalis Strains

Background: Enterococcus faecalis has intrinsic resistance which aids its spread in the hospital environment. As a nosocomial pathogen with increasing resistance breaking its route of transmission and spread is therefore imperative. Aims: In this study, six brands of disinfectants and eight medicated soaps commonly used in health care facilities and at homes were investigated for anti-enterococcal activity against eleven strains of E. faecalis ten of which are vancomycin-resistant. Place and Duration of Study: This study was carried out in the Department of Microbiology, Ekiti State University, Nigeria between July, 2009 and February, 2010. Methodology: Standard microbiological methods were used to determine the effects of the disinfectant and soap samples on the strains of vancomycin-resistant E. faecalis. Results: Two of the disinfectants, NXD and ZGC that there is no correlation between resistance to antibiotics, vancomycin in particular and disinfectants. The disinfectants CRT, RBT, TMS and DTA, in that decreasing order, showed anti-enterococcal property while MSF and NVA showed the least effect on the enterococci. Strains recovered from the surface of the bland soap sample had confluent growth pattern which indicated the ability of the organisms to survive on its surface. Survival was least on CRT soap sample followed by DTL soap sample and most of the strains grew very well on the surfaces of most of the soap samples. Conclusion: This study shows that most of the disinfectants are not effective at the manufacturers’ recommended in-use concentrations, and also that pathogens can be transmitted through the common use or sharing of soaps contaminated with the carriers of antimicrobial-resistant pathogens.


INTRODUCTION
Enterococcus species are ubiquitous commensal inhabitants of the gastrointestinal tract of humans, animals and other invertebrates. They are frequently isolated from environmental sources such as soil, surface waters, and raw plant and animal products, where their intrinsic resistance enables them to survive and spread in the environment [1,2]. These organisms acquire antibiotic resistance and spread resistance genes to other species [3]. Multiple antibiotic-resistant enterococci (MRE) have emerged as a global threat to public health which threatens to compromise effective antibiotic treatment [4]. The application of biocides to heavily contaminated environments reduces health care-associated transmission of contagious diseases [5,6]. Health care facilities can be contaminated by nosocomial pathogens in hospitals and during medical practices [7,8].
Biocides are used extensively in hospitals and other health care settings for a variety of purposes. In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial infections [8,9]. A wide variety of active chemical agents or biocides have been used both in antisepsis and disinfection for the prevention of both endemic and epidemic infections and/or diseases [10,11]. Biocides have a broader spectrum of activity than antibiotics, yet less emphasis is laid on biocides compared to antibiotics, as biocides have multiple targets [12]. The widespread use of antiseptic and disinfectant products has led to microbial resistance and cross-resistance to antibiotics in particular.
Most biocides are used singly or in combination and they contain a variety of ingredients which acts on different parts of the target bacteria [13,14,15]. As with antibiotics and other chemotherapeutic agents, acquired resistance to antiseptics and disinfectants can arise either through mutation or the acquisition of genetic material in the form of plasmids or transposons. The role of plasmids in encoding resistance to biocides has been reported [16]. The resistance pattern of vancomycin-resistant E. faecalis to some common antimicrobials (biocides) was therefore investigated in this study.

Source of Disinfectants
Six brands of common disinfectants used in both household and hospitals were purchased from supermarkets in Ado-Ekiti and were coded as follows: DTL, ZAL, NXD, SVN, VGL and ZGC. Seven different germicidal soaps against E. faecalis which included CRT, DTA, DTL, MSF, NVA, RBT and TMS were also examined. The active ingredients of the disinfectants and soap used are shown in Tables 1 and 2 respectively.

Determination of Susceptibility of E. faecalis to Disinfectants
Five dilution levels of each of the disinfectants were prepared with manufacturers' recommended in-use dilutions as median. Two loopfuls of standardized broth of an 18 hour culture (in Müller-Hinton broth, Oxoid, Basingstoke, Hampshire, UK) with approximately 5.0 x 10 7 cfu/ml were introduced into 2 ml of each of the dilution of the disinfectant. After contact for 10 minutes, 5% Tween 80 prepared in deionized water was added to neutralize the action of the disinfectant. The organism was streaked on freshly prepared Müller-Hinton Agar and incubated at 37ºC for 18 h. Both negative and positive control organisms were also similarly treated, plated and incubated as stated above. Development of colonies was taken as positive while plates without sign of growth were taken as negative.

Determination of Susceptibility of E. faecalis to Antiseptic Soaps
A 0.1 ml amount of standardized inoculum of the test organisms was introduced into 1 ml of 1.0% w/v of the soap solution and at time intervals, added to molten Müller-Hinton agar, plated and allowed to solidify. The plates were subsequently incubated at 37ºC for 24 h and evaluated thereafter according to the CLSI guidelines [17].

Determination of Survival of the Test Organisms on the Surface of the Soap
The ability of the isolates to survive on the surface of the soap samples was determined by inoculating standardized inoculum on the sterile surface (9.0 cm 2 ) of the soap samples, wrapped up in sterile aluminum foil. and incubated at 37ºC for 72 h. The inoculated surface of the soap was swabbed, plated on Bile aesculin agar (Oxoid), incubated at 37ºC for 24h and subsequently evaluated.

RESULTS AND DISCUSSION
Six brands of disinfectants commonly used in the health care facility and in homes were tested against strains of E. faecalis. Eleven strains of E. faecalis were used in this study (Table 3) out of which ten were vancomycin-resistant while vancomycin-sensitive E. faecalis ATCC 29212 served as control. The results of the present study indicate that the test organisms showed diverse resistance to the test agents; not all the disinfectants inhibited the growth of the test organisms. Dichlorometaxylenol and terpineol were present in two of the disinfectants, viz. ZGC and NXD which completely inhibited the test organisms at the manufacturers' recommended in-use concentrations. ZAL followed by VGL had the least anti-enterococcal effects. The finding in this study corroborates the report of Karatzas et al. [18] which showed that E. faecalis can adapt and develop resistance to extensively used antiseptics and disinfectants. Many reports of resistance often have parallel issues including inadequate cleaning, incorrect product use, or ineffective infection control practices, which cannot be overemphasized [19]. Hence the control of infectious diseases is inevitable and largely depends on the approaches employed to break the transmission chain [20].  (Table 4). The nature of the active ingredients and particularly their concentrations in biocides may explain why some are inhibitory while some are not [11,14]. Quite an array of chemical agents are presented or marketed as chemical floor disinfectants, each with different composition, activity and mode of action [15,21]. Enterococcus faecalis DMOF 53 followed by E. faecalis DMOF 47 were the most resistant strains while E. faecalis DMOF 21 showed the least resistance to the disinfectants. Vali et al. [16] and Noguchi et al. [22] reported that some acquired mechanisms of resistance have been shown to have clinical significance and disinfectants if not carefully selected may be carrier/reservoir of outbreak of nosocomial infections [8,23]. Except CRT, all the soap brands had just one active ingredient. Enterococcus faecalis DMOF 53 was the most resistant strain tested, followed by E. faecalis DMOF 47 while E. faecalis DMOF 21 and E. faecalis ATCC 29212 showed the least resistance to the disinfectants. This probably indicates that the active ingredients in the soap may be used to control the spread of the isolates investigated in this study.

R= Manufacturers' in use concentrations, + = growth, --= no growth
Among the soap samples tested CRT followed by DTL were very effective against the test organisms. Sharing soaps in health care facilities could lead to the spread of potentially harmful bacteria. Manufacturers' recommended in-use concentrations of most of the disinfectants were not effective, and hence would most probably not control proliferation and spread of the test organisms. The versatile nature, ability of the vancomycin-resistant E. faecalis to become resistant to adverse environmental conditions, possession of a large arsenal of pathogenic factors, intrinsic resistance to many antimicrobial agents and high propensity to transfer genetic materials, may as well explain resistance to the biocides investigated. This claim is however still open to further investigation.

CONCLUSION
We conclude that most of the disinfectants (biocides) examined were not effective at the inuse concentrations that were recommended by their respective manufacturers. Ability of most test organisms on the surface of soap samples also shows their poor activity and ineffectiveness against the test organisms. The use of biocides with poor efficacy may only give a false sense of security but represent potential reservoirs and or sources of transmission of antimicrobial-resistant pathogens.