The efficacy of cetylpyridinium chloride glove coatings against Staphylococcus epidermidis and Staphylococcus aureus

international

Biodeterioration

& Biodegradation, Vol. 39, No. 0

PII:

SO964-8305(96)00037-6

I (1997)

l-7

1997 Elsevier Science Limited

All rights reserved. Printed in Great Britain 0964s8305/97 $17.00 + 0.00

ELSEVIER

The Efficacy of Cetylpyridinium Chloride Glove Coatings Against Staphylococcus epidermidis and Staphylococcus aureus E. L. Prince,” C. Perez-Giraldob & L. H. G. Morton” aDepartment of Applied Biology, University of Central Lancashire, Preston, UK ‘DPTO Microbiologia, Facultad de Medicina, Extremadura University, Badajoz, Spain

(Received 20 October 1995; revised version received 14 February

1996; accepted 23 July 1996)

A study was undertaken to determine whether cetylpyridinium chloride (CPC) glove coatings are effective against the common skin-inhabiting bacteria Staphylococcus epidermidis and Staphylococcus aureus. The time course of their activity in the presence and absence of glove material was assessed. The in vitro MIC of CPC against each of the test organisms was determined as 0.3 ug ml-’ A spectrophotometric assay was used to measure the CPC concentration in each of 20 unused surgical gloves, which was found to be highly variable, but always well in excess of the MIC against the test organisms. The time course of CPC activity was determined in vitro in the presence and absence of glove material, and it was found that the activity of CPC was significantly reduced when glove material was present. The time course of CPC activity was also determined using glove cultures, at initial inoculum levels of lo4 or 10’ cfu ml-‘, and it was found that at the lower inoculum level, Staphylococcus epidermidis was rendered non-viable in 50% of gloves tested within 15 min, and Staphylococcus aureus within 2 h. Reductions in cell counts were less dramatic at the higher inoculum level, S. epidermidis was rendered non-viable in 50% of gloves tested within 2 h, but S. aureus was rendered non-viable in only 10% of gloves tested in the same time, although the viable counts of most of the other treatments were significantly reduced. 0 1997 Elsevier Science Limited

been carried out to determine the incidence of perforations in surgical gloves, either before use (Paulssen et al., 1988; Ballbach et al., 1972; Korniewicz et al., 1994), or arising during the course of normal usage (Furuhashi & Miyamae, 1976; Church & Sanderson, 1980; Brough et al., 1988; Korniewicz et al., 1989). The results of these studies demonstrate that unused gloves are not uncommonly perforated before use, indeed the American Society for Testing and Materials Standard for surgical gloves (ASTM, 1977) permits 1.5% to contain holes yet still be acceptable for sale (Beck & Nora, 1977). During surgery, the rate of glove perforation depends upon the type of operation being performed, but can occur in as many as 75% of cases in procedures such as abdominal mass closure (Brough et al., 1988), often without the knowledge of the personnel involved. Although the use of antiseptic hand-washing agents prior to glove donning reduces the viability of the

INTRODUCTION The use of gloves during surgical procedures serves two purposes: to protect the surgeon from contamination by blood or other exudates from the patient, and to protect the patient from transfer of microorganisms from the surgeon’s hand (Paulssen 1988). The AIDS epidemic has also et al., prompted health care personnel to question the barrier effectiveness of gloves used in clinical settings (Korniewicz et al., 1994). The growing concern for the need to protect health care workers against blood-borne pathogens is further highlighted by the introduction of an emergency standard test method which involves exposing protective clothing to a surrogate virus challenge (ASTM, 1992). Protection is conferred by the provision of a physical barrier, and the et?ective functioning of gloves is therefore compromised if this barrier is breached. A number of studies have 1

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E. L. Prince et al.

skin flora, the antiseptic concentration available from a washed skin surface is insufficiently high to provide adequate protection against microbial contaminants resulting from glove perforation. (Modak et al., 1992). The use of antiseptic-coated gloves, which are coated on their inner surfaces with a layer of cetylpyridinium chloride (CPC), theoretically provides additional security by bringing about a further reduction in the viability of the skin flora than would result from the use of hand washing alone. Regrowth of the flora is also inhibited during lengthy operations, indeed Newsom et al. (1988) report that when CPC-coated gloves are used, the postoperative levels of skin contamination are almost always lower than those recorded after the preoperative scrub. Martin et al. (1988) confirm the antimicrobial properties of CPC-coated gloves against a monoculture of Staphylococcus aureus, after an incubation period of 18 h. There are, however, no reports of the time course of activity of CPC-coated gloves, although this ought to be an important parameter for consideration given that the durations of surgical procedures vary considerably. The present study was therefore undertaken in order to evaluate the antibacterial efficacy of CPC-coated gloves and in particular the time course of their activity, in order to assess the potential security against cross-infection provided by their use in surgical procedures of various durations. Staphylococcus aureus and Staphylococcus epidermidis were chosen as test organisms since these are amongst the most predominant and persistent staphylococci isolated from human skin (Kloos & Musselwhite, 1975).

MATERIALS AND METHODS

gloves) were available for this work. These gloves were manufactured and supplied by LRC Products Limited (London, UK). Spectrophotometric determinations of CPC concentration in ‘Biogel’ gloves Initial calibration studies were carried out using a preparation of cetylpyridinium commercial chloride (Sigma C-9002). This was used to prepare a range of dilutions of CPC in distilled water, at concentrations ranging from 0.625 to 200 ng ml-‘. The dilutions were scanned between the wavelengths of 190 and 900nm using a PerkinElmer Lambda 5 UVjVIS spectrophotometer, and were found to absorb strongly at a wavelength of 258.9nm. Absorbance values recorded at this wavelength were therefore used both to prepare a calibration curve, and in all subsequent determinations of unknown CPC concentrations. Ten-millilitre aliquots of distilled water were dispensed into each of twenty individual unused gloves, which were then massaged for 30s and subsequently left to stand for 1Omin at ambient temperature. The contents were then removed and CPC concentration was determined spectrophotometrically by reference to the calibration curve. Susceptibility of test organisms to CPC in vitro The minimum inhibitory concentration (MIC) of CPC against the test organisms was evaluated using an initial inoculum of IO4 colony forming units per ml (cfu ml-‘). All determinations were performed using nutrient broth as the liquid medium, incubated for 24 h at 37°C. The MIC was defined as the lowest concentration of CPC which prevented the development of turbidity in the broths.

Microorganisms CPC activity in the presence of glove material Cultures of Staphylococcus aureus (Strain no. NCTC 657 1) and Staphylococcus epidermidis (Strain no. NCIMB 4276), obtained from the culture collection at the University of Central Lancashire, were used throughout this programme of work. Gloves Sterile surgical gloves containing CPC as the active ingredient (Regent Biogel starch free surgeons’

Samples of glove material (1.0 cm*) were sterilised by autoclaving, after first removing the coating of CPC by repeated washing in distilled water. An overnight broth culture of S. epidermidis in nutrient broth (LabM) was diluted in quarter strength ringers solution (Oxoid) to provide an approximate cell density of lo5 cfu ml-‘. Twentymillilitre aliquots of the suspension were dispensed into sterile glass bottles and CPC was added to

Efficacy of CPC

provide concentrations of 0.5, 2.0 or 8.0 ug ml-‘. Eight replicates were provided at each concentration; four containing 1.5 g of washed glove material and the remainder with no further additions. A further four replicates containing neither CPC nor glove material were provided as controls. The samples were incubated at 37°C for 4h and the viability of the inocula was periodically assessed by viable count estimations. Quarter strength Ringers solution (Oxoid) was used as a diluent for viability estimations throughout this programme of work as preliminary experiments had shown an inactivator to be unnecessary. Glove cultures Inocula of the test organisms used were prepared as overnight broth cultures in nutrient broth (LabM) and then diluted with quarter strength Ringers solution to provide approximate cell densities of either lo4 or lo5 cfu ml-‘. The viability of the inocula was determined prior to each experiment by plating appropriate dilutions in quarter strength Ringers solutions (Oxoid) onto nutrient agar. The antimicrobial activity of the gloves was determined by dispensing 10 ml aliquots of the above inocula into individual gloves, which were then tied off at the cuff and incubated at 37°C for periods of up to 4 h. At intervals throughout the experiment, small samples of broth were removed from the gloves and dilutions thereof were plated onto nutrient agar in order to determine the viability of the inocula. Twenty gloves were inoculated with S. aureus and 24 with S. epidermidis.

RESULTS Spectrophotometric determination of CPC concentration in ‘Biogel’ gloves The CPC concentrations recorded varied from 24.6 to 110.8 pg ml-‘, with a mean value of 60.43 and a standard deviation of 23.6.

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CPC activity in the presence of glove material Figure 1 shows the activity of various concentrations of CPC against S. epidermidis both in the presence and in the absence of glove material. The control replicates, containing no CPC, showed an increase in mean viable count from 8 x lo4 to 9 x lo5 cfu ml-’ over the 4 h duration of the experiment. No such increase was recorded in the case of test replicates containing CPC. At a CPC concentration of 0.5ug ml-‘, there was no evidence of proliferation of test organisms either with or without glove material. At a CPC concentration of 2 ug ml-‘, the test organisms were rendered non-viable within 2 h in the absence of glove material and again showed no evidence of proliferation when glove material was present. At a CPC concentration of 8 ug ml-‘, the test organisms were rendered non-viable within 2 h in the presence of glove material and within 15 min in its absence. Glove cultures Figure 2 shows the results of the glove culture experiments. There was again no evidence of proliferation of the test organisms at either of the levels inoculum indeed initial employed, substantial reductions in the viable counts were recorded over the course of the experiment, particularly using the lower initial inocula. At this inoculum level, S. epidermidis was rendered nonviable within 15 min in 50% of gloves tested, and in 70% of gloves tested after 4 h. S. aureus was rendered non-viable in 50% of gloves tested after 2 h, and in 90% of gloves tested after 4 h. At the higher inoculum level, the reductions in viable counts were less dramatic, although there was still no evidence of proliferation of the test organisms. S. epidermidis was rendered non-viable within 2 h in 50% of gloves tested and in 60% after 4 h. S. aureus was rendered non-viable in only 10% of gloves tested after 2 h, but the viable counts of most of the other replicates were significantly reduced.

DISCUSSION Susceptibility of test organisms to CPC in

vitro

The MIC for both S. epidermidis and S. aureus was found to be 0.3 ug ml-‘.

The results of the spectrophotometric determination of CPC concentration suggested considerable variation between individual gloves,

E. L. Prince et al.

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Fig. 1. Activity of CPC against S. epidermidis in vitro at concentrations of 0.5, 2.0 and 8.0 pg ml-‘. Results shown are the means of four replicates of each treatment.

although even the lowest concentration found was considerably in excess of the MIC value against the extraction the test organisms. Furthermore, method used will inevitably result in an underestimation of the amount of CPC present, since not all the coating will be removed by washing with 10ml of water. It is also perhaps the inappropriate to refer to somewhat gloves ‘concentration’ of CPC in individual without first considering the relevance of this parameter in vivo. When gloves are ‘in-use’ the activity of the CPC coating against the skin flora of the wearer will be exerted at the glove-skin interface, where the CPC will dissolve in the layer of sweat trapped inside the glove. Unless the

volume of sweat produced is greater than 10 ml, the actual concentration of CPC at the glove-skin interface will be higher than suggested by the spectrophotometric determination. The finding that CPC activity is reduced in the presence of glove material is unremarkable as many biocides exhibit this effect, usually due to adsorption of the biocide to a surface, thereby reducing its available concentration. However, although the presence of glove material appears to reduce the bactericidal activity of CPC, the bacteriostatic activity does not seem to be affected. Thus, at a CPC concentration of 0.5 ng ml-‘, i.e. close to the MIC of the test organisms, bacteriostasis is achieved whether or not glove

Efficacy of CPC

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material is present. At a concentration of 2.0 ug ml-’ CPC was bacteriostatic in the presence of glove material, but bactericidal in its absence. However, at a concentration of 8 ug ml-‘, i.e. well below the concentration found in the gloves by the spectrophotometric determination, CPC was bactericidal in the presence of glove material and was able to reduce the viability of S. epidermidis to zero within 2 h. The glove culture experiments were carried out using 10 ml aliquots of liquid cultures, and the concentration of CPC in these experiments would

therefore be of the order suggested by the spectrophotometric assay, since this was performed in the same volume of liquid. Again, however, the in-use concentration at the gloveskin interface is likely to be higher than the concentration evaluated in the glove culture experiments, for the reasons explained above. The results of the glove culture experiments show that the CPC within the gloves was bacteriostatic at both of the initial inoculum levels used, although the bactericidal activity was less pronounced at the higher inoculum level.

E. L. Prince

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However, it should be considered that the coating is intended to prevent regrowth of the skin flora on post-scrub hands and the initial inoculum in viva ought therefore to be lower than the levels employed in the present study. Using a contact plate technique, Newsom et al. (1988) counted less than 10’ viable colonies on post-scrub swabs taken from hands washed with soap and less than 10 colonies when chlorhexidine or povidone-iodine scrubs were used. Holloway et al. (1990) used a glove juice technique to evaluate the effectiveness of a

chlorhexidine scrub and found a mean of 2.3 x lo3 viable bacteria per hand in a survey of 95 individuals. Under these conditions the use of CPC-coated gloves would clearly be effective in preventing regrowth of staphylococci post-scrub and would have the added advantage of exerting a bactericidal activity against organisms still viable post-scrub. Since this bactericidal activity is exerted within 15 min, it is clear that the use of CPC-coated gloves ought to be advantageous even for procedures of relatively short duration, at these inoculum levels. The present study,

Efficacy

however, was limited to in vitro experimentation, and took no account of the potential interactions between CPC glove coatings and the wearer’s hands. In particular, there is a possibility that residual hand-washing agents on the wearer’s hands might significantly affect the activity of CPC in vim. It might therefore be expedient to follow up the present study with such as glove juice additional experimentation, tests, in order to investigate this possibility.

of CPC

7

Church, J. & Sanderson, P. (1980). Surgical gloves puncture. J. Hospital Infect., 1, 84. Furuhashi, M. & Miyamae, T. (1976). Effect of pre-operative hand scrubbing and influences of pinholes appearing in surgical rubber gloves during operation. Bull. Tokyo Med. Dental Uni., 26, 73-80. Holloway, P.M., Platt, J.H., Retbrouck, G., Lilly, H.A., Mehtar, S. & Drabu, Y. (1990). A multi-centre evaluation of two chlorhexidine-containing formulations for surgical hand disinfection. J. Hosp. Znfect., 16, 151159. Kloos, W.E. & Musselwhite, M.S. (1975). Distribution and persistence of Staphylococcus and Micrococcus species and other aerobic bacteria on human skin. Appl. Environ. Micro&o/., 30(3), 381-387.

REFERENCES

Korniewicz, D.M., Laughton, (1989). Integrity of vinyl

B.E., Butz, A. & Larson, E. and latex procedure gloves.

Nursing Res., 38(3), 144146.

American Society for Testing Materials (1997). Standard specification for rubber and latex examination gloves. In Annual Book af ASTM Standards 37, l-10. Philadelphia: The American Society for Testing of Materials. American Society for Testing Materials (1992). Emergency standard test method for resistance of protective clothing materials to penetration by blood-borne pathogens using viral penetration as a test system. ASTM Designation :ES 22-92, l-7. Ballbach, R.L., Beavin, P. & Walters, S.H. (1972). A study of testing methods for the detection of defects in disposable latex and plastic gloves. J. Assoc. Official Anal. Chem., 55(5), 1074-1080. Beck, W.C. & Nora, P.F. (1977). ASTM Standard for surgical gloves. AORN J., 25(5), 869-872. Brough, F.J., Hunt, T.M. & Barrie, W.W. (1988). Surgical glove perforations. Brit. J. Surgery, 75, 110-l 17.

Korniewicz, D.M., Kirwin, M., Cresci, K., Sing, T., Choo, T.E., Wool, M. & Larson, E. (1994). Barrier protection with examination gloves: Double versus single. Am. J. Infect. Control., 22(l), 12-15.

Martin, M.V., Dunn, H.M., Field, E.A., Field, J.K., Hibbert, S.A., McGowan, P. & Wardle, I. (1988). A physical and microbiological evaluation of the re-use of non-sterile gloves. Brit. Dental J., 165, 321-324. Modak, S., Sampath, L., Miller, H.S.S. & Millman, I. (1992). infectious inactivation of pathogens by Rapid chlorhexidine-coated gloves. Infect. Control and Hosp. Epidemiol., 13(8), 46347 1. Newsom, SW., Rowland, C. & Wells, F.C. (1988). What is in the surgeon’s glove? J. Hosp. Infect., ll(Supplement A), 244259. Paulssen, J., Eidem, T. & Kristiansen, R. (1988). Perforations in surgeon’s gloves. J. Hosp. Infect.. 11, 82-85.