writings:school:bacterial_inhibition_soap

Antibacterial inhibition of a hospital soap versus consumer off-the shelf soaps

Authors
Alan Trick & Matthew Strauss1)
Class
BIO 112 University of the Fraser Valley
Date
2016 April 07

Introduction

Soap is an important staple of good hygiene but only recent years have shown how necessary it is for good health. As a cleansing agent and purification ritual, humans constantly seek better ways of achieving cleanliness[1]. As a cleaning tool, soap was first written about by Greek physician Galen (130–200 AD) and the chemist Gabiribne Hayyan in the eighth century[2]. However simple physical cleanliness isn’t the only effect of washing. Science has since learned the impact of washing hands in regards to disease prevention. Simple washing can reduce diarrhea risk by 47% and avert approximately 1 million diarrhea-related deaths[3]. Even in unsanitary conditions, hand washing can have a positive interrupting effect on diseases such as Shigella[4]. In 1874, Hungarian physician Ignaz Semmelweis discovered that washing hands could reduce streptococcal puerperal sepsis from 22% to 3%[5].

The necessity of commercial antibacterial soap is debated and even so, 76% of liquid soaps and 29% of bar soaps were found to contain antibacterial agents[6]. Recent advances in antibacterial products have produced a phenomena where antibacterial resistant strains develop from overuse[7]. This is a worrying trend as super-bugs can develop in such conditions[8]. Not all bacteria are bad but those that are tend to deal severe blows to infected individuals—thus antibacterial resistant soaps are necessary for individuals who come into close proximity of harmful bacteria. Doctors, patients, nurses, and staff who work in clinics and hospitals are the most likely workers to contract a bug due to their proximity.

Hospitals should be among the cleanest locations as delicate and susceptible patients come into close proximity with potentially contagious bacteria from sick patients. Surveys reveal almost 6–8% of infections are acquired in hospital[9]. Infections acquired from hospitals can harm patients, extend stays and consume resources[10]. Even with this knowledge, some studies find that doctors do not wash their hands as often as is advised[11]. It appears staff believe they wash between patients 73% of the time; the reality was observed to be only 9% of visits had a wash[12]. The unfortunate prevalence of negligent hand-washing only reinforces the need for effective soap in dealing with the plethora of potentially harmful bacteria hospital staff interact with.

When antibiotic soaps are used expansively, their effects can permeate throughout the ecological and social system and find their way back to humans. While daily use antibacterial soaps are debatable due to the potential of creating superbugs, hospitals are surely a place where sterility is desirable[8]. This experiment will not compare the primary activity of soaps and surfactants in removing detritus and pathogens from skin; rather, it will compare various soaps antibacterial resistances. Using gram negative Escherichia coli and gram positive Bacillus subtilis bacteria, soaps with varying active ingredients will be compared. If preventing bacterial infections are of primary importance in the hospital environment, then Deb Hygenipak, a hospital sourced soap, should have a stronger bacterial resistance against both gram negative and gram positive bacteria than the commercially available brands tested.

Full names of products and possible active ingredients can be found in table 3 and table 4 in the appendix.

Procedure

The soaps were tested using an agar disk diffusion protocol provided by the University of the Fraser Valley[13]. Overall, 3 trials were performed. Deionized water was used as a negative control and chloramphenicol disks were used as a positive control in order to ensure proper handling and execution of procedures. Chloramphenicol is a good positive control because it has high activity against gram-positive and gram-negative bacteria[14].

On the first week, 800 mL of tryptic soy agar was prepared, autoclaved for 20 minutes, and then cooled down in a water bath at 55°C. Then the agar was poured into 40 sterile Petri dishes using sterile technique, and then left on the bench to dry overnight. 10 test tubes of 5 ml of tryptic soy broth were also prepared and sterilized in the autoclave along with cotton swabs, deionized water, and filter paper disks.

The three trials were then performed on the second and third week. Using sterile technique, two bacterial cultures (B. subtilis and E. coli) were transferred from starter plate cultures into 1 tube of broth each. They were then incubated overnight at 30°C (for B. subtilis) and 37°C (for E. coli).

After the bacteria had grown, they were transferred to Petri dishes along with soap samples and controls. The bacterial samples were transferred with sterile technique using cotton swabs. The swab was streaked across the whole plate, the plate rotated 90°, and then streaked once more. Soap samples and the negative control were applied to the filter paper disks separately in small dishes. The negative control and the Deb Hygenipak Skin Cleanser were applied using one drop from a Pasteur pipette. The remaining soaps were deemed to be too viscous for a pipette. For these, the filter paper disks were mixed, using flamed forceps, in a thin layer of soap placed inside their dishes. The soap and control disks were then transferred to the Petri dishes using flamed forceps. For each soap and control, three dishes of E. coli and three dishes of B. subtilis were prepared. The Petri dishes were then incubated overnight at 30°C (for B. subtilis) and 37°C (for E. coli).

Finally, a gram stain was also performed on bacterial broths for the second and third tests. This was to ensure that our bacterial samples had not been swapped or mislabeled.

Results

Table 1: Zones of inhibition from disks on B. subtilis
Soap Brand Mean Diameter (mm) Standard Deviation
Deb Hygenipak 16 7.4
Live Clean 11 2.7
T36 19 5.8
Equate 20 2.9
Natural Concepts 50 13
Chloramphenicol 26 1.9
Deionized water 6 0
Table 2: Zones of inhibition from disks on E. coli
Soap Brand Mean Diameter (mm) Standard Deviation
Deb Hygenipak 7 1.3
Live Clean 6 0.33
T36 9 1.0
Equate 7 0.72
Natural Concepts 39 9.5
Chloramphenicol 25 1.3
Deionized water 7 0.79

Against both bacteria, Natural Concepts and the positive control chloramphenicol produced the largest zones of inhibition. Negative control deionized water produced either a minimal zone of inhibition or an unexpectedly large zone of inhibition. Due to either faulty procedure, the “tidal effect” of excessive soap sample bleeding into nearby zones or contamination, certain negative control zones were substantially large. Their relative closeness to Natural Concepts is theorized to be the cause as Natural Concepts produced some zones of inhibition that included the negative control disc. All soaps were less effective against E. coli with Deb Hygenipak, Live Clean, T36 and Equate all producing reliably minimal zones. Of note, the zones were visually smaller than those of the negative control, indicating lack of any gram negative antibiotic resistance. Against B. subtilis however, the four soaps had improved effect.

Of deviations, as zones of inhibitions increased, consistency in size decreased. Natural Concepts had a standard deviation of 13 at an average zone of 50 while Live Clean had a standard deviation of 0.33 with an average zone of 6.

The Deb Hygenipak produced modest zones against B. subtilis but had nearly no effect on E. coli.

Discussion

The results obtained did not support our hypothesis. This is both because of the effectiveness of off-the-shelf soap products and the ineffectiveness of the hospital soap against our culture of E. coli. On the outset, this result may be a little surprising and possibly concerning because of the risk of bacteria developing resistance to household antibacterial products and the importance of an aseptic environment in a hospital. However, the research done here is not conclusive, and further testing may be needed.

What our results do show is a significant variation in the inhibitory power of soaps. Triclosan, in our tests, was an extremely effective antibacterial agent. This should not be completely surprising since Assadian et al.[15] reported triclosan having an MIC between 0.5 μg/mL (for E. coli ATCC 25922) and 64 μg/mL (for E. coli AGT 11K). The Equate Hand Soap was also surprisingly effective against B. subtilis considering that the container did not market itself as an antiseptic. This may be due to the effectiveness of DMDM Hydantoin, a formaldehyde releaser[16].

Additionally, Live Clean gave the weakest results overall, despite having ingredients with known antibacterial function—a review by Mendel Friedman[17] noted that epigallocatechin gallate (an important component of tea extract) was more effective against gram-positive bacteria from Staphylococcus spp. (MIC 50–100 μg/mL) than against several gram-negative bacteria including E. coli (MIC 800 μg/mL). This seems to indicate that there is a significant difference between antibacterial substances with high activity, and those with low–moderate activity.

The varying standard deviations in our results are also worthy of mention. Natural Concepts had the largest standard deviations, a fact which is probably due to its large zone of inhibition, and the difficulty of measuring a 50 mm zone on one quadrant of a 150 mm Petri dish. Another test giving this soap its own Petri dish could probably produce better results. In the presence of B. subtilis Deb Hygenipak and T36 also had large standard deviations. This may be explained by the difficulty in interpreting unclear zones.

So, why wasn't the Deb Hygenipak soap effective against E. coli? To be fair the product's web page does not contain any particular antiseptic claims[18]. Still, the soap lists ingredients with known antibiotic activity. Shepherd et al.[19] observed the MIC of bronopol for E. coli to be 13 μg/mL, and Croshaw[20] states that bronopol is has greater activity against gram-negative bacteria than gram-positive.2) Possible causes can be put into two categories: either the expected ingredients were not effective, or the expected ingredients were not there.

The active ingredients of the Deb Hygenipak soap have a similar mechanism of action: oxidation of thiol groups and the formation of disulphide bonds [21][22]. This action is limited by anaerobic conditions, a low pH, or the presents of significant other thiol groups (such as from proteins in agar). None of these should have been an effect within our experiment. Additionally, a search of the literature was unable to find any examples of bronopol resistance.

If the ingredients are effective, then the other possibility was that they were not present (in sufficient quantity). Chemical degradation is a possibility, however, the Deb Hygenipak soap was stored in a specimen container, away from sunlight, for 2 weeks at room temperature. According to Matczuk et al.[23] bronopol should not significantly degrade in that time, particularly in the presence of citric acid (an ingredient of the soap).

A related explanation is that perhaps there wasn't enough bronopol in the soap to inhibit growth at the given concentrations. Since the ingredients list do not specify any amounts, it is not really possible to know without additional testing. In a related fashion, it is also possible that the Deb Hygenipak soap adhered strongly to the filter paper and poorly to the agar so that only a small portion of it actually effected the bacteria.

In the future, there are several experiments that could be performed that may help improve results, or shed light on the details of these soaps. One change would be to dry out the soap disks before placing them on the agar. This would reduce the possibility of a so-called “tsunami effect” where the rapid exit of liquid from the filter paper to the agar actually pushes bacteria away and results in a zone of inhibition. Another possibility would be to grow bacteria in suspension and then add different amounts of soap to test for the Minimum Inhibitory Concentration.

More involved tests could also be performed by modifying the soaps or the bacteria. For example, Storm et al.[24] notes that EDTA and polymyxin make the outer membrane of E. coli more permeable. Adding one of these may have some effect on on how the Deb Hygenipak inhibits E. coli.

The experiment performed here only tests one aspect of the several important factors in soap hygiene. One limitation of our study was that due to the laboratory environment and time only two non-pathogenic bacterial strains were tested. In particular, bronopol is important for its activity against Pseudomonas aeruginosa [20], a multi-drug resistant bacteria responsible for opportunistic infections in immunocompromised individuals[25]. A larger study, with bacterial samples that are more pertinent to what would be found in a hospital setting, is important for establishing a clearer picture of the situation.

Another limitation of our study was that we were not studying these soaps in precisely the way they were intended to be used. The most important action of hand soaps is to help reduce the ability of bacteria and other pathogens to adhere to the skin. This was not tested in our experiment.

Also relevant, is the ability of soap products to trigger allergies and cause dermatitis. Dermatitis is not just irritating—it also compromises the skin's ability to keep out foreign pathogens. Larson et al.[11] describe the problem in detail.

In conclusion, there is significant variation in the ability of soaps to inhibit bacterial growth. Products that claim to be antiseptic may not work as intended in small amounts or at low concentrations. Proper hand-washing technique is critical to hygene, both in the hospital and laboratory, and at home.

Acknowledgements

Deb Hygenipak, the hospital soap used in this experiment, was provided by Fraser Valley Regional Hospital. The remaining soaps and chloramphenicol were provided by the University of the Fraser Valley.

Appendix

Table 3: Abbreviations of soaps used
Abbreviation Full name
Deb Hygenipak Deb Hygenipack Pure Unscented Foaming Skin Cleanser
Live Clean Live Clean (Sweet Pea) Moisturizing Liquid Hand Soap
T36 T36 Antiseptic Hand Sanitizer
Equate Equate Original Hand Soap
Natural Concepts Natural Concepts Antibacterial Liquid Soap
Table 4: Possible active ingredients of soap products
Soap Brand Active Ingredients (in order of amount)
Deb Hygenipak Bronopol, Citric Acid, Methylchloroisothiazolinone, Methylisothiazolinone
Live Clean Plant Extracts (Camellia Sinensis, Lavandula Angustifolia, Chameinilla Recutita Flower, Rosmarinus Officinalis, Calendula Officinalis Flower, Viola Odorata Flower/Leaf, Lathyrus Odoratus Flower), Sodum Benzoate
T36 Ethanol, Benzalkonium Chloride
Equate DMDM Hydantoin
Natural Concepts Triclosan, DMDM Hydantoin

References


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10. a Emmerson, A.M. and Enstone, J.E. and Griffin, M. and Kelsey, M.C. and Smyth, E.T.M., 1996. The Second National Prevalence Survey of infection in hospitals—–overview of the results. Journal of Hospital Infection, 32(3), Elsevier, pp.175–190.
11. a, b Larson, Elaine L and 1994 APIC Guidelines Committee and others, 1995. APIC guidelines for handwashing and hand antisepsis in health care settings. American Journal of Infection Control, 23(4), Mosby, pp.251–269.
12. a Tibballs, James, 1996. Teaching hospital medical staff to handwash. The Medical Journal of Australia, 164(7), pp.395–398.
13. a Avril Alfred, 2016. The clean (or dirty?) truth: Evaluating the effectiveness of hand soaps or household cleaners against bacteria. University of the Fraser Valley.
14. a Sood, Smita, 2016. Chloramphenicol—A Potent Armament Against Multi-Drug Resistant (MDR) Gram Negative Bacilli?. Journal of Clinical and Diagnostic Research, 10(2), JCDR Research & Publications Private Limited, DC01.
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17. a Mendel Friedman, 2007. Overview of antibacterial, antitoxin, antiviral, and antifungal activities of tea flavonoids and teas. Molecular Nutrition & Food Research, 51(1), Wiley Online Library, pp.116–134.
19. a Shepherd, Julia A and Waigh, Roger D and Gilbert, Peter, 1988. Antibacterial action of 2-bromo-2-nitropropane-1, 3-diol (bronopol). Antimicrobial Agents and Chemotherapy, 32(11), American Society for Microbiology, pp.1693–1698.
20. a, b Croshaw, Betty and Groves, MJ and Lessel, B, 1964. Some properties of bronopol, a new antimicrobial agent active against Pseudomonas aeruginosa. Journal of Pharmacy and Pharmacology, 16(S1), Wiley Online Library, pp.127T–130T.
21. a Bowman, W.R. and Stretton, R.J., 1972. Antimicrobial activity of a series of halo-nitro compounds. Antimicrobial Agents and Chemotherapy, 2(6), American Society for Microbiology, pp.504.
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23. a Matczuk, M. and Obarski, N. and Mojski, M., 2012. The impact of the various chemical and physical factors on the degradation rate of bronopol. International Journal of Cosmetic Science, 34(5), Wiley Online Library, pp.451–457.
24. a Storm, D.R. and Rosenthal, K.S. and Swanson, P.E., 1977. Polymyxin and related peptide antibiotics. Annual Review of Biochemistry, 46(1), Annual Reviews, pp.723–763.
25. a Nedeljković, Natasa Stanković and Tiodorović, Branislav and Kocić, Branislava and Cirić, Vojislav and Milojković, Marko and Waisi, Hadi, 2015. Pseudomonas aeruginosa serotypes and resistance to antibiotics from wound swabs.. Vojnosanitetski Pregled, 72(11), pp.996–1003, ISSN 0042-8450.
1)
who may be referred to as HIS GLORY AND GREATNESS, LORD OF ALL THE ABBOTSFORD, FIRST OF HIS NAME, CONQUERER OF B SUBTILIS AND E COLI, HIS MAJESTY MATTHEW
2)
It should be noted that this study was paid for by The Boots Company which invented bronopol.
writings/school/bacterial_inhibition_soap.txt · Last modified: 2016/04/21 01:48 by trick