Topical antimicrobial therapy

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Chapter 8.13


Topical antimicrobial therapy


K. Bergvall1 (Chairperson), K. Varjonen2 (Secretary)


1 Djurakuten i Stockholm, Kungstensgatan 58, 11329 Stockholm, Sweden


2 Faculty of Veterinary Medicine and Animal Science, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden


Kerstin Bergvall (Sweden) welcomed everyone to the workshop on topical antimicrobial therapy. The workshop was intended to be an interactive session with four invited speakers followed by a discussion after each subject. The four speakers and their topics were introduced.


Kerstin Bergvall reminded the audience that although bacteria have been around for 3.5 billion years, penicillin was discovered as recently as 1928 by Alexander Fleming. It was only 30 years later that the first report of methicillin-resistant Staphylococcus aureus (MRSA) was published. With the emergence of antibiotic resistance, nonantibiotic treatment protocols for infectious diseases have become increasingly interesting and important topics for discussion. The first speaker of the session, Ralf Mueller, was invited to make his presentation.


Antibacterial shampoos – can they relieve/reduce bacterial burden? (R. Mueller)


Ralf Mueller (Germany) presented the results of a study, ‘Antibacterial activity of hair shafts after shampooing’,1 and some additional work on antimicrobial activity of biocides.


In one study the residual antibacterial activity on keratinocytes of a chlorhexidine and phytosphingosine shampoo was investigated and compared to a shampoo vehicle. Canine keratinocytes were harvested before and after shampooing. The cells were cultured and bacterial growth was compared between the two treatment groups before and after shampooing. No difference was seen between the placebo and active ingredients with regard to the residual antibacterial activity on keratinocytes after shampooing.


Cytological samples from the dogs in the study were taken before shampoo treatment was started and repeated 4 weeks later. Ralf Mueller was unsure whether the dogs were shampooed once or twice weekly. The bacterial count on cytology was reduced significantly in both the placebo (shampoo vehicle) and the active ingredient shampoo treatment groups, indicating that mechanical removal of bacteria by shampooing plays a significant role in the effectiveness of shampoos in treatment of bacterial pyoderma. Ralf Mueller commented that based on this study, any shampoo used properly should reduce bacterial numbers.


Ralf Mueller pointed out that there are many possible problems to consider when evaluating shampoo studies. There are very few randomized controlled studies that look at effects in vivo. It is difficult to extrapolate from in vitro studies as concentrations of the ingredients on the skin may differ and incubation times may also differ in the clinical settings. Many clinical studies do not have a control group, or they have a control group that is not shampooed and, therefore, underestimates the effects from mechanical actions.


Another study1 aimed to investigate whether there is a residual antibacterial activity on the hair shaft after shampooing. Previous studies have shown that there are bacterial colonies on the hair shafts as well as the skin surface. Forty-two healthy dogs from the Institute of Physiology at Ludwig-Maximilians University, Munich, Germany, were used in the study. The dogs were beagles, foxhounds and beagle-mixed breed dogs. They were shampooed twice weekly for 2 weeks. A 10-minute shampoo, 10-minute rinsing protocol was used. The following shampoos were evaluated: chlorhexidine 0.8% (Actibac®, Ceva), chlorhexidine 2%/miconazole 2% (Malaseb®, Dermcare), chlorhexidine 3% (Pyohex®, Dermcare), chlorhexidine 4% (Hexocare®, Alfavet), ethyl lactate 10% (Etiderm®, Virbac), benzoyl peroxide 2.5% (Peroxyderm®, Vetoquinol) and a control vehicle (Dermazyme®, Ceva).


The dogs were shampooed twice weekly, four times in total. Two shampoos per dog were used: the right half of the dog was shampooed with one shampoo, the left half of the dog with another shampoo, and a stripe in the middle was left untreated to avoid cross-contamination. One group was treated with ethyl lactate on one side and chlorhexidine/miconazole on the other; one group with 3% chlorhexidine on one side, 2.5% benzoyl peroxide on the other; one group with 3% chlorhexidine and a 3% chlorhexidine conditioner afterwards on one side and 0.8% chlorhexidine on the other side; and one group with 4% chlorhexidine on one side and control on the other side. Hair samples were taken with a clipper from the left and the right sides of the thorax before the first shampoo therapy, after the last shampoo therapy once the dog was dry, and then 2, 4 and 7 days later.


The hair was dried for 10 minutes and then 0.02 g of hair was placed onto agars coated in 100 μL of 0.3 McFarland Standard dilution of Staphylococcus pseudintermedius (21284) and incubated at 37°C for 24 hours. The inhibition zones were measured by measuring the longitudinal and perpendicular inhibition zones around the hair sample. All samples were done in duplicates. The groups were compared using Kruskal-Wallis and Dunn post-test, P < 0.05.


The results of the study were as follows. No inhibition zones were seen before the study. A small inhibition zone was seen around the control group. Ralf Mueller believes this was likely caused by a small bacterial inhibition from the surfactant in the shampoo base. Large inhibition zones were seen around three groups: the Pyohex®, Malaseb® and the 3% chlorhexidine plus conditioner. Smaller inhibition zones were seen in the 0.8% chlorhexidine group, even less in the 4% chlorhexidine, and no inhibition was seen in the ethyl lactate and benzoyl peroxide groups. At day 17 the results were very similar but, at that time there was no residual activity of the base. Large inhibition zones were seen in the Pyohex®, Malaseb® and the 3% chlorhexidine plus conditioner groups, very little inhibition was seen in the remaining two chlorhexidine groups. No inhibition was seen with benzoyl peroxide. Two dogs treated with ethyl lactate had inhibition zones at day 17 but not directly after shampooing. The researchers concluded that there are differences in the residual antibacterial activity depending on the shampoo. The differences are not solely dependent on the concentration of the ingredient because the 2% and 3% chlorhexidine had much higher inhibition zones than the 0.8% and 4% chlorhexidine. This result was found to be compatible with the results of previous in vitro studies, including one by Lloyd and Lampert.2 The different inhibition zones were thought possibly to be due to differences in the diffusion activities of the shampoos. One possible explanation is that some of the ingredients, such as benzoyl peroxide, may not diffuse into the agar and, therefore, no inhibition zone occurs.


Based on the results it was concluded that some shampoos have residual antibacterial activity and others do not, and that the formulation of the shampoo is more important than its concentration of active ingredients. Ralf Mueller pointed out that if you believe the hair shafts play a role in the carriage of bacteria, shampoos that are manufactured to provide residual activity on hairs should be used.


Credit was given to Isabell Kloos, a doctoral student who had performed the study, and all the people involved, the Institute for Nutrition, Institute for Infectious Medicine and Zoonoses, and the dermatology team at the university in Munich.


Paul Bloom (USA) asked, what is Pyohex?


Ralf Mueller answered that it is a chlorhexidine 3% shampoo.


Ralf Mueller then asked the audience whether they believe that shampoos can have a residual antibacterial activity. Six people raised their hands. He then asked how many believed hair shafts played a role, and a few people in the audience felt that this was likely.


Ralf Mueller finished his presentation by concluding that much more work was needed in this area. These kinds of studies are a start to determine whether there is a difference between shampoos. Unfortunately, he had found it very difficult to get funding from companies for these kinds of studies, and this study is one of the first shampoo studies funded by a shampoo company, Dermcare. He believes that it should be important for companies that market their products to veterinarians to have their products evaluated, and provide evidence that their products perform according to the claims they make. He hopes for more studies in the future.


Kerstin Bergvall thanked Ralf Mueller for his talk and invited Nobuo Murayama to make his presentation.


Antibacterial shampoos/washing and staphylococci (Nobuo Murayama)


Nobuo Murayama (Japan) introduced the subject of his talk and presented the results of several studies. He reported that in their hospital they have experienced methicillin-resistant Staphylococcus pseudintermedius (MRSP) in two-thirds of cases (113/170).3 These staphylococci show multidrug resistance including high levels of resistance towards ofloxacin (84.1%), lincomycin (99.2%), clindamycin (92.0%) and minocycline (65.5%). Therefore, alternative treatments, including topical antimicrobial ingredients such as benzoyl peroxide, ethyl lactate and chlorhexidine, need to be considered.


Previous studies have reported clinical improvement of 60–70% with use of benzoyl peroxide in cases of canine superficial pyoderma by two clinical researchers.4,5 There are some disadvantages with benzoyl peroxide including its antiseborrhoeic effect and propensity to cause irritation. It may thus be difficult to perform frequent shampooing with benzoyl peroxide. In one study with ethyl lactate a 77% clinical improvement of canine superficial pyoderma was reported,5 but this has not been shown in other studies.


Nobuo Murayama and his team investigated the in vitro efficacy of different concentrations of benzoyl peroxide, ethyl lactate and chlorhexidine against S. pseudintermedius. In their study a 100 times dilution of benzoyl peroxide showed growth of S. pseudintermedius within 15 minutes of contact time. A 500 times dilution of 10% ethyl lactate showed growth of S. pseudintermedius within 15 minutes. On the other hand, a 1000 times dilution of 2% chlorhexidine showed no growth of bacteria within 15 minutes. They concluded that chlorhexidine is more effective against S. pseudintermedius using a low concentration and a short exposure time compared to benzoyl peroxide and ethyl lactate.


Nobuo Murayama and his team next wanted to compare the clinical efficacy of chlorhexidine to other products. A recently published study by Loeffler et al. reported on the clinical efficacy of 3% chlorhexidine gluconate compared to 2.5% benzoyl peroxide.6 In this study, 70% of cases of pyoderma treated with chlorhexidine showed good clinical improvement whilst 20% of benzoyl peroxide-treated patients showed good clinical improvement.


In their study, Nobuo Murayama and his team compared 2% chlorhexidine acetate and 10% ethyl lactate and looked at resolution of scales, papules/crusts, erythema and pruritus. Treatment was done twice weekly for 1 week. Chlorhexidine significantly improved skin lesions compared to ethyl lactate.7


In another study, 2% chlorhexidine acetate and 4% chlorhexidine gluconate were compared. Both showed the same efficacy for canine superficial pyoderma.8 A 2% chlorhexidine acetate shampoo treatment of canine superficial pyoderma associated with cephalexin-resistant S. intermedius group was evaluated. The dogs were shampooed every 2 days for 2 weeks. Seventy-five percent of cases in the study had their lesions resolved or showed clinical improvement.8


Nobuo Murayama shared some clinical tips that he recommends to improve success with shampoo therapy. His group compared three different doses of chlorhexidine acetate for treatment efficacy. Group 1 received 57 mL/m2, group 2 received half the dosage of group 1, and group 3 received one-third of the dosage of group 1. The results showed that groups 1 and 2 showed the same degree of improvement for canine superficial pyoderma. Based on this study, Nobuo Murayama reported that they use a coin measurement when explaining to owners how much shampoo to use. An area of shampoo the size of a Japanese 500 yen coin per two hand-sized areas on the dog’s body equals about 29 mL/m2 as used for group 2 in the study. Coins in other currencies like a quarter US dollar or a 2 euro coin are almost the same size.


The shampoo contact time was then examined. A 1-minute contact time and 10-minute contact time of chlorhexidine were compared and almost the same improvement could be seen for canine superficial pyoderma.


Nobuo Murayama concluded that bathing with high concentrations of chlorhexidine is an effective therapy for canine superficial pyoderma. However, clinical response is not seen in all cases. One possible explanation for the inefficacy of chlorhexidine is the presence of multidrug efflux pumps.These pumps are associated with antimicrobial resistance encoded by antiseptic resistance genes including qacA, qacB and smr. In humans, S. aureus has been shown to have antiseptic resistance genes and this has also been found within equine, bovine and feline staphylococcal strains. Antiseptic resistance genes were not detected in 100 strains of S. pseudintermedius from cases of canine superficial pyoderma.


Nobuo Murayama discussed other factors that could cause chlorhexidine to be ineffective. Inefficacy can be caused by organic matter and poor penetration into biofilms. S. aureus has recently been shown to produce biofilms. Chlorhexidine treatment of impetigo and furunculosis in humans who have biofilm formation does not show good efficacy. Recently S. pseudintermedius has also been shown to produce biofilms. Poor efficacy could also be explained by lack of owner compliance. Nobuo Murayama thanked the audience for their attention.


Kerstin Bergvall thanked Nobuo Murayama for his presentation. She asked whether he would recommend the use of a detergent prior to chlorhexidine to help remove organic matter.


Nobuo Murayama felt that this needs to be investigated. He recommends that owners thoroughly wet the dog with warm water before using chlorhexidine shampoo, but he does not usually recommend the use of degreasers.


Ralf Mueller

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Jun 13, 2017 | Posted by in INTERNAL MEDICINE | Comments Off on Topical antimicrobial therapy

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