ABSTRACT
Background
The antibacterial efficacy of chlorhexidine shampoo is directly affected by formulation and bathing factors.
Hypothesis/Objective
To evaluate the in vitro antibacterial efficacy of chlorhexidine‐containing shampoos at various dilutions and to compare their lathering ability.
Animals
No animals were utilised in this study.
Materials and Methods
Eight chlorhexidine‐containing shampoos, three non‐chlorhexidine shampoos, and a 2% chlorhexidine gluconate solution were tested against one American Type Culture Collection (ATCC) meticillin‐sensitive Staphylococcus pseudintermedius and one institutional meticillin‐resistant S. pseudintermedius isolate. The effect of formulation on minimum inhibitory concentration (MIC) was determined using a broth microdilution method. The first dilution that had no visible growth and four preceding dilutions were plated on blood agar to determine the minimum bactericidal concentration (MBC). Lathering ability and lather stability were assessed using a modified cylinder shake method. MIC and MBC were compared for the dilution ratio and chlorhexidine gluconate/digluconate concentration using a Kruskal–Wallis test with Bonferroni correction (p < 0.001).
Results
All products had a detectable MIC. Statistically significant differences between MIC and MBC were observed between shampoos that were not based on chlorhexidine concentration alone. Two non‐chlorhexidine shampoos had no detectable MBC. Over time, all shampoos had a significant decrease in lather height. Lathering ability significantly differed between some shampoos.
Conclusions and Clinical Relevance
This preliminary study suggests that shampoo formulation and not just chlorhexidine concentration impacts efficacy. Further investigation with more robust numbers of bacterial isolates and large‐scale head‐to‐head clinical trials is required to determine if the reported in vitro variance has clinical significance.
Keywords: bathing, chlorhexidine, lather, pyoderma, shampoo, Staphylococcus pseudintermedius
Background: The antibacterial efficacy of chlorhexidine shampoo is directly affected by formulation and bathing factors. Hypothesis/Objective: To evaluate the in vitro antibacterial efficacy of chlorhexidine‐containing shampoos at various dilutions and to compare their lathering ability. Conclusions and Clinical Relevance: This preliminary study suggests that shampoo formulation and not just chlorhexidine concentration impacts efficacy. Further investigation with more robust numbers of bacterial isolates and large‐scale head‐to‐head clinical trials is required to determine if the reported in vitro variance has clinical significance.
ZUSAMMENFASSUNG
Hintergrund
Die antibakterielle Wirksamkeit von Chlorhexidin Shampoo wird direkt von der Formulierung und den Badefaktoren beeinflusst.
Hypothese/Ziele
Das Ziel dieser Studie war eine Evaluierung der in vitro antibakteriellen Wirksamkeit eines Chlorhexidin‐enthaltenden Shampoos bei unterschiedlichen Verdünnungen, und ein Vergleich der schäumenden Fähigkeiten.
Tiere
Es wurden in dieser Studie keine Tiere eingesetzt.
Materialien und Methoden
Acht Chlorhexidin‐enthaltende Shampoos, drei nicht‐Chlorhexidin enthaltende Shampoos und eine 2%ige Chlorhexidin Glukonat Lösung wurden gegenüber einem Methicillin‐sensitiven Staphylococcus pseudintermedius der American Type Culture Collection (ATCC) und einem institutionellen Methicillin‐resistenten S. pseudintermedius Isolat getestet. Die Auswirkung der Formulierung auf die minimale Hemmkonzentration (MIC) wurde mittels Bouillon Mikrodilutionsverfahrens bestimmt. Die erste Verdünnung, die kein sichtbares Wachstum zeigte, sowie vier vorhergehende Verdünnungen wurden auf Blutagar ausgestrichen, um die minimale bakterizide Konzentration (MBC) zu bestimmen. Die Fähigkeit zu Schäumen und die Stabilität des Schaumes wurden mittels modifizierter Zylinder Schüttelmethode erfasst. MIC und MBC wurden in Bezug auf Verdünnungsgrad und Chlorhexidin Gluconate/Diglukonat Konzentration mittels Kruskall‐Wallis Test mit Boniferroni Korrektur (p < 0,001) verglichen.
Ergebnisse
Alle Produkte zeigten eine messbare MIC. Statistisch signifikante Unterschiede zwischen MIC und MBC wurden bei den Shampoos beobachtet, die nicht nur auf Chlorhexidin basierten. Zwei Nicht‐Chlorhexidin Shampoos zeigten keine messbare MBC. Mit Zunahme der Zeit zeigten alle Shampoos eine Abnahme der Schaumgröße. Die Fähigkeit zu Schäumen war zwischen einigen Shampoos sehr unterschiedlich.
Schlussfolgerungen und klinische Bedeutung
Diese vorläufige Studie zeigt, dass Shampoo Formulierungen und nicht nur Chlorhexidin Konzentrationen die Wirksamkeit beeinflusst. Es sind weitere Untersuchungen mit mehreren stabilen Zahlen von bakteriellen Isolaten und Head‐to‐Head klinische Studien in einem großen Rahmen nötig, um festzustellen, ob die berichtete in vitro Varianz eine klinische Signifikanz aufweist.
摘要
背景
含氯己定香波的抗菌效果直接受到配方和洗浴因素的影响。
假设/目的
评估不同稀释度下含氯己定香波的体外抗菌效果，并比较其起泡能力。
动物
本研究未使用动物。
材料与方法
测试了 8 种含氯己定香波、3 种非氯己定香波和一种 2% 葡萄糖酸氯己定溶液，对象为一个美国类型培养物保藏中心(ATCC)甲氧西林敏感假中间型葡萄球菌株和一个机构分离的耐甲氧西林假中间型葡萄球菌株。通过肉汤微量稀释法测定配方对最低抑菌浓度(MIC)的影响。将无可见生长的首个稀释度及其前四个稀释度接种于血琼脂上，以确定最低杀菌浓度(MBC)。利用改良圆筒摇动法评估起泡能力和泡沫稳定性。使用 Kruskal–Wallis 检验结合 Bonferroni 校正(p < 0.001)比较稀释比例和氯己定葡萄糖酸盐/二葡萄糖酸盐浓度下的 MIC 和 MBC。
结果
所有产品均有可检测的 MIC。不同香波之间 MIC 和 MBC 的差异具有统计学意义，而不仅仅取决于氯己定浓度。两种非氯己定香波未检测到 MBC。随着时间推移，所有香波的泡沫高度显著下降。部分香波之间的起泡能力差异显著。
结论与临床相关性
该初步研究表明，香波的配方而不仅仅是氯己定的浓度会影响其疗效。需要通过更大量的细菌分离株和大规模的临床对照试验来进一步研究，以确定所报道的体外差异是否具有临床意义。
Résumé
Contexte
L’efficacité antibactérienne du shampooing à la chlorhexidine est directement influencée par sa formulation et les facteurs liés au bain.
Hypothèse/Objectif
Évaluer l’efficacité antibactérienne in vitro de shampooings contenant de la chlorhexidine à différentes dilutions et comparer leur pouvoir moussant.
Animaux
Aucun animal n’a été utilisé dans cette étude.
Matériaux et méthodes
Huit shampooings contenant de la chlorhexidine, trois shampooings sans chlorhexidine et une solution de gluconate de chlorhexidine à 2 % ont été testés contre un isolat de Staphylococcus pseudintermedius sensible à la méticilline provenant de l’American Type Culture Collection (ATCC) et un isolat institutionnel de S. pseudintermediusrésistant à la méticilline. L’effet de la formulation sur la concentration minimale inhibitrice (CMI) a été déterminé à l’aide d’une méthode de microdilution en bouillon. La première dilution ne présentant aucune croissance visible et les quatre dilutions précédentes ont été étalées sur une gélose au sang afin de déterminer la concentration bactéricide minimale (CBM). La capacité de moussage et la stabilité de la mousse ont été évaluées à l’aide d’une méthode de secouage cylindrique modifiée. La CMI et la CBM ont été comparées pour le rapport de dilution et la concentration en gluconate/digluconate de chlorhexidine à l’aide d’un test de Kruskal‐Wallis avec correction de Bonferroni (p < 0,001).
Résultats
Tous les produits avaient une CMI détectable. Des différences statistiquement significatives entre la CMI et la CBM ont été observées entre les shampooings qui n’étaient pas basés uniquement sur la concentration en chlorhexidine. Deux shampooings sans chlorhexidine n’avaient pas de CBM détectable. Au fil du temps, tous les shampooings ont présenté une diminution significative de la hauteur de la mousse. La capacité moussante différait considérablement entre certains shampooings.
Conclusions et pertinence clinique
Cette étude préliminaire suggère que la formulation du shampooing, et pas seulement la concentration en chlorhexidine, a un impact sur l’efficacité. Des recherches supplémentaires avec un nombre plus important d’isolats bactériens et des essais cliniques comparatifs à grande échelle sont nécessaires pour déterminer si la variance in vitro rapportée a une signification clinique.
要約
背景
クロルヘキシジンシャンプーの抗菌効果は、直接製剤や入浴要因に影響を受ける。
仮説/目的
本研究の目的は、さまざまな希釈度によるクロルヘキシジン含有シャンプーのin vitroでの抗菌効果を評価し、泡立ち能力を比較することであった。
被験動物
本研究に供した動物はいなかった。
材料および方法
8つのクロルヘキシジン含有シャンプー、3つのクロルヘキシジ非含有シャンプー、2%グルコン酸クロルヘキシジン溶液をアメリカ培養細胞系統保存機関(ATCC)のメチシリン感受性Staphylococcus pseudintermedius分離株1種および施設内メチシリン耐性S. pseudintermedius分離株1種に対して試験した。最小発育阻止濃度(MIC)に対する製剤の効果は微量液体希釈法を用いて測定した。目視で増殖が認められなかった最初の希釈液およびその前に作成した4つの希釈液を用いて最小殺菌濃度(MBC)を測定した。泡立ち能および安定性は改良シリンダーシェイク法を用いて評価した。MICおよびMBCについて、希釈率およびグルコン酸クロルヘキシジン/ジグルコン酸クロルヘキシジン濃度を比較した。比較にはボンフェローニ補正を施したクルスカル・ウォリス検定を用いた(p < 0.001)。
結果
全ての製剤において検出可能なMICが認められた。クロルヘキシジン濃度だけに基づかないシャンプー間で、MICおよびMBC間に統計的な有意差が認められた。クロルヘキシジン非含有シャンプー2製剤ではMBCは検出できなかった。全てのシャンプーにおいて経時的な泡立ちの高さの減少を認めた。泡立ち能はいくつかのシャンプー間で有意に異なった。
考察および臨床的意義
本予備試験はクロルヘキシジン濃度だけでなく、シャンプー製剤が有効性に影響することを示唆している。報告されたin vitroでの変動が臨床的意義を持つかどうかを判断するには、より堅牢な細菌分離株数を用いた追加調査および大規模な直接比較臨床試験が必要である。
Resumo
Contexto
A eficácia antibacteriana do shampoo de clorexidina é diretamente afetada pela formulação e pelos fatores relacionados ao banho.
Hipótese/Objetivo
Avaliar a eficácia antibacteriana in vitro de shampoos contendo clorexidina em diferentes diluições e comparar sua capacidade de formação de espuma.
Animais
Nenhum animal foi utilizado neste estudo.
Materiais e Métodos
Oito shampoos contendo clorexidina, três shampoos sem clorexidina e uma solução de gluconato de clorexidina a 2% foram testados contra um isolado de Staphylococcus pseudintermedius sensível à meticilina da American Type Culture Collection (ATCC) e um isolado institucional de S. pseudintermedius resistente à meticilina. O efeito da formulação na concentração inibitória mínima (CIM) foi determinado pelo método de microdiluição em caldo. A primeira diluição, que não apresentou crescimento visível, e as quatro diluições anteriores foram semeadas em ágar sangue para determinar a concentração bactericida mínima (CBM). A capacidade de formação de espuma e a estabilidade da espuma foram avaliadas usando um método de agitação de cilindro modificado. A CIM e a CBM foram comparadas quanto à razão de diluição e à concentração de gluconato/digluconato de clorexidina usando um teste de Kruskal‐Wallis com correção de Bonferroni (p < 0,001).
Resultados
Todos os produtos apresentaram CIM detectável. Diferenças estatisticamente significativas entre CIM e CBM foram observadas entre os shampoos que não foram baseados apenas na concentração de clorexidina. Dois shampoos sem clorexidina não apresentaram CBM detectável. Ao longo do tempo, todos os shampoos apresentaram uma diminuição significativa na formação de espuma. A capacidade de formação de espuma diferiu significativamente entre alguns shampoos.
Conclusões e Relevância Clínica
Este estudo preliminar sugere que a formulação do shampoo, e não apenas a concentração de clorexidina, impacta a eficácia. Investigações adicionais com números mais robustos de isolados bacterianos e ensaios clínicos comparativos em larga escala são necessárias para determinar se a variância in vitro relatada tem significância clínica.
RESUMEN
Introduccion
La eficacia antibacteriana del champú con clorhexidina se ve directamente afectada por la formulación y los factores relacionados con el baño.
Hipótesis/Objetivo
Evaluar la eficacia antibacteriana in vitro de champús con clorhexidina en diversas diluciones y comparar su capacidad de formación de espuma.
Animales
No se utilizaron animales en este estudio.
Materiales y métodos
Se analizaron ocho champús con clorhexidina, tres champús sin clorhexidina y una solución de gluconato de clorhexidina al 2% frente a un aislado de Staphylococcus pseudintermedius sensible a la meticilina de la Colección Americana de Cultivos Tipo (ATCC) y un aislado institucional de S. pseudintermedius resistente a la meticilina. El efecto de la formulación sobre la concentración mínima inhibitoria (MIC) se determinó mediante microdilución en caldo. La primera dilución sin crecimiento visible y las cuatro diluciones anteriores se sembraron en agar sangre para determinar la concentración mínima bactericida (MBC). La capacidad y estabilidad de la espuma se evaluaron mediante un método modificado de agitación en cilindro. Se compararon la MIC y la MBC para la proporción de dilución y la concentración de gluconato/digluconato de clorhexidina mediante la prueba de Kruskal‐Wallis con corrección de Bonferroni (p < 0,001).
Resultados
Todos los productos presentaron una MIC detectable. Se observaron diferencias estadísticamente significativas entre la MIC y la MBC entre champús que no se basaron únicamente en la concentración de clorhexidina. Dos champús sin clorhexidina no presentaron MBC detectable. Con el tiempo, todos los champús presentaron una disminución significativa de la altura de la espuma. La capacidad de formación de espuma difirió significativamente entre algunos champús.
Conclusiones y relevancia clínica
Este estudio preliminar sugiere que la formulación del champú, y no solo la concentración de clorhexidina, influye en la eficacia. Se requieren más investigaciones con un número más robusto de aislados bacterianos y ensayos clínicos comparativos a gran escala para determinar si la diferencia in vitro reportada tiene relevancia clínica.
1. Introduction
Canine pyoderma treatment is the most common reason for antimicrobial use in small animal practice [1], and Staphylococcus pseudintermedius is the most common bacterial pathogen of canine pyoderma [2]. Antimicrobial shampoos are a recommended first‐line therapy for canine superficial pyoderma [3, 4]. The efficacy of chlorhexidine as a topical antiseptic to treat superficial S. pseudintermedius skin infections has been well‐established [5, 6]. Chlorhexidine shampoos contain varying chlorhexidine salts (gluconate/digluconate, acetate and hydrochloride salts) and concentrations ranging from 2% to 4% [7, 8]. Anecdotally, opinions within the dermatology community on the efficacy of certain chlorhexidine concentrations have been based on prior work that evaluated the efficacy of four commercially available chlorhexidine‐containing formulations, not simply chlorhexidine concentration in a standardised vehicle [9]. The current peer‐reviewed consensus for an appropriate chlorhexidine concentration in a shampoo to use in the management of canine pyoderma is ≥ 2% [4].
Although a shampoo may contain antimicrobials known to be effective, the efficacy of those active ingredients is influenced by the amount of water used to dilute the product, rinsing time and volume of water used, patient hair coat length, presence of organic material, contact time and shampoo formulation [9, 10, 11, 12]. The antimicrobial efficacy of the active ingredients in a shampoo is directly influenced by other ingredients in the shampoo's formulation [9, 10, 12]. Inactive ingredient properties such as electrical charge can influence the efficacy of active ingredients such as chlorhexidine, which is a divalent, cationic biguanide agent. Some concurrent ingredients, such as imidazoles or Tris‐EDTA, may work in an antagonistic or synergistic manner with chlorhexidine, or have an individual bactericidal effect [13, 14, 15]. Shampoo formulations can contain surfactants (lathering agents, cleansing agents and conditioners), thickeners, softeners, preservatives, fragrances and colouring additives. Surfactants reduce surface tension and increase a shampoo's ability to form lather. Some surfactants may bind chlorhexidine and decrease its efficacy through the formation of micellar binding because surfactants are amphophilic molecules with both a hydrophilic and lipophilic part [9, 11, 12]. It has never been investigated whether shampoos with increased lathering ability reduce the efficacy of chlorhexidine gluconate/digluconate. Lathering ability is one of the most important product qualities that people care about when selecting shampoo for their own hair care [16]. Therefore, the lathering ability of a chlorhexidine‐containing shampoo may positively or negatively influence a pet owner's experience and perception of pet bathing, which could increase or decrease compliance.
Previous publications evaluating the antimicrobial activity of chlorhexidine‐containing shampoo investigated formulations that are not used in the United States or are no longer available [9, 10, 12, 17]. The first aim of the present study was to determine the in vitro efficacy of differently formulated commercially available chlorhexidine gluconate/digluconate‐containing shampoos against meticillin‐sensitive and meticillin‐resistant S. pseudintermedius (MSSP and MRSP) isolates. The second aim of this study was to compare the lathering ability and lather stability of these shampoos. Given the chemical nature of chlorhexidine, the varying content of active and inactive ingredients of commercially produced shampoos and prior in vitro studies, we hypothesised that shampoo formulations would differ in their antimicrobial efficacy, not simply based on chlorhexidine concentration alone. Additionally, given anecdotal evidence and the authors' personal experiences, we hypothesised that there would be significant differences in lathering ability and that this would not influence antimicrobial efficacy.
2. Materials and Methods
2.1. Determination of Minimum Inhibitory and Bactericidal Concentration
A total of eight chlorhexidine gluconate/digluconate‐containing shampoos (Malaseb, Miconahex+Triz, TrizCHLOR 4, BioHex, Douxo S3 PYO, KETOCHLOR, ChlorhexiDerm 4%, Davis Chlorhexidine), one chloroxylenol shampoo (Universal Medicated), two non‐chlorhexidine shampoos (DermaLyte, Allergroom) and a standardised 2% chlorhexidine gluconate solution were evaluated. The products tested in this study were chosen because they contain a variety of percentage chlorhexidine gluconate/digluconate products with variable added ingredients and represent the array of products available in the United States. A chloroxylenol shampoo was included for evaluation, as some veterinary surgeons prescribe this shampoo as an antibacterial alternative to chlorhexidine‐containing shampoo. Other non‐chlorhexidine shampoos were selected because they are commonly used, nonmedicated general‐purpose cleansers that are a negative control for antibacterial efficacy and a positive control for lathering ability. Antimicrobial products tested are listed in Table 1 with their active ingredients. The minimum inhibitory concentration (MIC) of the shampoo formulations was determined using a modified broth microdilution method published previously [17, 18, 19]. One laboratory isolate of MSSP (isolate no. ATCC 49051) and one institutional MRSP were cultured for 24 h at 37°C on sheep blood agar (SBA) plates. The MRSP isolate was obtained from the institution's microbiology laboratory and confirmed as meticillin‐resistant both by demonstrating oxacillin resistance on sensitivity testing following CLSI guidelines and genotypically by using a commercially available latex agglutination test (Oxoid PBP2′ Latex Agglutination Test Kit; Thermo Fisher Scientific) to identify the PBP2a protein encoded by the mecA gene, the presence of which characterises meticillin resistance. The isolates were incubated on SBA plates for 24 h. Bacterial colonies were then suspended in sterile phosphate‐buffered saline and adjusted to a turbidity of 0.5 McFarland standard using a nephelometer (SENSITITRE Nephelometer; Trek Diagnostics). Preliminary experiments determined the resulting 0.5 McFarland bacterial suspension resulted in a final concentration of 1.7 × 108 colony‐forming units (cfu)/mL for the S. pseudintermedius isolates.
TABLE 1.
Antimicrobial products evaluated with their main active ingredients.
| Products | Main active ingredient(s) and original concentration(s) |
|---|---|
| 2% Chlorhexidine solution (VetOne) | Chlorhexidine gluconate 20,000 mg/L |
| Universal medicated shampoo (Vetoquinol) | Chloroxylenol 20,000 mg/L, salicylic acid 20,000 mg/L, sodium thiosulfate 20,000 mg/L |
| Davis chlorhexidine shampoo (Davis Manufacturing) | Chlorhexidine gluconate 20,000 mg/L |
| KETOCHLOR (Virbac) | Chlorhexidine gluconate 23,000 mg/L, ketoconazole 10,000 mg/L |
| BioHex (VetBiotek) | Chlorhexidine gluconate 20,000 mg/L, miconazole nitrate 20,000 mg/L, MicroSilver BG 1000 mg/L |
| Malaseb (Dechra Veterinary Products) | Chlorhexidine gluconate 20,000 mg/L, miconazole nitrate 20,000 mg/L |
| Miconahex + Triz (Dechra Veterinary Products) | Chlorhexidine gluconate 20,000 mg/L, miconazole nitrate 20,000 mg/L, TrizEDTA (Tromethamine, USP; Disodium EDTA) concentration not listed |
| Douxo S3 PYO (Ceva Animal Health) | Chlorhexidine digluconate 30,000 mg/L, ophytrium 5000 mg/L |
| ChlorhexiDerm 4% (Elanco) | Chlorhexidine gluconate 40,000 mg/L |
| TrizCHLOR 4 (Dechra Veterinary Products) | Chlorhexidine gluconate 40,000 mg/L, TrizEDTA (Tromethamine, USP; Disodium EDTA) concentration not listed |
Shampoo dilutions of 1:2 were prepared by mixing 1 mL of shampoo with 2 mL of Mueller–Hinton Broth (MHB) in a 16 × 100 mm sterile glass tube. Thorough mixing was performed with a vortex and repeated inversion. Shampoo serial dilutions were performed in glass tubes to create a 1:16 shampoo dilution. Sterile 96‐well cell culture plates (Nunc MicroWell 96‐Well, Nunclon Delta‐Treated Flat‐Bottom Microplate; Thermo Fisher Scientific) were used to prepare serial two‐fold dilutions of each shampoo and chlorhexidine solution in triplicate as described previously [17, 18]. Each well was filled with 50 μL of MHB. The first serial dilution was performed by transferring 50 μL of the 1:16 shampoo dilution to a well of 50 μL of MHB. Mixing was achieved with pipetting. Then, 50 μL was removed to mix with the following well and repeated. To each well, 50 μL of microbial suspension was added to yield shampoo dilutions from 1:64 to 1:8,388,608. All microbial suspensions were used within 30 min of preparation. A positive control (microbial suspension in MHB) and two negative controls (MHB only and shampoos diluted in MHB) were included. Plates were incubated at 37°C for 24 h, then inspected for visible growth. If visible growth was present up to and including the 1:64 dilution well, then lower dilutions were evaluated in triplicate on an individual basis as described above for dilutions of 1:4 to 1:32. MICs were recorded as the first well with no visible growth. The well with visible growth and four preceding dilutions were plated on SBA for 18 h by pipetting 50 μL aliquots from the wells onto a plate and using a sterile loop to streak evening across the entire plate. Minimum bactericidal concentrations (MBCs) were recorded as the lowest dilution that completely prevented growth after incubation on sheep blood agar. The dilution ratio was defined as the proportion of solute (shampoo) to solvent (MHB). For the tested shampoos, a higher dilution ratio for MIC or MBC indicated greater in vitro antimicrobial efficacy.
2.2. Lathering Ability and Lather Stability
Lathering ability and lather stability were performed using a modified cylinder shake method [20]. At room temperature, a 10% shampoo solution was made by adding 0.5 mL of shampoo to 4.5 mL of sterile distilled water in a 16 × 100 mm sterile glass tube, which was inverted 10 times. The height of the lather generated was measured from the water line at 0, 1, 5, 10, 15, 30 and 60 min. This was performed in triplicate for each shampoo by three different individuals. Lather stability was calculated by dividing the decrease in median lather height (mm) from the 0 min time point by the time interval (min) from the 0 min time point.
2.3. Statistical Analysis
All data were evaluated for normality with the Shapiro–Wilk test. All of the MIC and MBC data (dilution ratio and chlorhexidine concentration) were non‐normal in distribution, and comparisons were made between the shampoos with the Kruskal–Wallis test, including pairwise comparisons for post hoc analysis using the Bonferroni correction. Three non‐chlorhexidine shampoos (Allergroom, DermaLyte and Universal Medicated Shampoo) were excluded from MIC/MBC chlorhexidine gluconate/digluconate concentration comparison because they do not contain chlorhexidine. Lathering ability and lather stability data for all shampoos were combined to evaluate changes over time with a Friedman test. Individual shampoos were evaluated for decreasing lathering ability with a Friedman test. Differences in lathering ability based on the shampoo were compared with the Kruskal–Wallis test, including pairwise comparisons for post hoc analysis. A p‐value of < 0.05 was considered significant. Pearson correlation was performed between lathering ability and MIC/MBC chlorhexidine gluconate/digluconate concentration and dilution ratio. statistics IBM SPSS STATISTICS v27 (IBM Corp) was used for all statistical evaluations.
3. Results
3.1. Control Data
In all experiments, the microbial suspensions incubated in MHB resulted in confluent growth. There was no microbial growth in the wells containing MHB or shampoos diluted in MHB only.
3.2. Minimum Inhibitory Concentration
All products had a detectable MIC (Table 2) and there were no statistical differences between the MIC for MSSP and MRSP isolates (p = 0.785 and p = 0.862, respectively), and thus, the results were combined for comparison between tested products. Products are listed in order of lowest MIC to highest MIC in Table 2. The MICs of five chlorhexidine‐containing shampoos were significantly lower than those of three non‐chlorhexidine shampoos (Table 3). The MICs of some of the chlorhexidine shampoos, even with similar chlorhexidine concentrations, were significantly different. MICs ranged from 1.22 to 9.77 mg/L chlorhexidine gluconate/digluconate.
TABLE 2.
Minimum inhibitory and bactericidal concentration for tested products.
| Product | MIC dilution ratio/(chlorhexidine gluconate/digluconate mg/L) | MBC dilution ratio/(chlorhexidine gluconate/digluconate mg/L) | ||
|---|---|---|---|---|
| MSSP | MRSP | MSSP | MRSP | |
| Malaseb | 1:8192–1:16,384 | 1:8192–1:16,384 | 1:4096–1:8192 | 1:4096 |
| 2.44–1.22 | 2.44–1.22 | 4.88–2.44 | 4.88 | |
| BioHex | 1:8192–1:16,384 | 1:8192–1:16,384 | 1:4–1:8 | 1:4–1:8 |
| 2.44–1.22 | 2.44–1.22 | 5000–2500 | 5000–2500 | |
| Miconahex + Triz | 1:8192 | 1:8192–1:16,384 | 1:4096 | 1:4096 |
| 2.44 | 2.44–1.22 | 4.88 | 4.88 | |
| TrizCHLOR 4 | 1:8192 | 1:8192 | 1:4096 | 1:4096 |
| 4.88 | 4.88 | 9.77 | 9.77 | |
| ChlorhexiDerm 4% | 1:8192 | 1:8192 | 1:2048 | 1:2048 |
| 4.88 | 4.88 | 19.53 | 19.53 | |
| KETOCHLOR | 1:4096 | 1:4096–1:8192 | 1:2048 | 1:2048 |
| 5.62 | 5.62–2.81 | 11.23 | 11.23 | |
| Douxo S3 PYO | 1:4096 | 1:4096 | 1:1024–1:2048 | 1:2048 |
| 7.32 | 7.32 | 29.30–14.65 | 14.65 | |
| Davis chlorhexidine shampoo | 1:2048–1:4096 | 1:2048 | 1:1024–1:2048 | 1:1024 |
| 9.77–4.88 | 9.77 | 19.53–9.77 | 19.53 | |
| 2% Chlorhexidine solution | 1:2048–1:4096 | 1:2048 | 1:512–1:1024 | 1:1024 |
| 9.77–4.88 | 9.77 | 39.06–19.53 | 19.53 | |
| Universal medicated shampoo | 1:512 | 1:512 | 1:8–1:16 | 1:8–1:16 |
| NA | NA | NA | NA | |
| Allergroom | 1:512 | 1:512 | NA | NA |
| NA | NA | NA | NA | |
| DermaLyte | 1:512 | 1:512 | NA | NA |
| NA | NA | NA | NA | |
Note: MIC and MBC are reported in dilution ratio (solute [shampoo]:solvent [MHB]). The corresponding chlorhexidine concentration of the MIC/MBC is reported in the row below in red as chlorhexidine gluconate/digluconate (mg/L). Products are listed in order of lowest MIC to highest MIC.
Abbreviations: MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; MRSP, meticillin‐resistant Staphylococcus pseudintermedius ; MSSP, meticillin‐sensitive Staphylococcus pseudintermedius ; NA, not available.
TABLE 3.
Significant differences found when comparing MIC dilution ratios and chlorhexidine concentration between tested products against Staphylococcus isolates.
| Products with lower MIC | DermaLyte | Allergroom | Universal medicated shampoo | 2% Chlorhexidine solution | Davis chlorhexidine shampoo | Douxo S3 PYO | KETOCHLOR | ChlorhexiDerm 4% | TrizCHLOR 4 | Miconahex + Triz | BioHex | Malaseb | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| DermaLyte | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.821 | 0.006* | 0.006* | 0.003* | < 0.001* | < 0.001* | |
| Chlorhexidine concentration | |||||||||||||
| Allergroom | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | 0.821 | 0.006* | 0.006* | 0.003* | < 0.001* | < 0.001* | ||
| Chlorhexidine concentration | |||||||||||||
| Universal medicated shampoo | Dilution ratio | 1.000 | 1.000 | 1.000 | 0.821 | 0.006* | 0.006* | 0.003* | < 0.001* | < 0.001* | |||
| Chlorhexidine concentration | |||||||||||||
| 2% Chlorhexidine solution | Dilution ratio | 1.000 | 1.000 | 1.000 | 0.720 | 0.720 | 0.390 | 0.100 | 0.100 | ||||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.003* | 0.001* | 0.001* | |||||
| Davis chlorhexidine shampoo | Dilution ratio | 1.000 | 1.000 | 0.718 | 0.718 | 0.389 | 0.102 | 0.102 | |||||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | 1.000 | 0.003* | 0.001* | 0.001* | ||||||
| Douxo S3 PYO | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | ||||||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | 0.017* | 0.005* | 0.005* | |||||||
| KETOCHLOR | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |||||||
| Chlorhexidine concentration | 1.000 | 1.000 | 0.390 | 0.153 | 0.150 | ||||||||
| ChlorhexiDerm 4% | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | ||||||||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | 1.000 | |||||||||
| TrizCHLOR 4 | Dilution ratio | 1.000 | 1.000 | 1.000 | |||||||||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | ||||||||||
| Miconahex + Triz | Dilution ratio | 1.000 | 1.000 | ||||||||||
| Chlorhexidine Concentratio | 1.000 | 1.000 | |||||||||||
| BioHex | Dilution ratio | 1.000 | |||||||||||
| Chlorhexidine concentration | 1.000 | ||||||||||||
| Malaseb | Dilution ratio | ||||||||||||
| Chlorhexidine concentration | |||||||||||||
Note: Comparisons were made between the shampoos with the Kruskal–Wallis test including pairwise comparisons for post hoc analysis using the Bonferroni correction. A p‐value of < 0.05 was considered significant: *p < 0.05 (in green). Nonsignificant p‐values appear in red. When looking at the comparisons, the product name at the top of the column has the lower MIC.
Abbreviation: MIC, minimum inhibitory concentration.
3.3. Minimum Bactericidal Concentration
For all products tested, there were no statistical differences between the MBC for MSSP and MRSP isolates (p = 0.856–0.921), so results were combined for comparison between tested products. Two non‐chlorhexidine shampoos (Allergroom, DermaLyte) had no detectable MBC, so they were excluded from MBC dilution ratio comparisons. There were significant differences in MBC dilution ratios and chlorhexidine concentrations between some of the chlorhexidine‐containing products (Table 4). MBCs ranged from 2.44 to 5000 mg/L chlorhexidine gluconate/digluconate.
TABLE 4.
Significant differences found when comparing MBC dilution ratios and chlorhexidine concentration between tested products against Staphylococcus isolates.
| Products with lower MBC | Universal medicated shampoo | BioHex | 2% Chlorhexidine solution | Davis chlorhexidine shampoo | Douxo S3 PYO | KETOCHLOR | ChlorhexiDerm 4 | TrizCHLOR 4 | Miconahex + Triz | Malaseb | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Universal medicated shampoo | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | 0.470 | 0.470 | < 0.001* | < 0.001* | < 0.001* | |
| Chlorhexidine concentration | |||||||||||
| BioHex | Dilution ratio | 1.000 | 1.000 | 0.287 | 0.119 | 0.119 | < 0.001* | < 0.001* | < 0.001* | ||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | 0.110 | 1.000 | < 0.001* | < 0.001* | < 0.001* | |||
| 2% Chlorhexidine solution | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | 0.010* | 0.017* | 0.010* | |||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | 1.000 | 0.003* | 0.004* | 0.003* | ||||
| Davis chlorhexidine shampoo | Dilution ratio | 1.000 | 1.000 | 1.000 | 0.041* | 1.000 | 0.041* | ||||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | 0.029* | 0.042* | 0.029* | |||||
| Douxo S3 PYO | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |||||
| Chlorhexidine concentration | 1.000 | 1.000 | 0.097 | 0.140 | 0.097 | ||||||
| KETOCHLOR | Dilution ratio | 1.000 | 1.000 | 1.000 | 1.000 | ||||||
| Chlorhexidine concentration | 1.000 | 1.000 | 1.000 | 1.000 | |||||||
| ChlorhexiDerm 4% | Dilution ratio | 1.000 | 1.000 | 1.000 | |||||||
| Chlorhexidine concentration | 0.005* | 0.008* | 0.005* | ||||||||
| TrizCHLOR 4 | Dilution ratio | 1.000 | 1.000 | ||||||||
| Chlorhexidine concentration | 1.000 | 1.000 | |||||||||
| Miconahex + Triz | Dilution ratio | 1.000 | |||||||||
| Chlorhexidine concentration | 1.000 | ||||||||||
| Malaseb | Dilution ratio | ||||||||||
| Chlorhexidine concentration | |||||||||||
Note: Allergroom and DermaLyte had no detectable MBC and were not included in this table. Comparisons were made between the shampoos with the Kruskal‐Wallis test including pairwise comparisons for post hoc analysis using the Bonferroni correction. A p‐value of < 0.05 was considered significant: *, p < 0.05 (in green; nonsignificant p‐values in red). When looking at the comparisons, the product name at the top of the column has the lower MBC.
Abbreviations: MBC, minimum bactericidal concentration; NA, not available.
3.4. Lathering Ability and Lather Stability
At the 0 min time point, there were significant differences in median lather height between multiple products (Table 5; Figure 1). The Pearson correlation between lathering ability and MIC/MBC chlorhexidine gluconate/digluconate concentration and the dilution ratio showed low or no correlation (R‐values from −0.430 to 0.420).
TABLE 5.
Significant lathering ability differences at the 0 min time point.
| Products with higher lathering ability | Davis chlorhexidine shampoo | KETOCHLOR | Miconahex + Triz | ChlorhexiDerm 4 | Universal medicated shampoo | 2% Chlorhexidine solution | BioHex | Douxo S3 PYO | Malaseb | Allergroom | TrizCHLOR 4 | DermaLyte |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Davis chlorhexidine shampoo | 1.000 | 0.129 | 0.083 | 0.277 | 1.000 | 0.041* | 0.002* | 0.011* | < 0.001* | < 0.001* | < 0.001* | |
| KETOCHLOR | 0.180 | 0.116 | 0.377 | 1.000 | 0.058 | 0.004* | 0.016* | < 0.001* | < 0.001* | < 0.001* | ||
| Miconahex + Triz | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |||
| ChlorhexiDerm 4% | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | ||||
| Universal medicated shampoo | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |||||
| 2% Chlorhexidine solution | 1.000 | 0.700 | 1.000 | 0.057 | 0.034* | 0.020* | ||||||
| BioHex | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | |||||||
| Douxo S3 PYO | 1.000 | 1.000 | 1.000 | 1.000 | ||||||||
| Malaseb | 1.000 | 1.000 | 1.000 | |||||||||
| Allergroom | 1.000 | 1.000 | ||||||||||
| TrizCHLOR 4 | 1.000 | |||||||||||
| DermaLyte |
Note: Differences of lather height at the 0 min time point were compared with Kruskal–Wallis test including pairwise comparisons for post hoc analysis. A p‐value of < 0.05 was considered significant: *p < 0.05 (in green; nonsignificant p‐values appear in red). When looking at the comparisons, the product name at the top of the column has the taller lather height.
FIGURE 1.
Lathering ability of chlorhexidine‐containing shampoos. Lathering ability was evaluated using a modified cylinder shake method [20]. A 10% shampoo solution was inverted 10 times in a sterile glass tube. The height of the lather generated was measured from the water line at 0, 1, 5, 10, 15, 30, and 60 min. This was performed in triplicate for each shampoo by three different individuals. Median values are reported, and error bars represent the standard deviation.
4. Discussion
This preliminary in vitro study reports on the MIC and MBC of eight chlorhexidine gluconate/digluconate‐containing shampoos, three non‐chlorhexidine shampoos and a 2% chlorhexidine gluconate solution against one laboratory MSSP and one institutional MRSP isolate. MICs and MBCs for all tested products met chlorhexidine concentrations (500–40,000 mg/L) that are well tolerated and used clinically [21, 22]. This preliminary work suggests that chlorhexidine gluconate/digluconate‐containing shampoo efficacy may be influenced by formulation. Observed MICs and MBCs for the formulations were not simply based only on the chlorhexidine gluconate/digluconate concentration contained in the shampoo. MICs of chlorhexidine gluconate/digluconate were significantly lower for shampoos with 2% chlorhexidine gluconate and 2% miconazole nitrate (Malaseb, Miconahex + Triz and BioHex), even when compared to 2% chlorhexidine gluconate solution, 2% chlorhexidine gluconate shampoo (Davis Chlorhexidine) and 3% chlorhexidine digluconate shampoo (Douxo S3 PYO). Two 4% chlorhexidine gluconate shampoos (ChlorhexiDerm 4%, TrizCHLOR 4) performed the same as three 2% chlorhexidine gluconate and 2% miconazole nitrate shampoos (Malaseb, Miconahex + Triz and BioHex) with significantly lower MICs and higher dilution ratios compared to the two control shampoos (DermaLyte, Allergroom) and a shampoo containing chloroxylenol (Universal Medicated Shampoo). Likewise, MBC chlorhexidine concentrations were significantly lower for two 2% chlorhexidine gluconate and 2% miconazole nitrate shampoos (Malaseb, Miconahex + Triz) compared to 2% chlorhexidine gluconate solution, 2% chlorhexidine gluconate shampoo (Davis Chlorhexidine) and 4% chlorhexidine gluconate shampoo (ChlorhexiDerm 4%). Therefore, the antimicrobial efficacies of chlorhexidine‐containing shampoos evaluated in this study could not be ranked according to their chlorhexidine gluconate/digluconate concentration alone. Owing to varying active and inactive ingredients along with formulation variations, the performance of chlorhexidine‐containing products evaluated in this study cannot simply be extrapolated and applied to other products containing similar chlorhexidine or other active ingredient concentrations. This is a common misconception and misinterpretation of these types of studies. The overinterpretation and extrapolation of preliminary studies of this nature need to be avoided: the results of this study are preliminary and inherent to the products evaluated.
Antimicrobial shampoos are a recommended first‐line therapy for canine superficial pyoderma and may be as effective as systemic antibiotics [4, 23]. With great concern for antimicrobial resistance in veterinary medicine, emphasis should be placed on appropriate at‐home bathing recommendations and techniques to ensure successful topical therapy and mitigate areas for potential topical therapy failure. Appropriate contact time, shampoo selection, shampoo dosing and shampoo dilution are all essential for the success of topical antimicrobial therapy. This preliminary work may suggest that some shampoo formulations retain better antibacterial efficacy when diluted during at‐home bathing. During at‐home pet bathing, excessive water use or using too little shampoo may dilute the concentration of the shampoo's active ingredients, affecting antimicrobial efficacy. Chlorhexidine‐containing shampoo dosing has been recommended at 19 mL/m2 of body surface area [24]. No study has ever investigated pet owners' shampoo or water usage during at‐home pet bathing. Shampoos may or may not be diluted to concentrations beyond MIC/MBC efficacy during at‐home pet bathing, and the dilution of shampoos used during at‐home pet bathing needs to be studied.
Interestingly, one 2% chlorhexidine gluconate and 2% miconazole nitrate shampoo with 0.1% MicroSilver BG (BioHex) had a similar significant dilution ratio and chlorhexidine concentration for MIC, yet performed significantly differently for MBC from the other shampoos containing 2% chlorhexidine gluconate and 2% miconazole nitrate. This suggests that this product (BioHex) contains an ingredient that is influencing MBC in vitro. This interaction may be related to the ingredient, metallic silver particles (MicroSilver BG), positive ion surface charge [25, 26] competing with chlorhexidine to bind anionic portions of bacteria. Alternatively, this interaction could be a consequence of other inactive ingredients creating a biofilm‐like effect or protective vesicle. This effect may or may not be clinically relevant in vivo and needs to be investigated further.
When comparing the lathering ability to chlorhexidine gluconate/digluconate efficacy of the shampoo formulations tested, it was not supported that increased lathering ability had decreased antimicrobial efficacy. Inversely, shampoos with poor lathering ability did not have superior antimicrobial efficacy. No statistically significant associations were detected between lathering ability and MIC/MBC chlorhexidine gluconate/digluconate concentration or dilution ratio, although this interpretation can be confounded by other inactive and active ingredients such as Tris‐EDTA or imidazoles. Surfactants that reduce surface tension and increase a shampoo's ability to form lather may not always interfere with the active ingredient efficacy. Thus, antimicrobial efficacy of chlorhexidine‐containing shampoos cannot be estimated based on their lathering ability. A consumer survey on hotel toiletries reported lathering ability as the number one most important feature of hotel shampoo that consumers care about [16]. Even when comparing responses between consumer gender, age group or socioeconomic status (consumers that stay at economy versus luxury hotels), a shampoo that lathers was the most important shampoo feature compared to scent, bottle size, colour, shampoo texture, bottle design, packaging and labelling [16]. Shampoos with more lathering ability may prevent product overuse and ensure adequate shampoo dispersion across the body. Well‐lathering shampoos also may ensure client compliance for bathing owing to a positive client experience and an enjoyable perception of at‐home bathing. Interestingly, it is well recognised in humans and less defined in dogs that surfactants could have a potential negative impact on epithelial barrier function with atopic dermatitis, which is another factor for consideration and further investigation [27].
This preliminary investigation has several important limitations. One major limitation is that only one laboratory isolate of MSSP and one institutional isolate of MRSP were tested, which may not be entirely representative of all MSSP or MRSP isolates because antimicrobial susceptibility can vary between bacterial isolates and geographical regions [19]. Although low numbers of bacterial isolates were tested, there were no significant differences between MIC or MBC for the MSSP and MRSP isolates, which likewise have been reported in a prior investigation [28]. When evaluating our preliminary data to try and evaluate similar shampoos such as Malaseb and Miconahex + Triz to account for formulation differences in an appropriately powered study for the MIC values with an alpha of 0.05 and a power of 0.90, we determined that a sample size of 171 isolates would be required to find a significant difference between the two products when adequately accounting for type I error. This number of isolates for adequate powering is similar to a prior calculation derived from a study looking at ear solutions [10]. However, the powering value that we calculated could have greatly varied in the range of four to 643 isolates, depending on the product comparisons required, using the same predetermined power calculation values. When looking at similar comparisons with the MBC data, the preliminary data had a significant outlier with BioHex shampoo; including BioHex, the number of isolates required to show significant differences between the various products could range from four to 8016 isolates, yet excluding BioHex resulted in a large yet lesser range (4‐399 isolates). Either way, as the products behaved likewise in this preliminary report, it would take a larger number of isolates to determine if a significant difference in the shampoo formulations exists. Regardless, it is important to reiterate that the data here relate to these evaluated formulations and not simply the active ingredient values. Further, it would be inappropriate to extrapolate the activity of these evaluated formulas to other products with similar active ingredients if there is any variation in the formulation or production process. Another limitation is that in vitro studies may not represent efficacy in vivo. This in vitro study design allowed for evaluation of current chlorhexidine gluconate/digluconate‐containing shampoos in standardised conditions. In vitro studies may not mimic the clinical contact times during bathing and do not account for the presence of organic material such as inflammatory exudates and sebum, which affect shampoo antimicrobial efficacy.
5. Conclusions
As shampoo formulations change throughout the years, efficacy has been presumed based on the reported antimicrobial efficacy of chlorhexidine surgical scrub or other shampoo formulations with the same chlorhexidine concentration. This preliminary study supports in vitro antimicrobial performance varying significantly between different commercial antimicrobial shampoos and is not dependent on just chlorhexidine concentration. Results cannot be applied to other products with similar chlorhexidine concentrations owing to differing shampoo formulations. Lathering ability varied between chlorhexidine‐containing antimicrobial shampoos. Some chlorhexidine gluconate/digluconate‐containing shampoos lathered as well as non‐chlorhexidine control shampoos. There was no correlation between lathering ability and antimicrobial efficacy. Further investigations with robust numbers of bacterial isolates and large‐scale head‐to‐head clinical trials are needed to determine if in vitro variance has clinical significance.
Author Contributions
Emily E. Binversie: conceptualisation; data curation; formal analysis; funding acquisition; investigation; methodology; writing – original draft. Jason B. Pieper: conceptualisation; formal analysis; funding acquisition; investigation; methodology; project administration; writing – review and editing. Darren J. Berger: conceptualisation; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; supervision; writing – review and editing.
Conflicts of Interest
J.B.P. has received speaking honoraria from Virbac Animal Health, Vetoquinol, and CEVA for continuing education lectures. D.J.B. is a current employee of Elanco Animal Health and has also received a speaking honorarium from Virbac Animal Health in the last 5 years.
Supporting information
Table S1: Lathering ability.
Acknowledgements
The authors thank the Iowa State University Veterinary Diagnostic Laboratory for use of space and access to research facilities.
Funding: This project was funded through an American College of Veterinary Dermatology Resident Research Grant and in part by Iowa State University's endowed German Shepherd Dog Memorial Fund.
Study Presentation: This study abstract was presented at the North American Veterinary Dermatology Forum, Orlando (FL), 27–30 April 2025.
Data Availability Statement
The data that supports the findings of this study are available in supporting information of this article.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1: Lathering ability.
Data Availability Statement
The data that supports the findings of this study are available in supporting information of this article.