Evaluation of the use of paired modified Wright’s and periodic acid Schiff stains to identify microbial aggregates on cytological smears of dogs with microbial otitis externa and suspected biofilm

Abstract

Background Micro‐organisms associated with canine otitis externa (OE) may cause biofilm‐associated infections (BAI). A key component of biofilm is microbial aggregate and extracellular polymeric substance (EPS). Periodic acid Schiff (PAS) can stain polysaccharide EPS in human otitis media with effusion, but this has not been tested in canine OE. There is no cytological definition for microbial aggregate, and definitive methods for identifying BAI in a clinical setting in canine OE have not been defined. Objectives To establish whether PAS stain can identify polysaccharide matrix on cytological smears; and to determine the reproducibility of identification of microbial aggregates within a discrete area of stained matrix, using paired modified Wright’s and PAS‐stained smears. Animals Forty privately‐owned dogs presenting to a dermatological referral practice. Methods and materials In this prospective, cross‐sectional study, three investigators independently and blindly classified 40 paired modified Wright’s–PAS slide sets into groups: aggregate‐associated infection (AAI) and non‐AAI (n = 27); and control (n = 13). Agreement between investigators for presence of AAI was measured using Fleiss’ kappa statistic (FK). Agreement between investigators and dermatologists for presence of AAI upon cytological evaluation, and suspected BAI based on clinical examination, was measured using Cohen’s kappa statistic. Results The matrix was confirmed to stain PAS‐positive. Interinvestigator agreement for AAI was very good using PAS (0.82 FK) and fair using modified‐Wright’s (MW) (0.33 FK). Reproducible cytological features associated with AAI were the presence of: three or more distinct aggregates (0.76 FK); discrete areas of PAS‐positive matrix (0.70 FK); and the presence of high‐density material (0.70 FK) using PAS stain. Conclusion PAS can stain the extracellular matrix on otic smears, and a novel protocol for reproducible identification of cytological features such as microbial aggregates has been established.

Emerging Approaches for Mitigating Biofilm-Formation-Associated Infections in Farm, Wild, and Companion Animals

“…Staphylococcus pseudintermedius and P. aeruginosa are frequently the pathogens responsible for canine otitis externa (OE) in dogs [ 92 ]. This disease is associated with the development of biofilm [ 93 ]. Furthermore, cytological smears stained with periodic acid–Schiff (PAS) and modified Wright’s stain also support the presence of biofilm [ 3 ].…”

The Role of Biofilms in the Pathogenesis of Animal Bacterial Infections

“…OE is a frequently reported disorder in dogs, associated with infections by Staphylococcus pseudintermedius ( S. pseudintermedius ) and P. aeruginosa in addition to yeast pathogens [ 19 ]. Cytological observations of microbial aggregates and filamentous veil-like materials in clinical practice have earlier been indicative of the presence of biofilms [ 20 ]. In smears from human otitis media with effusion, periodic acid-Schiff (PAS) has been used to visualize polysaccharide biofilm matrix components.…”

“…In a study by Parnell-Turner et al to identify biofilms in dogs with OE, several investigators studied cytological smears from both patients and controls stained with PAS and modified Wright’s stain in a blind test [ 20 ]. The term microbial aggregate was defined as: “(i) a distinct, dense mass of apparently connected micro-organisms; and (ii) different micro-organisms that come into focus across two or more focal planes”.…”

“…( a – d ) 100× (oil) objective, bar = 10 μm. Reprinted from [ 20 ], Copyright (2021), with permission from John Wiley and Sons.…”

Managing recurrent otitis externa in dogs: what have we learned and what can we do better?

“…On modified Wright-Giemsa stained cytology (i.e. rapid in-clinic stains) they form fine pink-cerise veil or net-like material embedding the neutrophils and organisms (figure 8), although Periodic Acid Schiff (PAS) 48 can be used as a more specific stain. However, optimising culture techniques to identify biofilm-forming ability from clinical samples would help clinicians when planning treatment in cases where the biofilm is not clinically or cytologically obvious 3 .…”