Biofilm Models: Different Ways of Biofilm Characterization and Drug Discovery.

The ability of bacteria to develop biofilms and its added effect on antimicrobial resistance have been a concern for both animal and human medicine. The need to understand biofilm biology has been addressed with the help of three biofilm models, i.e., in vitro, ex vivo, and in vivo. Due to the implications of animal welfare involved in in vivo models, this article is mainly focused on in vitro and ex vivo study models to analyze biofilm biology. In in vitro biofilm models, the microtiter plate and Calgary biofilm device are the most commonly used techniques for biofilm analysis. Quantification of the biofilm biomass generated by these two techniques can be assessed with the help of a crystal violet assay. Although in vitro biofilm models help advance understanding of the biology of biofilm and are easy to perform, they fail to address certain important questions, such as the importance of the substrate on which biofilm grows and the interaction between the organisms and the substrate. To address this concern, an ex vivo model can be utilized to characterize the behavior and characteristics of biofilms on different substrates. Ex vivo biofilm models are considered a bridge between the in vitro and in vivo biofilm models. Although neither of the currently available biofilm assessment models is considered the gold standard, they have significantly increased understanding of biofilm behavior. Further studies are warranted to develop more refined biofilm models. © 2023 Wiley Periodicals LLC. Basic Protocol 1: In vitro biofilm models for microtiter plate/crystal violet assay for biofilm growth assessment Basic Protocol 2: Crystal violet assay/tissue culture plate method for testing of antibiofilm agents Alternate Protocol: Calgary biofilm device to determine biofilm susceptibility to antimicrobial agents Basic Protocol 3: Ex vivo biofilm skin models: canine/porcine skin explants.

Ultrastructural Analysis of Differences in the Growth and Maturation of Staphylococcus pseudintermedius Biofilm on Biotic and Abiotic Surfaces.

Biofilms are extremely complex yet systematic microbial structures. Studies comparing the differences in their growth on living and nonliving surfaces by electron microscopy are limited. Therefore, the purpose of this study was to ultrastructurally investigate the differences in the growth and development of Staphylococcal biofilm on polycarbonate filters and canine skin explants. Using scanning and transmission electron microscopy (SEM and TEM), Staphylococcus pseudintermedius was incubated for 6, 12, 24, 48, and 72 h. It was observed that similar amounts of exopolymeric substance (EPS) were deposited on the biofilm on both surfaces, but the biofilm on the skin explants was primarily flat, whereas the biofilm on the membrane developed a multilayered plateaued look. Microcolony formation was only observed on the membrane filter during the early stages of biofilm development. On the membrane biofilms, EPS was observed to be deposited in a distinctive pattern. EPS deposition on the membrane surface was observed to peak before it declined, but on the explant, a constant increase was observed at all time points. Cell exposure to the environment on both the membrane filters and explants differed depending on the stage of biofilm formation. On both the membranes and the skin explants, there was a perceptible difference between the biofilm growth patterns and speeds. The results of this study suggest that data extrapolated from studies on biofilm bactericidal compounds performed on abiotic surfaces (such as polycarbonate filters) may not be entirely applicable to biofilm growing on biotic surfaces (e.g., skin) due to ultrastructural variations revealed in this study. IMPORTANCE Biofilm has been recognized as an important source of antimicrobial resistance. These sessile microbial colonies tend to attach and grow on every surface, biotic and abiotic, and they account for approximately 80% of chronic and recurrent infections. Biofilms are not all alike; they have different structures and microbial compositions. This high variability allows for differences in the production of exopolymer substances, affecting antimicrobial penetration. No studies have been published that simultaneously compare the structure of biofilms grown on abiotic (in vitro) and biotic (ex vivo) surfaces. To identify treatment alternatives, it is essential to understand the differences between biofilms. The results of the study show how biofilm structures and compositions are dependent on the substrate on which they grow.

Prevalence of mecA, mecC and Panton-Valentine-Leukocidin Genes in Clinical Isolates of Coagulase Positive Staphylococci from Dermatological Canine Patients.

Coagulase positive Staphylococci (CoPS) are the leading cause of canine cutaneous and otic infections. Virulence factors associated with Staphylococci include the expression of mec and panton-valentine leukocidin (pvl) genes. Methicillin-resistance (MR) is commonly associated with mecA gene expression, although a recently identified variant, mecC, has been reported. This study aims to evaluate the prevalence of mecA, mecC and pvl genes in 232 clinical isolates of CoPS collected from dogs with pyoderma. A multiplex PCR, and Kirby-Bauer disk diffusion susceptibility test for cefoxitin was performed for all isolates. PBP2a agglutination test was performed on 127 isolates. Standard MRSA isolates were used as positive controls. The mecA gene was identified in 149/232 isolates (64.2%): 116 S. pseudintermedius, 30 S. coagulans and three S. aureus. The pvl gene was present in only 1 isolate of S. pseudintermedius (0.4%), whereas no isolates carried the mecC gene. 34 isolates were resistant to cefoxitin (14.6%) and they were all mecA positive. The results of this study show an MR prevalence of 64.2% confirming concerns about antibiotic resistance in veterinary medicine. In conclusion, this is the first study analyzing the prevalence of mecC and pvl in comparison to mecA, in a large cohort of CoPS clinical isolates from dogs with pyoderma. A multimodal surveillance on the prevalence of mecC and pvl in veterinary medicine is essential to appropriate antimicrobial management.

Effect of Nanosulfur Against Multidrug-Resistant Staphylococcus pseudintermedius and Pseudomonas aeruginosa.

Multidrug resistance (MDR) has significantly increased in the past decades and the use of nanotechnology has opened new venues for novel treatments. Nanosulfur is a potent antimicrobial agent and a cheaper alternative to other nanomaterials. However, very few studies have been published on its activity against MDR organisms. Therefore, the goal of this in vitro study was to assess cytotoxicity, antimicrobial, and anti-biofilm activity of nanosulfur (47 nm, orthorhombic) against clinical isolates of MDR Staphylococcus pseudintermedius (SP) and Pseudomonas aeruginosa (PA) in planktonic and biofilm state using canine skin explants. Nanosilver (50 nm, spherical) was tested as a comparative control. Concentrations between 1866.7 and 0.11 µg/mL of both nanoparticles were tested. The ultrastructure of nanosulfur was assessed via electron microscopy. Both types of nanoparticles showed no direct cytotoxicity on a canine keratinocyte cell line. In the planktonic phase, nanosulfur was able to inhibit or kill (6-log10 reduction of CFU) 7 of 10 MDR-SP isolates at 233.3 µg/mL, whereas, when in biofilm state, 6 of 10 isolates were killed at different concentrations (233.33 to 1866.7 µg/mL). Nanosilver did not show any antimicrobial or anti-biofilm activity at any concentrations tested. Both types of nanoparticles were ineffective against MDR-PA in either state. Ultrastructurally, nanosulfur was present in individual nanoparticles as well as forming larger nanoclusters. This is the first study showing an antimicrobial and anti-biofilm activity of nanosulfur for MDR-SP in absence of cytotoxicity. Nanosulfur has the potential to be used in veterinary and human medicine as effective, safe, and cheap alternative to antimicrobials and anti-biofilm agents currently available. KEY POINTS: • Nanosulfur is a better alternative than nanosilver to treat MDR-Staphylococci. • Nanosulfur is an effective agent against MDR-Staphyloccocal biofilm. • Canine skin explant model is reliable for testing anti-biofilm agents.

Evaluation of the effects of chlorhexidine digluconate with and without cBD103 or cCath against multidrug-resistant clinical isolates of Staphylococcus pseudintermedius.

BACKGROUND: Because of the increased incidence of multidrug-resistant (MDR) bacteria, the use of disinfectants over antibiotics has been encouraged. However, the interactions between disinfectants and host local immunity are poorly understood. OBJECTIVE: To assess the effects of chlorhexidine digluconate (Chx), with and without selected host defence peptides (HDPs), against MDR Staphylococcus pseudintermedius (MDR-SP). METHODS AND MATERIALS: Ten clinical isolates of MDR-SP were tested, using a modified microbroth dilution method. Four two-fold dilutions of 2% Chx and 1 µg/mL the HDPs synthetic canine ß-defensin 103 (cBD103) or cathelicidin (cCath) were tested alone or in combination. Colony counts after 5, 15, 30 and 60 min, and a minimum inhibitory concentration (MIC) after 24 h were recorded. Friedman followed by Dunn's multiple comparison tests with significance of P < 0.05 were used for statistical analysis. Synergy, additivity/neutrality or antagonism were calculated. RESULTS: Growth was not inhibited by either HDP alone. An MIC of 0.312 µg/mL Chx was achieved for nine of the isolates. One isolate had an MIC of 0.078 µg/mL Chx. A MIC90 (in nine of 10 isolates) of 0.312 µg/mL was seen for Chx in combination with either HDP. Synergy was seen in the combination Chx/cCath used at the highest concentrations of Chx (0.624 µg/mL and 0.312 µg/mL) after 30 and 60 min incubation. Additivity/neutrality was seen for most of the other concentrations and times of incubation. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest a synergistic/additive effect between Chx and HDPs in dogs. Further studies evaluating the mechanisms behind this effect are needed.