Biofilm Production Capability of Clinical Aeromonas salmonicida Subspecies salmonicida Strains under Stress Conditions

Abstract

Biofilm formation of clinical isolates of Aeromonas salmonicida subspecies salmonicida was compared using scarce (minimal M9 and ABTG w/o amino acids) and enriched nutrient media (Tryptone Soya broth) at 8 °C, 16 °C, and 25 °C using direct enumeration of viable cells in biofilm (log CFU), crystal violet staining (ODc) of the formed biofilm biomass, and liquid–air border biofilm formation (pellicle test). Whole-genome sequencing (WGS) was performed with the usage of an Oxford nanopore system by Genomics and Transcriptomics Labor Düsseldorf (Heinrich-Heine-Universität Düsseldorf, Germany). A bioinformatic analysis was performed with the usage of Geneious Prime® 2023.0.4 (Biomatters, Inc., Boston, MA, USA). All data were analyzed using Statistica software version 13.0, and changes in biofilm production in correlation to changes in the type of nutritional medium and temperature were compared between groups using a one-way ANOVA analysis and Tukey’s test. All isolates formed biofilms in minimal M9 at 8 °C and 16 °C, and nine isolates failed to form biofilms in minimal M9 at 25 °C. In an ABTG medium, all isolates produced biofilms at 8 °C; however, three isolates at 16 °C and seven isolates at 25 °C failed to form any biofilms. Significant biofilm formation was observed in TSB at all temperatures. Some strains that formed a good biofilm in solid–liquid interface did not have the ability to form a pellicle (liquid–air border biofilm), and vice versa. In all cases of nutritional medium and temperature changes, there were statistically significant differences in the intensity of biofilm production, especially in the detected number of viable cells inside biofilms (log CFU, p < 0.005). Multiple biofilm-production genes, including polar flagella (filM) LuxR family (transcriptional regulators) and VapA family of histidine-kinase-associated genes, were sequenced from all studied isolates. Genetic differences based on geographical origin were not observed among the isolates. Significant variations in isolates’ ability to form biofilms were observed, possibly due to epigenetic factors. The optimal temperature for biofilm formation of A. salmonicida subspecies salmonicida in scarce media was 8 °C, and the majority of isolates were not capable of biofilm formation at 25 °C without enriched nutrient media.