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Article

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

by
Ksenija Aksentijević
1,
Aleksandra Daria Rajewska
2,
Konrad Wojnarowski
3,
Paulina Cholewińska
3,
Malgorzata Korzeniowska
2,
Peter Steinbauer
4,
Dušan Palić
3 and
Dusan Misic
2,*
1
Department of Microbiology, University of Belgrade, Bulevar Oslobodjenja, 18, 11000 Belgrade, Serbia
2
Department of Functional Food Products Development, Wrocław University of Environmental and Life Sciences, Chełmońskiego St. 37, 51-630 Wroclaw, Poland
3
Chair for Fish Diseases and Fisheries Biology, Ludwig-Maximilians-Universität München, Kaulbachstr. 37, 80539 Munich, Germany
4
Fish Health Service Section, Bavarian Animal Health Service, Senator-Gerauer-Str. 23, 85586 Poing, Germany
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(20), 9365; https://doi.org/10.3390/app14209365
Submission received: 13 August 2024 / Revised: 8 October 2024 / Accepted: 10 October 2024 / Published: 14 October 2024
(This article belongs to the Special Issue New Insights into Marine Ecology and Fisheries Science)
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Abstract

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Although this is a fundamental microbiological study, its results indicate the necessity of changing the approach to fish ponds maintenance and focusing all hygiene measures on the eradication of biofilms and not on a simple disinfection. Also, the practical laboratory approach to testing biofilms of A. salmonicida subspecies salmonicida should be changed.

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.

1. Introduction

Knowledge about Aeromonas (A.) salmonicida is continuously updated. Complicated and intensively fluctuating taxonomy inside the Aeromonas genus [1,2,3] lasted until the beginning of the 21st century, when five subspecies were officially established: salmonicida, smithia, achromogenes, masoucida, and pectinolytica [4]. A division of A. salmonicida to “typical” and “atypical” representatives is frequently applied even if a clear scientific basis to justify this differentiation is lacking. For example, the A. salmonicida subsp. salmonicida has been positioned as the typical representative of the genus, with the reference to its strict pathogenicity for salmonid fish, unlike other A. salmonicida subspecies that are opportunistic pathogens and have a wide range of hosts, including occasionally immunocompromised humans [5,6]. Therefore, for a long time, A. salmonicida was not considered a human pathogen because it cannot multiply at 37 °C. Regardless, in the last few years, sporadic cases of human infections with A. salmonicida have been reported, including bacteremia, endocarditis, eye, skin, and surgical wound infection, but without information about which subspecies of A. salmonicida caused the disease, which indicates the necessity of precise molecular subtyping of this causative agent [4,7,8,9,10]. So far, not a single case of human infection with A. salmonicida subspecies salmonicida has been recorded. In addition, it was recently proven that the pathogenicity of A. salmonicida subsp. salmonicida can be associated not only with salmonid fish but also with a wide range of other fish species like cod, black rockfish, sailfin sandfish, turbot, and goldfish [2,6]. The appearance of beta-haemolytic, sometimes pigmentated colonies on 5% sheep blood agar [6] was also considered to be typical only for subsp. salmonicida. Again, it was reported that other A. salmonicida subspecies, as well as A. hydrophila, can sporadically have identical colonies as A. subsp. salmonicida, which depends on the cultivation temperature [11]. Psychrophilic conditions for the optimal multiplication of A. salmonicida subsp. salmonicida is another basis on which it is considered a typical representative of the genus [12,13]. Yet, in recently published papers on this subject, there is the confusion in the interpretation of what are psychrophilic and what are mesophilic conditions—majority of authors arbitrarily declared temperatures of 22–28 °C as psychrophilic for A. salmonicida subsp. salmonicida [1,13,14], thus, ignoring the basic microbiological psychrophilic norm of a maximum 18 °C; and mesophilic conditions were declared as between 10 °C and 30 °C [6,15]. Indeed, the most researched habitat of A. salmonicida subsp. salmonicida are cold waters (from 2 °C in winter, to 18 °C in summer) [6]; however, recent findings clearly show the ability of A. salmonicida subsp. salmonicida to grow and multiply in a wider temperature range, between 4 °C and up to 30 °C [7,11,13,16,17,18]. This indicates that A. salmonicida subsp. salmonicida is most likely a psychrotropic microorganism (0–30 °C) [2,12,15].
Biofilm, or “multicellular pattern of life organization in bacteria” is considered a higher evolutionary stage of life organization in bacteria, enabling them to successfully survive in the external environment and in unfavorable conditions [19,20,21]. It has been proven that a biofilm is a natural form in which bacteria live in all ecological environments, including open waters, i.e., that bacteria cannot survive in the external environment as unicellular forms but can survive exclusively united in a biofilm [22]. The basic hypothesis is that a biofilm is formed when bacteria are under stress—the lack of nutrients and during suboptimal temperatures [23,24]. Based on our insight, there is only one scientific publication that deals with precisely genotyped A. salmonicida subsp. salmonicida biofilms [25].
Aeromonas salmonicida has been proven to have a “social life” and participates in the construction of multispecies biofilms with other aquatic bacteria (although it is unclear which subspecies of A. salmonicida), and this is mostly found in biofilms in natural river and sea water environments [26]. It has also been proven that in such multispecies-tight environments, resistance genes from other bacteria are much easier to transfer to A. salmonicida [26].
It is still not well understood which genes are responsible for the synthesis of biofilm components in Aeromonas salmonicida subsp. salmonicida. Genes that are not strictly typical for A. salmonicida are mostly listed, and most often, these are the genes that encode the production of components/processes that are important not only for biofilm but also for other bacterial functions. Thus, the genes for the Type 1, Type 2, and Type 3 autoinducer (AI-1, AI-2, AI-3) system that encode Quorum sensing and swimming motility [26,27,28]; genes for the production of lateral flagella [29]; polar flagella (which are even considered essential for biofilm production in the genus Aeromonas); type IV pilli [14]; and transcriptional regulator [30] are most often cited in research. The vapA gene encodes an A-layer protein, which is of great importance for A. salmonicida to attach to a surface and which is the basic prerequisite for biofilm formation [25].
In this study, the experiment was designed to fit the hypothesis that Aeromonas salmonicida subsp. salmonicida produces stronger biofilms in nutritionally poor environments and at suboptimal temperatures [31]. Another goal of this study was to establish the optimal temperature of Aeromonas salmonicida subsp. salmonicida for the in vitro production of biofilms, whether it correlates with its natural environment (8 °C or 16 °C) or the one that is mostly used for its isolation and identification (25 °C). To the best of our knowledge, this is the first study to examine precisely typed Aeromonas salmonicida subsp. salmonicida clinical isolates of fish origin at lower temperatures and in scarce nutritional media with three methods, in comparison with rich media and elevated temperatures, during a prolonged incubation that lasted 7 days to determine its ability to produce biofilm. With the partial genome sequencing analysis, we believe this will contribute to a better understanding of the physiology and virulence of this still insufficiently investigated pathogenic agent which is exceptionally important for the aquaculture.

2. Materials and Methods

2.1. The Origin of the Clinical Isolates

The investigated A. salmonicida subspecies salmonicida isolates were collected from a symptomatic brook trout hybrid (Salvelinus alpinus x fontinalis) during disease outbreaks with clinical symptoms typical for furunculosis. The samples were collected from several aquaculture establishments in the region of Bavaria, Germany, in all the seasons (Spring, Summer, Autumn, and Winter), during the outbreaks of the furunculosis, between 2017 and 2020 (Figure 1).
Sampling locations were located within watersheds of Danube, Regen, Inn, Isar, and Main as adjacent rivers. Fish samples were delivered to the Bavarian Animal Health Service, Fish Health Service division (Tiergesundheitsdienst Bayern e. V./Fischgesundheitsdienst, Poing, Germany) as a part of a routine laboratory diagnosis/investigation of clinical fish cases reported by fish farmers.

2.2. Isolation and Identification of the Isolates

Conventional microbiological methods were applied in the isolation of the microorganisms. The samples were inoculated on a Columbia 5% sheep blood agar (CSBA), MacConkey (MCC) agar, and Tryptone soya broth (TSB) (Merck KGaA, Darmstadt, Germany) and incubated at 25 °C for 48 h. Colonies grown on the CSB agar and the MCC agar which were suspicious for A. salmonicida (colonies were selected on basis of shape (round) and beta-hemolytic activity on blood agar) were replated on new plates in order to obtain a pure culture. The clinical isolates were confirmed during original case investigations as Aeromonas salmonicida by MALDI-TOF (Bruker Daltonics, using proprietary database of Bavarian Fish Health Service, Poing, Germany). Comparative analysis of whole-genome sequences with a reference strain of the Aeromonas salmonicida subspecies salmonicida ATCC® 33658™ genome served to confirm subspecies salmonicida for each studied isolate. The studied sequences were uploaded to “figshare” repository and are publicly available under the following link: https://figshare.com/articles/dataset/AP_Science/27169116/1, accessed on 4 October 2024.

2.3. In Vitro Biofilm Investigations

An investigation was performed in nutritionally rich Tryptone soya broth (Oxoid, CM0129B, Basingstoke, United Kingdom) and in nutritionally poor M9 and ABTG minimal salts. A minimal M9 medium was made of a commercially available product (M9 minimal salts 5×, Difco, Becton Dickinson and Company, Le Pont de Claix, France, consisted of Na2HPO4 3.39%, KH2PO4 1.5%, NaCl 0.25%, NH4Cl 0.5%), with the subsequent addition of (in final concentrations) 0.4% glucose, 0.004 M MgSO4, and 0.00001 M CaCl2 (all provided by Supelco, Merck KGaA, Darmstadt, Germany). A minimal ABTG medium was made as previously described [32], and it consisted of 1 mM MgCl2, 0.1 mM CaCl2, 15.1 mM (NH4)SO4, 22 mM KH2PO4, 0.5% glucose, (all provided by Supelco, Merck KGaA, Darmstadt, Germany), 33.7 mM Na2HPO4 × 2H2O, 51 mM NaCl, 0.01 mM FeCl3, (all provided by Chempur, Piekary Śląskie, Poland), and 2.5 μg/mL Thyamin–Hydrochlorid (purity >98.5% Carl Roth, Karlsruhe, Germany).
The strains were grown on the Columbia 5% sheep blood agar (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h at 25 °C. Bacterial inoculums were prepared by suspending each strain in the appropriate medium to an optical density (OD) of OD600 0.08–0.1 (approx. 1 × 108 CFU/mL), after which it was diluted 1:10, and 100 µL of the prepared dilutions (1 × 107 CFU/mL) were inoculated into microtiter plates with 96 flat-bottom wells (neoCulture Cell and Tissue Culture Plates W96, neoLab Migge GmbH, Heidelberg, Germany). Each isolate was inoculated in twelve replicates. The incubation lasted 7 days in Binder climatic chambers (KBF-LQC 240, Binder, Tuttlingen, Germany) under controlled atmospheric conditions at 8 °C, 16 °C, and 25 °C and at 80% humidity, and it was performed in two independent experiments.
Aeromonas salmonicida ATCC 7965 (Microbiologics, St. Cloud, MN, USA) was used as a comparative control in both applied methods.

2.4. The Determination of the Total Viable Cell Counts in Biofilms

The original method used [19] has been slightly modified. After the finished incubation, the planktonic cells and the medium were carefully removed with a sterile Pasteur pipette. The wells were rinsed three times with the sterile physiological saline (100 μL in every rinsing). Detachment of the formed biofilms was performed by adding 100 μL of the sterile physiological solution to each well, and the microtitration plate was placed on a shaker (Heidolph Vibramax 100, Heidolph Scientific Products GmbH, Schwabach, Germany) for 5 min at 750 RPM. In a previously disinfected ultrasonic bath filled with sterile, distilled water mixed with ice, the microtiter plates were placed so that the water covered just the bottom of the plate. The sonication lasted 10 s at 37,000 Hz (Elmasonic S60, Elma Schmidbauer GmbH, Singen, Germany). The detached biofilm in the physiological solution was transferred to sterile microtitration plates, and the whole detachment procedure was repeated one more time so that the total volume of physiological solution with detached biofilm was 200 uL. After that, serial dilutions from 10−2 to 10−7 were made. From each dilution, 10 μL were inoculated on the TSA in six replicates and subsequently incubated at 25 °C under microaerophilic conditions for 72 h. The enumeration of grown colonies was performed according to the formulas described earlier [33,34]. Replicates from two successive dilutions with minimally 15 and maximally 300 grown colonies were enumerated. The calculation of total CFU/mL was performed according to the formula:
NCFU = (∑C)/(V × [n1 + (0.1 × n2)] × d)
where ∑C is the total number the grown colonies on all the replicates obtained from two successive dilutions, and V is the volume of inoculum in each replicate (mL); n1 is the number of replicates used from the first dilution; n2 is the number of replicates used from the second dilution; and d is the dilution factor corresponding to the first dilution. In addition to CFU/mL, the obtained results are also expressed as log (CFU/mL).

2.5. Determination of Biofilm Biomass by the Crystal-Violet-Staining Method in Microtitration Plates

A previously described and slightly modified method was applied [35]. After incubation, the medium with planktonic cells was removed with a plastic, sterile Pasteur pipette, and the wells on the microtiter plate were washed three times with sterile, distilled water. After drying at room temperature, 125 μL of 0.1%, v/v crystal violet (Merck KGaA, Darmstadt, Germany) was added in all wells for 20 min. The dye was solubilized in 150 μL of acetic acid (33%, v/v). Absorbance was measured at 550 nm on a Thermo Scientific Multiscan Sky. The wells containing only sterile TSB, ABTG, and M9 medium were blanks.
The disadvantages of this assay are the secondary variables (incubation times, decoloring stain, pH oscillations, measurement errors during pipetting, oscillations in temperatures during cultivation) and are eliminated by using standardized protocols and applying the GLP (good laboratory practice) standards, which includes regular calibrations of all measuring instruments by competent institutions and under control of the “Committee for the quality assurance” at the Faculty of Biotechnology and Food Science, Wroclaw University of Environmental and Life Sciences, Poland, where the in vitro experiments were conducted.

2.6. The Determination of Biofilm Production on the Liquid–Air Border (Pellicle Test)

A biofilm formed at the border of two aggregate states (usually liquid–air) is known as a “pellicle”. The pellicle test was performed as previously described [36] in polystyrene test tubes with 3.9 mL of the medium (M9, ABTG, TSB) and 100 μL of each A. salmonicida subsp. salmonicida isolate suspension equal to 0.5 McF standard (approx. 1–2 × 108 CFU/mL) prepared from the 18 h old cultures obtained on TSA. The tubes were incubated for 6 days at temperatures of 8 °C, 16 °C, and 25 °C. After incubation, the contents of the tubes were removed by pipetting, and each tube was washed three times with 5 mL of the sterile physiological solution. After washing, the test tubes were dried by placing them in inverted position for 3–5 h at room temperature. The pellicle (biofilm) formed at the edges of the border of two aggregate states (liquid–air) was fixed by adding 5 mL 96% ethanol for 20 min at room temperature. After fixing, alcohol was removed by casting, and tubes were dried in an inverted position at room temperature. Staining of the pellicle (biofilm) was performed with a 0.3% solution of crystal violet dye for 10 min at room temperature. The dye was washed out with sterile saline until all the dye solution has been washed away from the tubes. The tubes were then placed in an inverted position to dry at room temperature. Negative controls were tubes with used liquid media. The pellicle test was repeated three times for each isolate. The ability to create a pellicle was evaluated based on the following criteria (Figure 2): (a) strong pellicle producers—strains that form a solid, stable, thicker pellicle of 1 mm (+++); (b) moderate pellicle producers—strains that form a stable, medium pellicle of a thickness up to 1 mm (++); (c) weak pellicle producers—very thin pellicle, (thinner than 1 mm) (+); (d) strains that do not produce biofilm—strains that do not produce a visible pellicle. A digital caliper was used for the measurements of the formed pellicles.

2.7. Molecular Sequencing

Plasmid and genomic DNA extraction was performed by utilizing Nucleospin Microbial DNA and Nucleospin Plasmid kits (Macherey-Nagel, Düren, Germany) according to the protocols attached to the kits, with modifications. After isolation, genomic DNA samples were additionally cleaned with Nucleospin gDNA Clean-kit (Macherey-Nagel, Düren, Germany) according to the attached protocol. Prior to the library preparation samples were pooled in 1:1 ratio. Whole-genome sequencing (WGS) has been performed with usage of the Oxford nanopore system by Genomics and Transcriptomics Labor Düsseldorf (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). Bioinformatic analysis was performed with the usage of Geneious Prime® 2023.0.4 (Biomatters, Inc., Boston, MA, USA). The sequences were cleaned of duplicate sequences and trimmed. The obtained data were mapped with “Map to references” (References: NZ_LSGW00000000.1, Mapping using Minimap 2.24—Oxford Nanopore plugin, with annotation), which allowed for demonstrating the compliance of the analyzed sequences with the reference gene—Aeromonas salmonicida subsp. salmonicida strain (ATCC 33658). The samples were then filtered to analyze the presence of selected genes associated with biofilm formation. A phylogeny analysis was performed with Galaxy web platform using MAFFT (version 7.526) and IQ-TREE (version 2.3.6) tools.

2.8. Statistical Analyses

Data were analyzed using Statistica software version 13.0 (StatSoft Inc., Tulsa, OK, USA). The quantitative bacterial counts of A. salmonicida subsp. salmonicida strains and calculated CFU per milliliter underwent logarithmic transformation, after which changes in media and temperature were compared between groups using one-way ANOVA and Tukey’s test. The criterion for statistical significance was a p-value of 0.05.

3. Results

3.1. Investigated Isolates

Of all investigated fish samples, 94 A. salmonicida subsp. salmonicida isolates were collected. Out of this, 10 isolates that originated from representative aquaculture establishments were chosen for the biofilms study. Investigated isolates were marked as follows: F203, F352, F461, F410, FN1413, FN187, F303, F262, F710, and F458.

3.2. Results of the Biofilm Production

3.2.1. Biofilm Production in M9 Minimal Medium

All clinical isolates successfully formed biofilms in minimal M9, at 8 °C and 16 °C, but the strength of those biofilms differed significantly both in the number of viable cells (log CFU) and in the production of biomass (ODC in crystal violet staining). The strongest-detected biofilm produced by clinical strains in M9 at 8 °C was made by strain F187, with a 5.41 log CFU (ODc value: 0.0053), and the weakest-detected biofilm was made by F710, with a 3958 detected log CFU (ODc value: 0.0055). Also, the strains reacted differently to the increase in temperature from 8 to 16 degrees. Most of the isolates decreased biofilm production between 1.99% and 19.75% at 16 °C in comparison to 8 °C (p < 0.05) (Table S1, Supplemental Materials). Only two strains (F410 and F710) increased biofilms production by 5.85% and 7.37%, respectively, at 16 °C in comparison to 8 °C (p < 0.05). The largest number of strains, a total of 9, could not produce any biofilm at 25 degrees in the M9 minimal medium, that is, no viable cells were detected in the biomass (Table S1, 8 °C vs. 25 °C and 16 °C vs. 25 °C, p = 0.0001). Only ATCC 7965 and F410 were capable of forming biofilms at 25 °C in M9. The complete results of this section are presented in Figure 3 and Supplemental Table S1.
The ODc values (biomass) detected in biofilms at 8 °C and 16 °C were very low, ranging from 0.0017 to 0.0375, which is not in correspondence with a relatively high number of viable cells detected in biofilms. However, ODc values were also detected at 25 °C (ranging from 0.0059 to 0.0133), but given that at 25 °C, no viable cells were detected within biofilms in any repetition, these results obtained from the crystal violet method cannot be interpreted as an active (living) biofilm but as a deposit of dead cells or deposited products of planktonic cells (Table S1).

3.2.2. Biofilm Production in Minimal ABTG Medium

In the ABTG medium, the largest oscillations in the ability of A. salmonicida subsp. salmonicida to produce biofilms in relation to temperature changes occurred in the detection of viable cells (log CFU). At a temperature of 8 °C, all isolates produced relatively strong biofilms, with relatively high viable cells log CFU, ranging from 3.71 to 6.04 log CFU (Figure 4, Table S2). At 16 °C, in ABTG, three isolates (F461, F458 and F710) did not produce any detectable viable cells biofilm (100% reduction in comparison to 8 °C, p < 0.001). The five clinical isolates (F410, F203, F352, F262, FN 1413) produced weaker biofilms (lower log CFU) at 16 °C than at 8 °C (reduction rate from 9.77% to 30.97%, p < 0.05). Isolates F303 and ATCC 7965 produced a stronger biofilm at 16 °C in ABTG compared to 8 °C; the increase was 4.21% to 6.33% (p < 0.05). Four strains (ATCC and three clinical isolates) produced biofilm at 25 °C, but all of them except ATCC produced weaker biofilms compared to 16 °C and 8 °C (decreasing rate 4.1% to 17.34% p < 0.05). A total of seven isolates failed to produce any detectable biofilm at 25 °C in the ABTG medium (no viable cells detected). F187 successfully produced biofilm at all three temperatures—at 8 °C and 16 °C, with 5.24 log CFU, and at 25 °C, with 4.6 log CFU.
ODc values of the crystal violet method were also detected in all isolates, and at all temperatures, but they did not correlate with the detected number of viable cells in biofilms at 25 °C, which is considered a non-specific reading of cellular detritus. Numerical values of the detected biofilms (log CFU and ODc) are given in Table S2.

3.2.3. Biofilm Production in Tryptone Soya Broth

Clinical isolates produced strong biofilms in TSB at all temperatures. The strongest biofilm at 8 °C was produced by FN187 and F410 (8.67 log CFU, ODc values: 0.9348–1.1036) and the weakest by F710 (6.21 log CFU, ODc value: 0.5442) (Figure 5, Table S3). At 16 °C and 25 °C, all clinical isolates increased production of biofilms compared to 8 °C in the range of 1% to 9% (p < 0.05), except for ATCC, which reduced biofilm production at 16 degrees. The ODc values were high in all biofilms (in comparison with M9 and ABTG) and ranged from ~0.5 to >3.
In Tables S1–S3 (Supplementary Materials), all the numerical values obtained in measurements in all tests, ODcs, and log CFUs, as well as percentage changes in the intensity of biofilm formation in relation to changes in temperature and medium, were presented.

3.2.4. Comparative Presentation of Biofilm Production between M9, ABTG and TSB

Figure 6 shows the individual ability of every isolate to produce biofilms in M9, ABTG, and TSB. Changes in biofilm production in TSB in comparison to M9, and in TSB in comparison to ABTG, were highly significant in all isolates and for all temperatures (p = 0.0001), except for the ATCC 7965.

3.2.5. Results of the Pellicle Test (Air–Liquid Border)

  • Pellicle test in minimal M9 medium
In the minimal M9 medium at 8 °C, the largest number of strains, a total of 6, that successfully formed biofilms on the bottom of polystyrene microtiter plates, had no ability to form biofilms at the liquid–air interface in polystyrene tubes (strains F187, F410, F710, F203, F142, F303). Even at a temperature of 25 °C, seven strains were not able to form a biofilm at the liquid–air interface (strains F187, F410, F710, F142, F303, F262, F458). However, two strains that could not form a biofilm on the bottom of polystyrene plates in minimal M9 medium at 25 °C (F461, F352), successfully formed a relatively strong biofilm at the liquid–air interface.
  • Pellicle test in minimal ABTG medium
At temperatures of 8 °C and 16 °C, most strains produced a good pellicle in ABTG medium, while at 25 °C, 4 strains lost that ability.
  • Pellicle test in TSB medium
At 8 °C in TSB, most of the strains produced a good pellicle except for four strains (FN187, F410, F461, and F710) which produced a weak pellicle. At 16 °C, most strains produced a good pellicle, except strain F142, which lost the ability to produce a pellicle. At 25 °C, almost all strains produced a good pellicle, except strain F461, which lost the ability to produce a pellicle. The detailed results of the pellicle test are shown in Table 1.

3.2.6. Detected Genes Responsible for the Biofilm Production in Analyzed Aeromonas salmonicida subsp. salmonicida Strains

Molecular sequencing was conducted on selected strains (F352, F187, F410, F710, F262, F1413, and F458), and the following genes important for biofilms were found: pfs, luxR, lysR (Type 2 autoinducer (AI-2) system), flgB, flgC, flgE, flgF, flgL, flgM, flgN, flhA, fliE, fliJ, maf2 (Lateral flagellar gene cluster), fliM (polar flagella), flpD, flpJ, flpL, plsB, mshD (Type IV pili), mshI, mshJ, mshK, mshL, mshO, mshP, mshQ (adhesins), vgrG (The type VI secretion system (T6SS), cysB, asnC, nhaR, hfq and LysR family (transcriptional regulators), vapA which is the part of the A450 LPS O-antigen and A-layer gene cluster, helicase genes family (hrpA, recQ), rpoN, arsR (alternative sigma factor σ54), and family of phosphodiesterase associated genes (FOB40_RS00700). Tables including all relevant citations regarding the aforementioned genes’ roles in the creation of the biofilm in the case of Aeromonas genus have been added in the Supplemental Materials. Differences between analyzed strains were found in case of lysR gene and presented in a form of genetic distance between analyzed strains (Table 2). Differences were relatively small and show that studied strains are more closely related to each other than to the reference genome strain, which is also backed up by phylogeny tree (Figure 7) and which suggests that in the case of the studied genes, their roles have not been changed due to the changes in their sequences. In case of the other genes, no significant differences were detected.

4. Discussion

4.1. Optimal Conditions for Aeromonas salmonicida subsp. salmonicida to Form Biofilms

For decades, the professional literature has been asking the question of how the Aeromonas salmonicida subsp. salmonicida maintains in the fast and cold trout pond waters, or in cold, clean rivers, where the complete water change is very intensive and thus unsuitable for the multiplication of pathogenic bacteria [37]. Furunculosis and Aeromonas salmonicida subsp. salmonicida persist in trout ponds despite intensive zoo hygiene measures, application of biocides, desiccation, vaccination, and even antibiotic therapy, but the disease occasionally still breaks out, even during the winter seasons, and causes great economic damage [38]. Thus, Aeromonas salmonicida subsp. salmonicida biofilms may be the explanation for the persistent presence of furunculosis in cold and unfavorable environments. In the doctoral dissertation of Lin [25], the ability of A. salmonicida subsp. salmonicida to produce biofilms in elevated temperatures between 20 ° and 28 °C in the presence of PUFAs (polyunsaturated fatty acids) has been investigated using crystal violet method, but the author added amino acids in minimal M9 (M9 with amino acids is marked as CM9) medium and performed the investigation on only one strain. Other works on this subject were performed on A. salmonicida strains of unknown subspecies, or subspecies other than salmonicida, originating from a wide range of different species of fish, reptiles, amphibians, domestic and wild animals, birds, even humans, being conducted most often at mesophilic temperatures, between 20 ° and 30 °C, mostly in rich nutrient media like Tryptone soya broth, and at the most incubation lasted for 24 to 48 h [16,17,29,39,40,41,42].
In our research, scarce media as minimal M9 without addition of amino acids, with glucose as the only organic source of energy, and ABTG medium, also without amino acids, with a slightly larger selection of salts compared to M9, and also with glucose as the only source of C atoms, has been chosen. Based on our results, it can be said that the optimal temperatures for Aeromonas salmonicida subsp. salmonicida to form biofilm could be defined only in relation to the presence of nutrients. Thus, 8–25 °C were optimal temperatures for Aeromonas salmonicida subsp. salmonicida to form a biofilms in a nutritionally rich environment; 8 °C was optimal in nutritionally poor environment, and 16 °C was the temperature at which only certain strains could form a biofilm in nutritionally poor media. In nutritionally poor media, the largest number of strains of A. salmonicida subsp. salmonicida were not able to form biofilms at 25 °C. It should be emphasized that the in vitro methodology did not include dynamic conditions in fast-flowing waters; it was a static biofilm investigation, but it can be a base for further research in dynamic conditions that correspond to the natural habitat of A. salmonicida subsp. salmonicida.
Several studies have concluded that other bacteria that are cold-adapted in their natural environments create better biofilms in nutrient-poor media than in -rich media [43]. For example, Pseudoalteromonas haloplanktis TAC125 forms much better biofilms (higher biomass, more compact structure, higher number of viable cells in the biofilm, higher amount of eDNA) in biofilms incubated at 15 °C and 0 °C in poor GG medium (the only source of carbon are D-Gluconic acid sodium and L glutamic acid,) than in rich Brain Heart Infusion Broth [44]. At zero degrees, Pseudoalteromonas haloplanktis TAC125 formed a much better biofilm at the solid–liquid interface than a pellicle in GG medium, but at 15 degrees, it formed a better pellicle in GG.
Similarly, Listeria monocytogenes produced a higher biomass of biofilms with a higher number of viable cells in Brain Heart Infusion (BHI) broth diluted 10x than in BHI of normal concentration, at 30 °C [45]. Detected biofilms produced in poor media had a significantly different structure (more compact) than biofilms in rich media, which indicates a different organization of bacteria in stress conditions in order to survive more efficiently.
Even mesophilic pathogens, such as Salmonella enterica serotype Kentucky, for which the optimum temperature for growth is 37 °C, formed a better biofilm at 20 °C than at 37 °C. In addition, the tested salmonellae were able to grow relatively good biofilms even at both 4 °C and 10 °C on both plastic surfaces and silicone [46]. In a similar study, Salmonella Typhimurum and S. Infantis produced stronger biofilms at 20 °C than at 37 °C, with the authors proving in the very same study that the investigated salmonellae at 5 °C produced biofilms almost as good as at 37 °C [47]. The results obtained in our study, as well as the results of other authors that are similar, point to the basic assumption that biofilms are the predominant form of bacterial organization in an external environment [48] that is very often lacking in nutrients and in which low (sometimes extremely low) temperatures prevail for most of the year despite what bacteria survive successfully.

4.2. Reliability of Applied Methods in Aeromonas salmonicida subsp. salmonicida Biofilm Testing

A few authors proposed the formula to calculate the strength of biofilm production based on the obtained ODc values from the crystal violet method, and it is proposed that, based on the results of the calculation, strains should be categorized into strong, moderate, weak biofilm producers, and those that do not produce biofilm [35]. The vast majority of scientific works still apply the crystal violet method, but without this proposed categorization, just simply showing obtained ODc values [19]. In our research, we did not apply the mentioned formulas because the break point values between the biofilm categories still do not exist, so proposed calculations, which are basically arbitrary and give only approximate, descriptive results, could have led us to the wrong conclusion that Aeromonas salmonicida subsp. salmonicida does not produce any biofilm, because of the very low ODc values obtained in M9 and ABTG media. Also, in our research, ODc values oscillated irregularly, especially at low temperatures and in samples with no detected viable cells. From the obtained results, it can be concluded that crystal violet is not a reliable method for testing Aeromonas salmonicida subsp. salmonicida biofilms in nutritionally scarce media, at low temperatures. Lin [25] used crystal violet in PhD research, investigating only one Aeromonas salmonicida subsp. salmonicida strain in CM9 (M9 with added casamino acids), at elevated temperatures (20 °C, 23 °C, and 28 °C), incubated for only 12–24 h, and obtained relatively low ODc values at approx. 0.05 at 20 °C.
However, from the results obtained in our investigation, the direct enumeration of viable cells in the biofilm with the determination of log CFU proved to be a more reliable method, which successfully detected even weak, hardly detectable biofilms in scarce media.

4.3. Molecular Analysis

The bioinformatic analysis evidenced high uniformity, as all genes associated with biofilm were found in all the sequenced strains. Out of the analyzed genes, some play a more important role in the process of biofilm formation, and luxR genes can be regarded as such because it was proven that they are crucial for the quorum sensing which plays a vital role in the mechanisms of biofilm creation [49,50]. However, luxR is not only typical for Aeromonas but also for other genera of bacteria [49,50]. The finding of a group of genes, including fliM, which are responsible for encoding the polar flagella, which is again considered crucial for the initial stages of biofilm formation, is certainly highlighted in our study. The polar flagella is connected with a mechanosensation phenomenon (surface sensing), which is the bacteria’s ability to determine exactly where (how far) the surface is, and how it can approach it and attach itself to it [51]. Again, this factor is not only crucial for biofilms in Aeromonas salmonicida subsp. salmonicida but also in most other motile species, including Pseudomonas aeruginosa, Bacillus subtilis, Vibrio cholerae, V. parahaemolyticus, and P. mirabilis [52]. Although Lin [25] emphasized in a doctoral dissertation that the vapA gene, which encodes the production of the A-monolayer, is extremely important for the molecular differentiation between different isolates of A. salmonicida subsp. salmonicida, in our research, it was shown that all strains are identical based on the genetic sequences of vapA. The sequences (in the translation test) differed in 1 amino acid, which is considered an insignificant finding, on the basis of which it cannot be concluded that the strains had a different origin.
Genetical homogeneity between different strains of Aeromonas salmonicida subsp. salmonicida isolated across Bavaria and their similarity to the reference genome cannot explain the strong differences in the ability to produce biofilms between the tested strains, and this strongly emphasizes the important role of epigenetics, and thus gene expression, in the adaptation and biofilm creation processes of this pathogen.

4.4. Future Perspectives and Possible Applicability of the Obtained Results

As previously said, these are the results of a fundamental microbiological examination of the ability of clinical isolates of A. salmonicida subsp. salmonicida to produce biofilms in different (scarce) nutritional media and at low temperatures. It can be seen that the strains show different (individual) abilities to produce biofilm under different conditions and therefore the results cannot be generalized. However, in order to clarify the possible applicability of the obtained results, the following can generally be said: (a) At 25 °C, the largest number of isolates did not have the ability to produce biofilms in minimal media (inorganic salts, glucose as the only source of C atoms). Hypothetically, this would mean that in conditions of increased (summer) temperatures, it is most important to keep pond waters maximally clean, without organic matter pollution as much as possible, to avoid biofilm formation. (b) At temperatures of 16 °C, it was shown that the tested isolates are capable of forming biofilms on the surfaces (air–liquid interface) in media with increased inorganic salt concentration (ABTG) and with the addition of iron (FeCl3). In essence, it is very difficult to control the amount of iron in fish pond waters, and according to the literature data, there is always a lot of iron in fish ponds. It is well known that iron plays an important role for the virulence, including the formation of biofilms, in many types of bacteria, including members of the Aeromonas genus [53]. Reducing available iron is a good defensive option against all bacterial pathogens. Some authors suggest increased aeration of ponds to eliminate iron, but in essence, new iron control strategies in pond waters should be applied as a way of fighting against the maintenance of A. salmonicida subspecies salmonicida [54]. Also, water exchange should be accelerated, because pellicle is difficult to form in highly dynamic conditions. (c) At all temperatures in the TSB medium (rich medium) all isolates formed strong biofilms, which means that organic pollution is a crucial factor that enables biofilm formation in A. salmonicida subsp. salmonicida in fish ponds. (d) At low temperatures, almost all isolates showed an extraordinary ability to form biofilms at the bottom of wells in extreme, minimal nutritional conditions, which indicates the maintenance of this pathogen in the cold months and in clean (without organic pollutants) and dynamic waters.
All this points to the necessity of applying an innovative pond-disinfection strategy. The disinfection protocol must be aimed not at a one-time, simple disinfection but at multiple times with the use of disinfection protocols and biocides that are proven to be able to eradicate biofilms.

5. Conclusions

Under nutrient-poor conditions, the optimal temperature for the biofilm production was 8 °C for the majority of clinical isolates of A. salmonicida subsp. salmonicida. This can explain the successful maintenance of A. salmonicida subsp. salmonicida in clean and fast waters during the cold months. In nutrient-poor conditions, most clinical isolates of A. salmonicida subsp. salmonicida are not capable of producing biofilms at 25 °C. This indicates the need for fish ponds to remain with as little organic pollution as possible during the summer months in order to avoid the formation of biofilms. The temperature of 16 °C was optimal for almost all clinical isolates of A. salmonicida subsp. salmonicida for producing good biofilms at the liquid–air interface (pellicle) in poor media but with an increased concentration of iron. This feature may indicate the need to reduce free iron in the pond waters, as well as a quick water change in order to prevent the formation of biofilms on the surface. The temperature of 25 °C was the optimal temperature for the production of biofilms in rich media for the majority of clinical A. salmonicida subsp. salmonicida strains. Due to too low and highly oscillating ODc values that can lead to a false assessment about the biofilm production, the crystal violet is not a suitable method for examining biofilms in A. salmonicida subsp. salmonicida in scarce nutritional media at low temperatures. Although the clinical isolates are phylogenetically close, they showed significant differences in the production of biofilms in different environmental conditions, which can be attributed to epigenetic factors. Further investigations are needed to establish the causes for the different expression of biofilm formation in A. salmonicida subsp. salmonicida at different temperatures.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app14209365/s1, Table S1: Comparison of viable bacterial cells count (log CFU) and optical density (ODc) at 550 nm of biofilm biomass in M9 produced by A. salmonicida subspecies salmonicida (isolates are in order from highest to lowest log CFU value at 8 °C); Table S2: Comparison of viable bacterial cells count (log CFU) and optical density (ODc) at 550 nm of biofilm biomass in ABTG produced by A. salmonicida subspecies salmonicida (isolates are in order from highest to lowest log CFU value at 8 °C); Table S3: Viable bacterial cells count (log CFU) and optical density (ODc) at 550 nm of biofilm biomass in TSB produced by A. salmonicida subspecies salmonicida (isolates are in order from highest to lowest log CFU value at 8 °C); Table S4: List of genes with importance for the biofilm formation in Aeromonas salmonicida subspecies salmonicida. Genes marked with the bolded letters were investigated in this study.

Author Contributions

Conceptualization and experimental design, D.M.; analysis, D.M., K.A., D.P., K.W., A.D.R., P.C. and M.K.; internal funding acquisition, D.M.; investigation, D.M., K.A., D.P., K.W. and P.S.; methodology, D.M., D.P., K.W. and P.C.; internal project administration, M.K.; writing—original draft, D.M., K.A. and A.D.R.; writing—review and editing, M.K., D.P. and K.W.; sample collection, K.W., D.P. and P.S. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Narodowe Centrum Nauki, (Poland), grant number 2019/35/B/NZ9/02774 and MABVAC project by Bavarian StMELF (A/20/12), Bavaria, Germany.

Institutional Review Board Statement

Ethical opinion and approval were not needed for this study: samples from investigations of clinical cases were obtained following routine fish diseases diagnostics protocols by accredited veterinarians. Collected samples were stored as per requirements from Bavarian Animal Health Service and were anonymized prior to subsequent evaluation within the scope of this study. Therefore, the use of anonymized clinical isolates is not a subject to approval or reporting to the Ethics Commission for the experimental animals’ welfare protection of the Government of Upper Bavaria, the responsible authority.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Geographical locations (red dots) of fishponds from where samples of diseased fish were taken for this study.
Figure 1. Geographical locations (red dots) of fishponds from where samples of diseased fish were taken for this study.
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Figure 2. Scheme of measuring the strength of the created pellicle (biofilm at the liquid–air interface) in polystyrene test tubes.
Figure 2. Scheme of measuring the strength of the created pellicle (biofilm at the liquid–air interface) in polystyrene test tubes.
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Figure 3. Biofilm production of clinical A. salmonicida subsp. salmonicida in minimal M9 after 7 days. Legend: ODc (columns)—measured optical density of formed biofilms; log CFU (lines)—number of viable bacteria in biofilms; F203, F262, F303, F352, F410, F458, F461, F710, FN1413, FN187—clinical isolates. The isolates are arranged in order from the strongest (left) to the weakest (right) biofilm producer (based on log CFU obtained at 8 °C).
Figure 3. Biofilm production of clinical A. salmonicida subsp. salmonicida in minimal M9 after 7 days. Legend: ODc (columns)—measured optical density of formed biofilms; log CFU (lines)—number of viable bacteria in biofilms; F203, F262, F303, F352, F410, F458, F461, F710, FN1413, FN187—clinical isolates. The isolates are arranged in order from the strongest (left) to the weakest (right) biofilm producer (based on log CFU obtained at 8 °C).
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Figure 4. Biofilm production of clinical A. salmonicida subsp. salmonicida in minimal ABTG after seven days. Legend: ODc (columns)—measured optical density of formed biofilms; log CFU (lines)—number of viable bacteria in biofilms; F203, F262, F303, F352, F410, F458, F461, F710, FN1413, FN187—clinical isolates. The isolates are arranged in order from the strongest (left) to the weakest (right) biofilm producer based on log CFU obtained at 8 °C.
Figure 4. Biofilm production of clinical A. salmonicida subsp. salmonicida in minimal ABTG after seven days. Legend: ODc (columns)—measured optical density of formed biofilms; log CFU (lines)—number of viable bacteria in biofilms; F203, F262, F303, F352, F410, F458, F461, F710, FN1413, FN187—clinical isolates. The isolates are arranged in order from the strongest (left) to the weakest (right) biofilm producer based on log CFU obtained at 8 °C.
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Figure 5. Biofilm production of clinical A. salmonicida subsp. salmonicida in TSB after seven days. Legend: ODc (columns)—measured optical density of formed biofilms; log CFU (lines)—number of viable bacteria in biofilms; F203, F262, F303, F352, F410, F458, F461, F710, FN1413, FN187—clinical Aeromonas salmonicida subsp. salmonicida isolates. The isolates are arranged in order from the strongest (left) to the weakest (right) biofilm producer based on log CFU obtained at 8 °C.
Figure 5. Biofilm production of clinical A. salmonicida subsp. salmonicida in TSB after seven days. Legend: ODc (columns)—measured optical density of formed biofilms; log CFU (lines)—number of viable bacteria in biofilms; F203, F262, F303, F352, F410, F458, F461, F710, FN1413, FN187—clinical Aeromonas salmonicida subsp. salmonicida isolates. The isolates are arranged in order from the strongest (left) to the weakest (right) biofilm producer based on log CFU obtained at 8 °C.
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Figure 6. The ability of each tested A. salmonicida subsp. salmonicida clinical strain to produce biofilms in different media: a comparative view. Legend: M9, ABTG—minimal nutrient media; TSB—rich nutrient medium.
Figure 6. The ability of each tested A. salmonicida subsp. salmonicida clinical strain to produce biofilms in different media: a comparative view. Legend: M9, ABTG—minimal nutrient media; TSB—rich nutrient medium.
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Figure 7. Phylogenetic tree representing the genetic distance between tested samples based on WGS analysis.
Figure 7. Phylogenetic tree representing the genetic distance between tested samples based on WGS analysis.
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Table 1. Results of the pellicle test (biofilm production at the liquid–air border) in polystyrene tubes.
Table 1. Results of the pellicle test (biofilm production at the liquid–air border) in polystyrene tubes.
Pellicle Test at 8 °CPellicle Test at 16 °CPellicle Test at 25 °C
StrainM9ABTGTSBM9ABTGTSBM9ABTGTSB
FN187−++−++++−++++
F410−++++++++++−−+++
F461++++++++++++++−
F710−++++++++−−+++
F203−−+++−+++++++−+++
F142−++++++−−+++
F303−++++−++++−+++++
F262++++−+++++−+++++
F458+−+++++++++−+++++
F352+++++++−−+++++−+++
Legend: Strong biofilm producers—strains that form a solid, stable, thicker pellicle of 1 mm (+++), moderate biofilm producers—strains that form a stable, medium pellicle thickness up to 1 mm (++), weak biofilm producers—very thin pellicle, (thinner than 1 mm) (+), strains that do not produce biofilm—strains that do not produce a visible pellicle (−).
Table 2. Genetic distance between analyzed strains on basis of lysR gene.
Table 2. Genetic distance between analyzed strains on basis of lysR gene.
Investigated
Isolates/Strains		Reference Strain GCA_000196395.1F352F187F410F710F262F1413F458
Reference strain GCA_000196395.1 0.460.480.120.480.460.480.46
F3520.46 0.130.040.130.000.130.00
F1870.480.13 0.040.000.130.000.13
F4100.120.040.04 0.040.040.040.04
F7100.480.130.000.04 0.130.000.13
F2620.460.000.130.040.13 0.130.00
F14130.480.130.000.040.000.13 0.13
F4580.460.000.130.040.130.000.13
The strains in regard to vapA are totally identical, even with the whole reference genome (GCA_000196395.1) but differed in one amino acid from the standalone reference gene for vapA (KR704893.1). The overall whole-genome similarity between analyzed strains was found to be in the range of 95–98%, and the GC% content was in the range 58–60%, which corresponds with the standard values for this subspecies.
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Aksentijević, K.; Rajewska, A.D.; Wojnarowski, K.; Cholewińska, P.; Korzeniowska, M.; Steinbauer, P.; Palić, D.; Misic, D. Biofilm Production Capability of Clinical Aeromonas salmonicida Subspecies salmonicida Strains under Stress Conditions. Appl. Sci. 2024, 14, 9365. https://doi.org/10.3390/app14209365

AMA Style

Aksentijević K, Rajewska AD, Wojnarowski K, Cholewińska P, Korzeniowska M, Steinbauer P, Palić D, Misic D. Biofilm Production Capability of Clinical Aeromonas salmonicida Subspecies salmonicida Strains under Stress Conditions. Applied Sciences. 2024; 14(20):9365. https://doi.org/10.3390/app14209365

Chicago/Turabian Style

Aksentijević, Ksenija, Aleksandra Daria Rajewska, Konrad Wojnarowski, Paulina Cholewińska, Malgorzata Korzeniowska, Peter Steinbauer, Dušan Palić, and Dusan Misic. 2024. "Biofilm Production Capability of Clinical Aeromonas salmonicida Subspecies salmonicida Strains under Stress Conditions" Applied Sciences 14, no. 20: 9365. https://doi.org/10.3390/app14209365

APA Style

Aksentijević, K., Rajewska, A. D., Wojnarowski, K., Cholewińska, P., Korzeniowska, M., Steinbauer, P., Palić, D., & Misic, D. (2024). Biofilm Production Capability of Clinical Aeromonas salmonicida Subspecies salmonicida Strains under Stress Conditions. Applied Sciences, 14(20), 9365. https://doi.org/10.3390/app14209365

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