Resistance of the examined bacterial strains to various antibiotics is presented in Table 2. The obtained results showed that about 20–40% of the analyzed strains were resistant to 14 antibiotics, while < 20% of the strains were resistant to 20 antibiotics. The bacterial strains were also analyzed for multiple antibiotic resistance (MAR). Among the tested strains, Paenibacillus azoreducens, resistant to 35 of the investigated antibiotics (all tested antibiotics) belong to eight various chemical classes. Pseudomonas fragi, Stenotrophomonas rhizophila, and Sphingobacterium multivorum were resistant to 22, 21, and 17 antibiotics, respectively. About 20% of the tested bacteria showed a 2–5 MAR (i.e., resistance from 2 to 5 antibiotics of the 37 antibiotics tested), while 47% were resistant to more than 5 antibiotics. Detailed description of the antibiotic susceptibility of bacteria isolated from onsite WWT facilities is presented by Jałowiecki et al. .
Apart from the evaluation of antibiotic susceptibility profile of bacteria, the ability of selected strains to develop a biofilm on polystyrene microtiter dishes was assessed by using the CV method. This method allows to indirectly quantify the attached bacterial biomass (live and dead cells, polysaccharides). Obtained results showed that most of the analyzed strains (19) from total 30 were able to form biofilm after 72 h of incubation (16/30 after 24 h; Table 3; Fig. 1). Interestingly, Pseudomonas chlororaphis ss aurantiaca strain was able to produce biofilm only after 24 h of incubation. For example, Pseudomonas fulva, Mycobacterium maritypicum, Aeromonas bestiarum, Carnobacterium divergens, and Stenotrophomonas rhizophila strains were classified as strong biofilm producers. Streptococcus australis, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Paenibacillus azoreducens, and Enterococcus haemoperoxidus, for example, were unable to form biofilm even after 72 h of incubation. In Table 3, the relation between biofilm formation and surface activities measured by surface tension is presented. The low values of surface tension in range between 28 and 36 mN/m were observed in the bacterial strains which were not able to produce biofilm or be classified as weak biofilm producers, for example, S. australis, P. fluorescens, S. maltophilia, P. chlororaphis ss aurantiaca, CDC group II-H, Flavobacterium resinovorum, Mycobacterium brumae, Bacillus horti, Acinetobacter johnsonii, Enterococcus haemoperoxidus, Serratia marcescens ss marcescens.
The dispersal properties of biosurfactants have been shown to rival those of conventional inhibitory agents against bacterial and yeast biofilms. This makes them suitable candidates for use in new generations of microbial dispersal agents and for use as adjuvants for existing microbial suppression or eradication strategies . New insights into biofilm physiology have now enabled researchers to design more effective bacterial inhibition/ dispersal strategies. There are two main inhibitory strategies, based on the formulation of new antibiofilm compounds and the construction of biofilm-resistant surfaces . Some of the most promising candidates for the inhibition of bacterial biofilms have come from biological surface-active agents (biosurfactants), since they are characterized by strong anti-adhesive, antimicrobial, and biofilm disruption properties [2, 20]. Many authors demonstrated the abilities of biosurfactants to disperse a biofilm of pathogenic microbial species by decreasing the cells viability and reducing the bacterial adhesion properties. The possible mechanism of such actions is related to binding of the biosurfactants molecules to cell wall components or its surface, which results in severe changes in outer membrane hydrophobicity. The insertion of surfactants into bilayer structure of cell membrane may result in disruption of its integrity. As a response to the increasing concentration of fengycin, the reorganization of membrane lipids into bilayer discs composed of fengycin micelles and lipids from membrane has been reported. The negative influence of biosurfactants on both Gram-negative and Gram-positive strains is related to the releasing of LPS molecules from the outer membrane or to the formation of transmembrane pores, which results in increased permeability of the cell wall, respectively [23, 30]. Satputea et al.  discuss the various roles of biosurfactants molecules in association with biofilm formation.
Literature data about biofilm formation capabilities of onsite wastewater technology microorganisms are very scarce, although application of biofilms in removal, bioremediation or biotransformation of organic pollutants, heavy metals, pharmaceutical, or personal care products (PPCPs) is well documented . Microorganisms in biofilms are characterized by higher tolerance towards harsh environmental conditions compared with their free-floating counterparts. Van Houdt and Michiels  noticed that biofilm formation process depends on several factors such as the bacterial surface, the surface for attachment, and surrounding medium. In recent years, bacterial biofilms have been widely utilized to degrade, neutralize, and mineralize various contaminations in wastewater-activated sludge or as recently in onsite wastewater technology. Microorganisms in communities are able to persist in different metabolic states, which increase efficiency of xenobiotics degradation. Moreover, multicellular and porous structure of multispecies biofilm allow for nutrients transport or accumulation of metabolites from the environment. Bacteria in sessile mode of growth are also able to communicate through quorum sensing (QS) and to exchange genetic material. As Edwards and Kjellerup  point out in the case of several PPCPs removal, e.g., non-steroidal anti-inflammatory drug diclofenac, lipid regulator gemfibrozil, and chemotherapeutic agent trimethoprim, membrane biofilm reactor was more efficient than traditional activated sludge. It is widely known that occurrence and fate of antibiotics in the environment may lead to the selection of antibiotic-resistant bacteria (ARB) [24, 26]. As many authors highlight, WWTPs often constitute the main source of pharmaceuticals released into the environmental matrices. Conn et al.  reported about the presence of 30 different organic wastewater contaminants (OWCs) including endocrine-disrupting compounds, antimicrobial agents, heavy metals, or disinfectants. On the other hand, Stanford et al.  reported about excellent removal of nonylphenols, estrone (E1), 17β-estradiol (E2), estriol (E3), and 17α-ethinylestradiol (EE2) from five onsite wastewater systems with aerobic and anaerobic sand filters. Besides this, development of more effective technologies, which will prevent the propagation of antibiotic-resistant bacteria and antibiotic-resistance genes, is still necessary. A promising alternative for removal of antibiotics are bioremediation methods with the use of bacterial strains able to degrade xenobiotically. Simultaneously, many authors suggest that immobilization of bacterial consortia on both organic and inorganic carriers increases degradation capabilities and provides protection against harsh environmental conditions, e.g., acidification, heavy metals, or high osmotic pressure. One of the crucial factors for immobilization of bacteria is biofilm formation ability. Characterization and determination of several bacterial features like cell wall properties or autoaggregation are also valuable for effective whole cell immobilization and degradation studies.
All examined strains exhibited varied autoaggregation index increasing with the time of incubation (Table 4). Among biofilm-forming strains, the highest autoaggregation index was observed for M. brumae and Bacillus alcalophilus. Noteworthy, that some strains are able to form biofilm, e.g., A. johnsonii, Streptococcus criceti, and P. fulva showed, no aggregation abilities were characterized by low autoaggregative properties, e.g., S. rhizophila, B. horti, B. alcalophilus, Variovorax paradoxus, P. fulva, C. divergens, or Flavobacterium tirrenicum. Strong autoaggregating phenotype was observed also for Microbacterium flavescens, Microbacterium maritypicum (Fig. 2a), CDC group II-H, and A. bestiarum (Fig. 2b) strains. Beside this, the results of visual autoaggregation assay showed no visible flocs after given time of incubation. Simoes et al.  reported that some species of bacteria are not able to form flocs without the presence of other bacteria species. Aggregation is also considered as one of the essential steps in communication among microorganisms and ecological interactions, e.g., adaptation and succession, which lead to colonization and subsequent biofilm formation . In natural settings, multispecies biofilm is primarily a mode of bacterial growth, where auto- and coaggregation processes mediate the formation of multicellular matrix and juxtapositioning of bacteria near to taxonomically favorable species present within the biofilm. In recent years, the role of initial attachment and bacterial aggregation in biofilm formation has been increasingly highlighted, since autoaggregation and coaggregation capabilities of bacteria facilitated the attachment to inert and biotic surfaces. Involvement of bacterial surface components, particularly EPSs, LPSs, outer membrane proteins (OMPs), and flagella, in combination with microbial signals in autoaggregation and biofilm formation processes has been widely documented . Moreover, the absence of biofilm-associated structures indicates that cell hydrophobicity and auto- or coaggregation properties are crucial factors responsible for bacterial adherence . A positive correlation between autoaggregation and biofilm formation abilities was demonstrated, e.g., for Sinorhizobium meliloti strains  isolated from root nodules of alfalfa plants and Myroides odoratus isolated from fish Oreochromis mossambicus . Moreover, obtained results indicated that both phenomena were dependent on the same adhesive forces. Similar observations were made by Kos et al.  which observed the strong relationship between adhesion and aggregation ability of probiotic Lactobacillus acidophilus M92 strains. On the other hand, Basson et al.  observed no correlation between autoaggregation and biofilm formation for 29 Flavobacterium johnsoniae-like isolates. In this study, weak correlations were observed between biofilm formations after 24, 48, and 72 h of incubation and 2 h of autoaggregation (P = 0.05; r = 0.13; r = 0.21; r = 0.09), respectively. Similarly, weak or negative correlations were observed also between 24, 48, and 72 h of incubations and 24 h of autoaggregation (P = 0.05; r = 0.02; r = − 0.033; r = − 0.74), respectively. Aggregation of bacteria is one of the essential processes, which plays an important role in both biofilm formation and various ecological interactions. Microorganisms in biofilms are characterized by physiological and structural heterogeneity and diverse gene expression. Increased tolerance of biofilms to various environmental pollutions or toxic compounds and their ability to immobilize ensure higher degradation and accumulation capacity of biofilms compared with planktonic cells. Motility of bacteria, dependent on the presence of flagella or pili, is one of the crucial factors which mediate the adherence of bacteria to different surfaces and hence biofilm formation.
The results from motility test demonstrated that most of the analyzed strains (27/30), except Chryseobacterium balustinum, Paenibacillus azoreducens, and Sphingobacterium multivorum, were motile. In Fig. 3, 12 bacterial strains with different motility properties are presented (Fig. 3). Surface-associated structures such as pili and flagella are considered as important factors involved in bacterial motility. Lack of biofilm-forming capabilities in non-motile bacterial mutants is well documented . Our results also confirmed those findings; all three non-motile bacterial strains simultaneously were not able to from biofilm. Flagellar motility is one of the essential factors for initial contact between the bacterial cell and surface and biofilm formation capabilities under static culture conditions for several bacterial strains, e.g., Escherichia coli, Listeria monocytogenes, and Yersinia enterocolitica [1, 11]. Van Houdt and Michiels  reported that flagella may influence the attachment and biofilm formation via several different mechanisms due to the involvement in reaching the surfaces, facilitating growth and spread of a maturing biofilm, and finally flagella act as cell wall appendages mediating directly the attachment of bacteria to biotic and abiotic surfaces.