Fish Probiotics: Cell Surface Properties of Fish Intestinal Lactobacilli and Escherichia coli

The properties of intestinal bacteria/probiotics, such as cell surface hydrophobicity (CSH), auto-aggregation, and biofilm formation ability, play an important role in shaping the relationship between the bacteria and the host. The current study aimed to investigate the cell surface properties of fish intestinal bacteria and probiotics. Microbial adhesion to hydrocarbons was tested according to Kos and coauthors. The aggregation abilities of the investigated strains were studied as described by Collado and coauthors. The ability of bacterial isolates to form a biofilm was determined by performing a qualitative analysis using crystal violet staining based on the attachment of bacteria to polystyrene. These studies prove that bacterial cell surface hydrophobicity (CSH) is associated with the growth medium, and the effect of the growth medium on CSH is species-specific and likely also strain-specific. Isolates of intestinal lactobacilli from fish (Salmo ischchan) differed from isolates of non-fish/shrimp origin in the relationship between auto-aggregation and biofilm formation. Average CSH levels for fish lactobacilli and E. coli might were lower compared to those of non-fish origin, which may affect the efficiency of non-fish probiotics use in fisheries due to the peculiarities of the hosts’ aquatic lifestyles.


Introduction
Fish are an important component of aquaculture. The productivity of fish aquaculture is most dependent on the effective control of emerging fish diseases, which, in recent decades, has led to the widespread utilization of antimicrobials. This will eventually lead to the development and spread of antimicrobial-resistant pathogens [1][2][3]. Therefore, there is an urgent need to develop alternative methods for combating fish pathogens, reducing the accumulation of antibiotic residues in fish meat, and other related environmental problems [4]. Such methods include phage [5,6] and probiotic therapies [7][8][9][10][11][12]. According to the International Scientific Association for Probiotics and Prebiotics, probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host (https://isappscience.org/for-scientists/resources/probiotics) (accessed on 17 November 2019) [13]. The host might be a human [14][15][16], animal [17], plant, or soil [18]. In addition to lactobacilli, Escherichia coli (E. coli) are widely used as probiotics [19][20][21][22]. various local fish farms in different regions of Armenia (Gegharkunik, Ararat, and Armavir regions) were investigated. The samples were received from humanely euthanized fish. The isolates from the most diluted samples were Escherichia coli and lactobacilli. One Enterococcus strain was found among the predominant cultivable fish isolates. Fish from the farms were transported in thermal bags to the ANAU laboratory and processed within 2 h of acquisition. The entire intestines were isolated according to Floris and coauthors [55]. Samples were added to 0.1% (w/v) peptone and incubated at 37 • C overnight to enrich the number of microbes. Overnight samples were serially diluted 10-fold, then spread on deMan Rogosa and Sharpe agar (MRS) (Thermo Scientific Oxoid, Waltham, MA, USA) for the detection and enumeration of Lactobacillus and on Endo agar (Thermo Scientific Oxoid, Waltham, MA, USA) for the isolation and differentiation of E. coli. Plates were incubated at 37 • C for 24 h.
A total of 15 commensal E. coli and 5 Lactobacillus spp. isolates, all randomly selected and morphologically different, and Enterococcus spp. were investigated.

Identification of Fish Lactobacilli and E. coli Isolates
The bacterial isolates were characterized based on gram staining, morphology observation according to Bergey's manual of determinative bacteriology, and further confirmation by PCR. The isolates were cultured in Luria Bertani Broth, Miller (HiMedia, Maharashtra, India), at 37 • C for 24 h, and bacterial genomic DNA was extracted using QIAamp DNA Micro Kit (Qiagen, Hilden, Germany).

Probiotic Strains
Probiotic bacterial strains of fish/shrimp origin from the microbial collections of the Southern Federal University of Russia (Bacillus subtilis str. 1R, Bacillus amyloliquefaciens str. 4R, Bacillus amyloliquefaciens str. 5R and Bacillus cereus str. 6R) and the Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan (Lactococcus str. UZ-1, Lactiplantibacillus plantarum str. R3, Lactococcus str. UZ-2, Enterococcus faecium str. R2 and Pediococcus acidilactici str. N) were used in this study. The cell surface properties of biofilm formation ability, cell surface hydrophobicity, and auto-aggregation abilities were not assessed for the above-mentioned probiotics before the current investigations.
Probiotic strains of human/sheep/milk origin from the bank of the International Association for Human and Animals Health of Armenia (Lacticaseibacillus rhamnosus str. Vahe, Lactiplantibacillus plantarum str. ZPZ, Lacticaseibacillus rhamnosus str. E5-2, Lactiplantibacillus plantarum str. K1-3, E. coli str. ASAP-1 and E. coli str. ASAP 2-1) and Vitamax LLC, Armenia (L. acidophilus Er-2 str. 317/402 from the commercial probiotic "Narum Caps", L. acidophilus Er-2 str. 317/402 from the commercial synbiotic "NARUM CAPS FAST" and commercial synbiotic "NARUM TAB", https://mynarum.com/ (accessed on 28 December 2021)) were also used. For studying the effect of growth medium on bacterial cell surface properties, bacterial cultures were also grown in a mixed medium containing MRS and LB in a ratio of 1:3. Based on several trials, a 1:3 (MRS and LB) mixed medium was chosen as it supported the growth of all tested bacteria.

Cell
Cultures were centrifuged (1165× g for 15 min), washed twice, and resuspended in sterile phosphate-buffered saline (PBS, pH 7) to an optical density matching a 0.5 MacFarland standard (OD 600 ) to standardize the bacterial density at 10 8 CFU/mL. The OD 600 of each bacterial suspension (BS) was measured using a spectrophotometer (Stat Fax 3300, Awareness Technology, Palm City, FL, USA).

Cell Surface Hydrophobicity
Microbial adhesion to hydrocarbons was tested according to Kos and coauthors [56] with a slight modification: xylene was used as the hydrocarbon solvent in this test. Bacterial cultures were adjusted to optical density OD 600 = 0.5 (the number of Lactobacillus sp. and Escherichia sp. is 10 11 bacteria/mL of culture medium, and the number of Bacillus sp. is 10 8 bacteria/mL). Then, 1 mL of xylene was added to 1 mL of the bacterial suspension. After a 10 min incubation at room temperature, the two-phase suspension was mixed by vortexing for 2 min and incubated for an additional 20 min at room temperature. The hydrocarbon layer was removed completely, and the absorbance of the aqueous-phase cell suspension was measured at 600 nm (Stat Fax 3300, Awareness Technology). The cell surface hydrophobicity (CSH) was expressed as a percentage using the following formula: where ODB and ODA are the absorbances of the bacterial suspension before and after mixing with hydrocarbon, respectively.

Auto Aggregation Study
The aggregation abilities of the investigated strains were determined as described by Collado and coauthors [57]. The optical density (OD 600 ) of the homogenized bacterial suspension was recorded and then monitored again, 2 and 24 h after incubation at 37 • C under static conditions. The percent of aggregation was evaluated as follows: where A time represents the absorbance of the mixture at 2 and 24 h, and A 0 is the absorbance at the starting point.

Biofilm Formation
The ability of bacterial isolates to form a biofilm was determined by performing a qualitative analysis using crystal violet staining based on the attachment of bacteria to the surface of polystyrene [58]. Specifically, 200 µL of bacterial suspensions (OD 600 = 0.5), incubated overnight, was transferred into a polystyrene 96-well plate (Biomat, Ala, Italy) and incubated for 48 h at 37 • C. Then, 25 µL of 0.5% crystal violet was added to each well, and the plates were incubated for 15 min at room temperature. Next, the wells' contents were aspirated, and the empty wells were washed 3 times with PBS. Crystal violet extraction was performed using 96% ethanol, and biofilm formation abilities were evaluated photometrically at 540 nm (Stat Fax 2100, Awareness Technology, Perchtoldsdorf, Austria).

Statistical Analysis
All data obtained from the five independent experiments are expressed as mean ± standard deviation (SD). A t-test was performed at a 95% confidence interval in order to determine the statistical significance (p < 0.05). The results were confirmed by the Mann-Whitney test. The impact of growth medium on bacterial cell surface hydrophobicity was also evaluated by the chi-squared test, and Pearson's correlation statistics were applied to describe correlations between the bacterial membrane characteristics (excel 2016).

Bacteria Grown in MRS/LB Mixed Growth Medium
There were no significant differences in the levels of cell surface hydrophobicity between the fish probiotic strains of Bacillus and LAB origins (p > 0.05). According to Table 1, the levels of cell surface hydrophobicity of the investigated probiotic LAB and Bacillus spp. from the fish and shrimp gut microbiota and of the fish LAB isolates were statistically lower than that of the lactic acid probiotics of human/sheep/milk origin (0.14 ± 0.4% vs. 8.5 ± 6.7%, p < 0.05) ( Table 1). There were also no differences between the cell surface hydrophobicity levels of fish LAB and Bacillus isolates/probiotics and fish LAB and Bacillus isolates (0.14 ± 0.4% vs. 0.15 ± 0.56%, p > 0.05) ( Table 1).
As expected, the levels of cell surface hydrophobicity of the fish E. coli isolates were lower than those of the probiotics of human origin (0.01 ± 0.05% vs. 4.5 ± 2.4%, p < 0.05 (Table 1) (usually to screen probiotics, the hydrophobicity/biofilm formation ability is taken into account [46]).

Bacteria Grown in LB and MRS Growth Media
According to Table 1, the levels of cell surface hydrophobicity of the investigated probiotic LAB and Bacillus spp. isolated from the fish/shrimp gut microbiota and of the fish LAB isolates were statistically lower than those of the lactic acid probiotics of human/sheep/milk origin (1.11 ± 2.8% vs. 6.7 ± 8.25%, p < 0.05) ( Table 1). There were no differences between the cell surface hydrophobicity levels of fish LAB and Bacillus isolates/probiotics and fish LAB and Bacillus isolates (1.11 ± 2.8% vs. 2.39 ± 3.9%, p > 0.05) ( Table 1).
As expected, the levels of cell surface hydrophobicity of the fish E. coli isolates were lower than those of the E. coli probiotics from the gut microbiota of non-fish origin (1.07 ± 2.4% vs. 13.9 ± 4.8%, p < 0.05) ( Table 1). Additionally, the levels of cell surface hydrophobicity of the fish E. coli isolates were lower than those of sheep isolates with the lowest cell surface hydrophobicity of the non-fish isolates (1.07 ± 2.4% vs. 5.17 ± 1.15%, p < 0.05) ( Table 1). Overall, the average levels of cell surface hydrophobicity for the fish lactobacilli and E. coli were lower than those of non-fish origin (Table 1). E. coli str. ASAP-1 and E. coli str. ASAP 2-1) and Vitamax LLC, Armenia (L. acidophilus Er-2 str. 317/402 from the commercial probiotic "Narum Caps", L. acidophilus Er-2 str. 317/402 from the commercial synbiotic "NARUM CAPS FAST" and commercial synbiotic "NARUM TAB", https://mynarum.com/ (accessed on 28 December 2021)) were also used. * Predominated gut isolates from the sheep with the lowest cell surface hydrophobicity [59]. p 1 -comparison with the human/sheep/milk LAB probiotics (and Bacillus strains). p 2 -comparison of isolates and all of fish/shrimp LAB and Bacillus isolates. p 3 -comparison of fish isolates and probiotics of human origin (E. coli). p 4 -comparison of fish isolates and isolates of non-fish origin.
There were no significant differences in the levels of cell surface hydrophobicity between the fish E. coli and LAB isolates and the Bacillus and LAB probiotics (p > 0.05) ( Table 1). However, we did not take into account the cell surface properties of candidate probiotics when screening fish-, Bacillus-, and LAB-origin probiotics from the fish bacteria, which, perhaps, may have affected the conclusion concerning fish probiotics. LAB and E. coli isolated from the gut of Salmo ischchan had lower CSH levels than gut bacteria of non-fish/shrimp origin.

Biofilm Formation Ability
The results on bacterial biofilm formation abilities are given in Table 3. In contrast to the data obtained on cell surface hydrophobicity, biofilm formation abilities were significantly different for the fish probiotic strains of Bacillus and LAB origin (1.93 ± 0.87 D vs. 0.139 ± 0.035 D, p < 0.05). The average of fish Bacillus biofilm formation abilities was also higher than that of the LAB probiotics of non-fish origin (1.93 ± 0.87 D vs. 0.169 ± 0.01 D; p < 0.05). Moreover, according to Table 3, no significant differences were found in the fish LAB probiotics in comparison with the human/sheep/milk probiotics in terms of biofilm formation ability (0.139 ± 0.02 D vs. 0.169 ± 0.01 D, p > 0.05). Table 3. Comparative characteristics of biofilm formation ability X of lactic acid bacteria and E. coli isolated from the fish gut microbiota, D average ± standard deviation.  4ˆF ifteen commensal E. coli and five lactobacilli isolates from the fish gut microbiota, all randomly selected and morphologically different, were used in this study. p 1 -comparison with the fish Bacillus probiotics. p 2comparison of fish/shrimp LAB and non-fish/shrimp origin LAB probiotics. p 3 -comparison of fish/shrimp LAB isolates and fish/shrimp LAB probiotics. p 4 -comparison of fish isolates and probiotic isolates of non-fish origin. p 5 -comparison of fish E. coli isolates and isolates of non-fish origin.

Bacteria
Simultaneously, there were no differences between the bacterial biofilm formation abilities of fish LAB probiotics and fish LAB isolates (0.139 ± 0.035 D vs. 0.228 ± 0.07 D; p > 0.05) ( Table 3). Even though fish E. coli isolates were statistically different from humanorigin probiotic E. coli strains (average biofilm formation ability of all fish E. coli isolates vs. average biofilm formation ability of probiotic E. coli strains, as 0.24 ± 0.5 D vs. 1.02 ± 0.26 D; p < 0.05) (Table 3), the biofilm formation ability was evaluated to be the same as that of non-fish origin isolates of E. coli. An exception was found for one isolate, whose biofilm formation ability was statistically higher compared to other fish E. coli (1.021 ± 0.25 D vs. 0.24 ± 0.5 D, p < 0.05). No differences were observed in the degree of biofilm formation of the studied bacteria when grown in a mixed medium.

Bacterial Cell Auto-Aggregation
The results of cell aggregation in LB and MRS growth media are given in Table 4. Interestingly, the cell aggregation of fish E. coli did not have any describable specificity in comparison with that of probiotic E. coli strains (54.43 ± 8.41% vs. 57.45 ± 3.97%, p > 0.05). In comparison, the cell aggregation for fish LAB strains was statistically lower that of probiotic strains (61.02 ± 8.32% vs. 94.08 ± 3.33% and 95.67 ± 2.6%, p > 0.05). The Enterococcus 9-3 strain has the same level of auto-aggregation as the probiotic strain Enterococcus faecium R2. ifteen commensal E. coli and five lactobacilli isolates from the fish gut microbiota, all randomly selected and morphologically different, were used in this study. p-comparison with the probiotic strains.
The highest Pearson correlations were shown between biofilm formation ability and surface hydrophobicity, biofilm formation and auto-aggregation abilities, and autoaggregation and biofilm formation abilities for the E. coli probiotics with non-fish origin (|r| = 1). These associations were comparably weak in fish E. coli isolates. Interestingly, non-fish origin lactobacilli isolates and probiotics had a weak correlation between their cell surface hydrophobicity and auto-aggregation, and cell surface hydrophobicity and biofilm formation. However, the correlation between the auto-aggregation and biofilm formation was high for the fish lactobacilli (|r| = 0.982), which, against the background of the same ability to form biofilms, was probably due to the relatively low auto-aggregation of these lactobacilli (Table 5).
A weak relation was also discovered between the auto-aggregation and biofilm formation abilities of fish Bacillus spp. Other associations related to fish bacteria were even weaker (|r| < 0.2). Probiotic bacterial strains of fish/shrimp origin from the microbial collections of the Southern Federal University of Russia (Bacillus subtilis str. 1R, Bacillus amyloliquefaciens str. 4R, Bacillus amyloliquefaciens str. 5R and Bacillus cereus str. 6R) were used. 2ˆF ifteen commensal E. coli and five lactobacilli isolates from the fish gut microbiota, all randomly selected and morphologically different, were used in this study. 3ˆP robiotic strains of human/sheep/milk origin from the bank of the International Association for Human and Animals Health of Armenia (Lacticaseibacillus rhamnosus str. Vahe, Lactiplantibacillus plantarum str. ZPZ, Lacticaseibacillus rhamnosus str. E5-2, Lactiplantibacillus plantarum str. K1-3, E. coli str. ASAP-1 and E. coli str. ASAP 2-1) were also used. X Bacteria were grown in the growth medium: E. coli (LB medium); LAB and Bacillus (MRS broth). CSH-cell surface hydrophobicity. BF-biofilm formation ability. AGG-auto-aggregation ability. 0.45 < |r|< 0.75-moderately correlated relationship. |r| > 0.7-a fairly strong relationship. |r| < 0.45 weak relationship. r = 0-no relationship.

Cell Surface Hydrophobicity
It is well known that the growth medium affects the ability of lactobacilli to form biofilms [61,62]. The hydrophobicity of the cell surface determines the ability of bacteria to attach to the cell, although physical parameters such as Brownian motion, van der Waals attraction, gravitational forces, and the surface electrostatic effect cannot be ignored [63].
The present studies confirm that the hydrophobicity of the cell surface of the studied LAB, as well as E. coli cells, depends on the growth medium. However, no correlation between hydrophobicity and growth medium was found; the results were specific for different species (also probably for strains).
Various methods are used to regulate the hydrophobicity of the cell surface of probiotics. It can be assumed that the targeted selection of a growth medium for probiotics may be one of the major approaches for this purpose. It is possible that modulation of the cell surface hydrophobicity of probiotic bacteria by prebiotics can determine the effectiveness of synbiotic preparations.
It is also interesting that fish intestinal bacteria, particularly lactobacilli and E. coli, have low cell surface hydrophobicity, regardless of the growth medium (Table 1). In order to understand this phenomenon, additional studies will be required to elucidate the mechanisms of host-bacteria interaction in fish, as well as the influence of the environment on the fish intestinal microflora. The surface proteins of LAB can also affect the hydrophobic characteristics of the cell surface and are important in the processes of adaptation of the biophysical characteristics of the cell surface in response to environmental changes [64]. It is also known that surface proteins can participate in combating fish pathogens. For example, a protein extract can inhibit the adhesion of the pathogen to epithelial cells [65]. The results of our studies do not exclude the possible role of surface layer proteins in the hydrophobic characteristics of the cell surface of lactobacilli grown in various media. Based on the presented and published data, it can also be assumed that lactic acid bacteria and E. coli may have adaptive functions in the microflora of fish. On the other hand, the lower level of hydrophobicity of the cell surface of fish bacteria probably indicates the transitory status of these bacteria.
All the fish probiotic strains used in this study were selected as probiotics due to their antagonistic behavior towards fish pathogens [11,66]. In this case, the absence of significant differences in the levels of cell surface hydrophobicity between the fish E. coli and LAB isolates and Bacillus-and LAB-origin probiotics (p > 0.05) ( Table 1) allows for the assumption that the cell surface hydrophobicity of E. coli, and LAB/Bacillus strains might not play a significant role in combating fish pathogens. On the other hand, there is a statistically significant difference between the levels of cell surface hydrophobicity of fish-and non-fish-origin probiotics (p < 0.05) ( Table 1). The question of whether probiotics of non-fish origin with a higher cell surface hydrophobicity and the same antagonistic quality are more advantageous than those of fish origin is unanswered and needs further clarification/investigation. Moreover, if it is recommended to use non-fish-origin probiotics which have a non-beneficial cell surface hydrophobicity, considering the factors mentioned above, it might be possible to affect the strain hydrophobicity levels with other methods, including a change of environment.

Biofilm Formation Ability and Auto-Aggregation
Bacterial biofilms are communities of surface-attached bacteria that express distinct properties compared to their free-living counterparts, including increased antibiotic tolerance and metabolic capabilities [67]. They play an important role in the development and functioning of the host organism and protect it against pathogens [68]. For example, the investigations by Mirani and coauthors on multispecies biofilm formation from Pseudomonas aeruginosa, Staphylococcus aureus, and E. coli strains showed that E. coli dominated during the pre-biofilm stage. The authors reported that E. coli adapted to a biofilm lifestyle before S. aureus and P. aeruginosa. However, after adopting a biofilm lifestyle, P. aeruginosa gradually came to dominate the consortia and dispersed other species. This could be explained by the ability of P. aeruginosa to produce cis-2-decanoic acid, which can disperse or inhibit S. aureus and E. coli biofilms [69].
The presented studies show the highest correlations between biofilm formation ability and surface hydrophobicity, biofilm formation and auto-aggregation abilities, and autoaggregation and biofilm formation abilities for the E. coli probiotics with a non-fish origin, similar to the pathogenic microorganisms [70,71], which is expected if we take into account the requirements for probiotics. The results of our study on the cell surface properties of L. acidophilus strain INMIA 9602 Er-2 317/402 Narine are consistent with the literature data on the hydrophilic properties and poor biofilm formation ability of other Lactobacillus strains [71].
It is known that the surface of bacterial cells consists of many identical subunits that form a porous lattice layer. Surface layer proteins are found in many species of lactobacilli. The functions of these proteins are poorly understood, but there is evidence that some surface layer proteins have protective and enzymatic functions and can also mediate the adhesion of lactobacilli to host cells or extracellular matrix proteins [72].
It is possible that the biofilm formation ability, the degree of surface hydrophobicity, and auto-aggregation (the first stage of adhesion) [49,73] of fish microbiome bacteria are determined by the presence of specific proteins of the surface layer, which, in turn, may underlie the adaptive properties of fish. Probably, the specificities in cell surface and auto-aggregation properties of fish lactobacilli have a noticeable impact on fish adaptive properties. Lactobacilli are known to modify their surface structure in response to environmental factors; the correlation between auto-aggregation and biofilm formation abilities might show that both of these characteristics depend on the same physical adhesive forces.
This study on the properties of the cell surface of fish intestinal bacteria is important for determining the effectiveness of the use of probiotics in fish production and requires additional research to clarify how the characteristics of bacterial surfaces contribute to probiotic effects. This is also important for assessing the role of the bacterial factor in studies of "interacting" ecosystems [74].

Conclusions
The properties of intestinal bacteria/probiotics, such as cell surface hydrophobicity, auto-aggregation, and biofilm formation ability, play an important role in shaping the relationship between bacteria and the host. The current investigation on bacterial surface characteristics revealed a difference between probiotics of fish and non-fish origins. Interestingly, LAB and E. coli isolated from the intestines of fish had a low level of cell surface hydrophobicity, which was influenced by the growth medium. Salmo ischchan fish intestinal lactobacilli isolates also differed from non-fish origin intestinal lactobacilli/lactobacilli probiotics by their association between the auto-aggregation and biofilm formation abilities.
The bacterial auto-aggregation (Table 4) indicates that perhaps the auto-aggregation of lactic acid bacteria, in contrast to bacterial hydrophobicity, is important in the fight against pathogens. This could also apply to E. coli probiotics; unfortunately, we do not have fish/aquatic E. coli probiotics to make a general guess. Further research will be aimed at testing this hypothesis, as well as elucidating its mechanisms. It is also interesting that non-fish origin lactobacilli isolates and probiotics also had weak associations related to auto-aggregation-cell surface hydrophobicity and cell surface hydrophobicity-biofilm formation, while the auto-aggregation-biofilm formation associations were high for the fish lactobacilli (|r| = 0.982) ( Table 5). Against the background of the same ability to form biofilms (Table 3), this was probably due to the relatively low auto-aggregation of these lactobacilli (Table 4).
Unlike in other animal taxa, where host genetic factors play a central role in shaping the microbiota, the intestinal microbiota of fish is mainly determined by the environmental factors of the habitat. This, along with the results of current investigations, is important for the selection of fish probiotics and the regulation of appropriate biotechnological processes.
These investigations serve as a foundation for further, more profound studies of fish bacteria/probiotics.