The Effects of Antibiotics, Metals, and Biotic Interactions on the Assembly of Taxonomically Diverse Single and Mixed Species Biofilms

To better understand the assembly of the sturgeon egg microbiome, we purified six bacterial isolates from eggs and characterized their ability to form biofilms under the stress of tobramycin, with and without exogenous protein. In experiments with single species biofilms, tobramycin reduced the metabolic activity of all isolates and increased biofilm biomass of three. The addition of exogenous protein to the assay countered the inhibition of biofilm and metabolic activity by tobramycin of Pseudomonas sp., Brevundimonas sp., Flavobacterium columnare and mixed biofilms of Pseudomonas-F. columnare and Brevundimonas-Hydrogenophaga. Two of the isolates (Pseudomo-nas spp.) that produced antimicrobial activity, were effective at reducing biofilm formation by Brevundimonas, but enhanced biofilm formation in other isolates. Increasing concentrations of Mg 2+ had no effect on biofilm formation but Ca 2+ enhanced biofilm formation of Pseudomonas aeruginosa PA01 (pos-itive control) and Brevundimonas. Biofilm assembly by these two bacteria was inhibited by low concentrations of Ni 2+ . Mixed biofilms of Brevundimonas and Hydrogenophage consistently produced more robust biofilm than the strains in isolation, suggesting synergism. Established Brevundimonas biofilm appeared adept at recruiting pelagic Acidovorax and Hydrogenophaga into biofilm, suggesting that it plays an important role in the selection of species into the microbiome.


Introduction
The response of biofilm to stressors has been heavily investigated, primarily from the perspective of medicine and health. Biofilm is often viewed as an alternative life form of microbes that can provide some resistance to host defenses, antibiotics and other stressors [1]- [8]. In its most passive aspect, biofilm, by virtue of its macromolecular matrix, provides a barrier that protects the embedded cells from soluble stressors. For example, the cells of a P. aeruginosa biofilm are more resistant to tobramycin than planktonic cells [9] [10] and matrix encased bacteria cannot be opsonized as easily and are more resistant to polymorphonuclear cells [4] [11] [12]. There also appears to be "restricted penetration" of metals in biofilm matrices that accounts for an up to 600 times increase in resistance to toxic metals [13], although specific local conditions and time can play a large role [14] [15]. A more active role for biofilm has been demonstrated as well in the secretion of proteases, for example, that attack lactoferrin, a host-produced, bacterial growth-inhibiting protein [16]. More subtle is the strategy of Staphylococcus aureus biofilms that release lytic toxins that alter the differentiation of activated macrophages [17]. Biofilms have proven to be such a successful survival strategy that from 40% -80% of bacteria and archaea are thought to exist in quasi-structured matrix-bound communities [18].
Our work has focused on the biofilm of Lake Sturgeon (Ascipenser fluvescens) eggs as a model for biofilm assembly as well as for aspects of conservation. Regarding the former, eggs of aquatic vertebrates are expelled into the water in essentially a sterile state. Because these naive eggs are rapidly colonized by bacteria from the surrounding milieu, they provide an excellent view into the assembly of a natural, multispecies biofilm. With respect to conservation, mortality during early oncogenic stages can exceed 95% for many fish species [19] [20] [21]. Lake Sturgeon is an endangered ancient species of fish with high egg mortality, in part the result of microbial activity [22]. Some endangered species, like the lake sturgeon, receive an assist from hatchery-reared juvenile supplements to the natural populations. More favorable outcomes in the hatchery will come from understanding the "rules of assembly" of the egg-associated microbes and the nature and composition of beneficial versus parasitic consortia that colonize these eggs.
Initial investigations into the sturgeon egg-associated microbiome included the isolation and characterization of over 100 bacterial isolates from the surface of sturgeon eggs as well as the phylogenetic characterization of the communities from healthy and moribund eggs [23]. Microbial community analysis allowed us to tentatively identify 3 -4 phylotypes that were consistently present in healthy eggs and were also represented in our isolate collection [24]. These isolates included 1.) Acidovorax sp., a Betaproteobacteria in the Comamonadaceae family, 2.) Brevundimonas sp., an Alphaproteobacteria in the Caulobactereaceae family, 3.) Hydrogenophaga sp., a Betaproteobacteria, also in the Comamonadaceae family, and 4.) Massilia sp., a Betaproteobacteria in the Oxalobacteraceae family.
Isolate characterization included screening for the production of antimicrobial  [24] that led to the identification of two Pseudomonas spp. that produced strong antimicrobial activity against other members of the collection. More recent work has shown that three of these isolates, as well as the fish pathogen Flavobacterium columnare, formed substantially more robust biofilm in the presence of exogenous protein [25]. These isolates were utilized in this study to examine the effects of antimicrobials on biofilm assembly of single and double species biofilms, metabolic activity of tobramycin-stressed biofilms, and the resistance/sensitivity of biofilms to increasing concentrations of Mg 2+ , Ca 2+ and Ni 2+ . Several of our isolates appear to interact synergistically when co-founding a biofilm and these mixed biofilms were examined as well. Finally, our studies identified Brevundimonas as a strong and collegial initiator of biofilm formation.

Chemicals
All solutions were prepared using water from a MilliQ water purification system fed with building Reverse Osmosis water. All chemicals were of molecular biol-

Formation of Single-and Mixed-Species Biofilms
In order to study single species biofilm formation, 50 μL of an overnight bacteri- To investigate biofilm formation by double species mixed-cultures, 25 μL of overnight culture of each isolate was added to the well along with 100 µl of sterile R2Broth (3 -4 replicates per condition based on the experiment). These plates were also incubated as above. In all experiments, pre-and post-incubation optical density at 600 nm was measured to confirm growth within the broth.
In addition to single-and mixed-species biofilm formation, we studied the effect of one isolate on the established biofilm of another isolate. In these experiments, 50 μL of overnight culture of the first species was added to 100 μL of sterile R2Broth in one well of a 96-well plate. After 24 -48 hours of incubation the remaining pelagic cells and broth was removed, and the wells were washed x3 with sterile physiological saline. 50 μL of the second species was then added along with 100 μL of fresh sterile R2Broth and the plates were re-incubated for 24 -48 hours, depending on the experiment.

Tobramycin and Milk Protein Assays
Tobramycin has been identified as an antibiofilm compound by numerous in-  [31]. In addition, at this concentration the dominant mode of inhibition of Pseudomonas is at the level of the ribosome [32]. Because we were interested in the metabolic activity of biofilms, we tested the activity of the biofilm of all isolates and isolate combinations in the presence of 5 µg/mL tobramycin and found that activity was diminished in all cases. This indicated that tobramycin at 5 µg/mL was diffusing through the biofilm matrix and into cells. Regarding milk protein, previous work in our laboratory indicated that the addition of exogenous protein to cultures promoted substantial biofilm formation by some species [25]. Based on these studies, milk protein was tested at 2.5%. To determine if tobramycin or milk protein influenced biofilm formation of our isolates, wells of 96-well microtiter plates containing 100 µl R2Broth supplemented with either 2.5% milk protein or tobramycin (5 µg/mL) or both, were inoculated with single-or double-species mixtures and then incubated at 25˚C on a rotary shaker (100 RPM) for 48 hours.

Brevundimonas Titration Assays
We measured biofilm formation of Hydrogenophaga F14 and Acidovorax F19 in co-culture with different starting concentrations of Brevundimonas F16 to determine the dependence of biofilm formation on Brevundimonas.

Crystal Violet Assay
Biofilm biomass was measured using the crystal violet assay [37] [38]. Crystal violet is a triarylmethane dye that forms a bond with negatively charged molecules and polysaccharides on the surface of bacterial cells within the biofilm and/or the extracellular matrix [39]. companied by shaking at 100 RPM as above. Plates were read on a BioTek Epoch plate reader at 600 nm. Wells were read x3, averaged, and the uninoculated control wells were subtracted from the absorbance of the sample wells.

Statistics
The Student's t-test as implemented in Microsoft Excel (two sample assuming unequal variance) was used to evaluate dissimilarity between samples. In experiments where there was a mixed biofilm of two isolates, our null hypothesis was that the two populations of bacteria did not interact in any manner. In this way we compared two-isolate biofilms with the sum of the individual isolate biofilms.
A "P" value of 0.05 or less indicated that the mixed biofilm differed from our null hypothesis. When challenging a 24-hour established biofilm with a second isolate, we used a biofilm with identical total incubation time derived from simultaneous inoculation of the two isolates as the control. For testing the effects of stressors (tobramycin or milk protein), the cognate sample without stressor(s) was used as the control.

Results
Over ten years ago we began an investigation into the microbial communities that attach to sturgeon eggs. As a model system, aquatic vertebrates offer a unique view into the assembly and biological relevance of microbial communities on the surface of eggs. When essentially sterile eggs are released from the female, they are engulfed by an environment with 10 5 -10 7 bacteria per ml and are rapidly colonized. Experimentally the investigator can control the environment surrounding the egg, measure physiological parameters and egg survival and correlate these with the egg-associated microbial community that assembled. Our previous work indicated that the egg-associated community was significantly different from the aquatic community, varied depending on the water temperature, increased in density as the egg matured and maintained unique populations of bacteria when healthy [23] [42]. Moreover, we purified over 100 bacterial isolates from the sturgeon egg and phylogenetically identified them, as well as characterizing their capacity to form biofilm and produce antimicrobial agents. Several of our isolates, Acidovorax F19, Brevundimonas F16, Hydrogenophaga F14 and Massilia B13, were correlated with higher survival rates of hatchery eggs and two Pseudomonas isolates, C22 & D2, were identified as producers of antimicrobials as measured by soft agar overlay technique. How these isolates interact with each other and with the fish pathogen F. columnare in the formation of biofilm, and the resistance of this biofilm to antibiotics, are the subjects of these investigations.
In Figure 1, we present the effect of antimicrobial producing isolates Pseudomonas C22 and Pseudomonas D2 on biofilm formation. Under the standard conditions of our assay (growth in R2broth at 25˚C), biofilms produced by Pseudomonas C22 and D2, Hydrogenophaga and Massilia were lower than 0.1 A600 nm while Acidovorax and particularly Brevundimonas had relatively robust biofilms (Panel A). When isolates were challenged with Pseudomonas . Single-species biofilms and biofilm assembly in the presence of antimicrobial-producing isolates C22 and D2. "Isolate X >>> C22" indicates that the established biofilm of X was challenged after 24 hours by a fresh overnight culture of C22. Controls for the two-species biofilms (black bars) were the sums of cognate single-species biofilms (gray bars). Panel (B). Assembly of biofilm with two species (gray bars) and challenge of established single-species biofilms (black bars). Controls for the challenged biofilms (black bars) were the cognate two-species biofilms (gray bars). The sum of the single-species biofilms of Panel A served as the controls for two-species biofilms in Panel B (null hypothesis). The asterisks identify responses that were statistically different (p = 0.05 or less) from the controls. The arrows indicate an increase (↑) or decrease (↓) of absorbance compared to controls. Note the abscissa scale differences between the two panels.
C22, one of the antimicrobial-producing isolates, biofilm production varied greatly. Brevundimonas had substantially diminished biofilm while biofilms of Acidovorax, Hydrogenophaga and Massilia were modestly increased when incubated with Pseudomonas C22. When Pseudomonas D2 replaced C22, biofilm formation of both Acidivorax and Brevundimonas were reduced while that of Hydrogenophaga and Massilia were modestly increased. These changes were statistically relevant. As pointed out in the Material and Methods section, our  Regarding susceptibility of biofilm formation to tobramycin, only the isolate combination of Brevundimonas F16 and Hydrogenophaga F14 was unaffected.
All other isolates and combinations were statistically different from the controls.  [25]. All isolates and two isolate combinations had statistically confirmed increases in biofilm, upon the addition of milk protein. In experiments where both milk protein and tobramycin were present, there were two responses. Some isolates saw complete amelioration of tobramycin inhibition when milk protein was present (Pseudomonas PA01, Brevundimonas, F. columnare, Pseudomonas C22-F. columnare and Brevundimonas-Hydrogenophaga) while the remaining isolates and combinations saw a partial amelioration. Figure 3 presents the companion experiment to Figure 2 where the metabolic activities of single-and double-species biofilms were determined. There were The combination of measuring both biofilm biomass and metabolic activity provides unusual insights into the response of isolate and isolate combinations to changing environmental conditions. To better visualize the relationship between biofilm and metabolic activity, we computed the ratio of biofilm biomass (crystal violet staining) to metabolic activity (resazurin reduction) and plotted this as the fraction of the unamended cognate sample (Figure 4). Thus, the value for P. aeruginosa PA01 in the presence of tobramycin is 1.71, indicating that the biomass: metabolic activity ratio is 1.71 times P. aeruginosa PA01 in the absence of the antibiotic. In the case of PA01, the metabolic activity has been disproportionally reduced compared to the biofilm. Only two samples had values less than 1.0, F. columnare + tobramycin and Pseudomonas PA01 + milk protein, indicating that the biomass: metabolic activity ratio was less than the isolate without supplements. Most of the remaining samples had values less than 10, indicating that the biofilm biomass had increased relative to the activity. Several samples were greater than 10 times their cognate control. Tobramycin alone produced nearly a 10-fold increase in biofilm accompanied by a 10-fold reduction in activity of Acidovorax and Hydrogenophaga, while milk protein produced substantial increases in biofilm biomass that were not matched by increases in activity in Acidovorax, Hydrogenophagaand F. columnare. Finally, the combination of milk protein and tobramycin yielded substantial increases in F. columnare, Acidovorax F19 and Hydrogenphaga F14. Of particular note was the robust response of Hydrogenophaga F14 where the biofilm increased 20-fold, but the activity was diminished 5-fold.
The effect of magnesium, calcium, and nickel on the formation of biofilms is presented in Figure 5. These metals were selected based on previous studies that suggested either an enhancement, in the case of magnesium and calcium [35]   [36], or stimulation, in the case of nickel [34], of biofilm formation. In the concentration range of 100 µM -1 mM, Mg2 + had little effect on biofilm formation.
Only Hydrogenophaga F14 and the combination of Brevundimonas-Pseudomonas C22 responded with a modest concentration-dependent increase in biofilm. The response to calcium (1 µM -100 µM) was more complex. Pseudomonas C22, Acidovorax F19, and the combination Pseudomonas C22 with Brevundimonas F16 showed no effects with calcium, but biofilm formation by P. aeruginosa PA01, Brevundimonas F16 and Hydrogenophaga F14 were modestly enhanced at the higher concentrations. The combinations of Brevundimonas with Hydrogenophaga or Acidovorax were moderately inhibited at higher calcium concentrations. Nickel was tested because of reports of biofilm stimulation in E. coli by subinhibitory concentrations [34]. Under the conditions of our assays in R2Broth, Nickel had little or no effect on biofilm formation by Pseudomonas C22,  Hydrogenophaga F14, Acidovorax F19 and the combination of Pseudomonas C22 and Brevundimonas F16. Nickel was, however, inhibitory to biofilm formation by P. aeruginosa PA01, Brevundimonas F16 and mixed biofilms with Brevundimonas and Hydrogenophaga or Acidovorax. Brevundimonas was particularly sensitive at even the lowest concentration (100 µM NiCl 2 ).
In our investigations, Brevundimonas was an unusually robust biofilm former, particularly in the presence of exogenous protein. In addition, it facilitated biofilm formation in collaboration with other isolates, notably Hydrogenophaga and Acidovorax. To test its role in biofilm formation with these isolates, we titrated either Hydrogenophaga or Acidovorax with Brevundimonas in standard biofilm assays. The results are presented in Figure 6(A) and shows that the amount of biofilm of Hydrogenophaga or Acidovorax, when paired with Brevundimonas, was dependent on the starting concentration of Brevundimonas.  was detected with Ethanol-killed Brevundimonas in the absence of a second strain. However, moderately enhance biofilm formation by Hydrogenophaga and Acidovorax was detected in the presence of Ethanol-killed Brevundimonas, although there was little concentration dependence. Note that in these experiments the controls to establish statistical relevance were the single-species biofilms of Hydrogenophaga or Acidovorax, inasmuch as Ethanol-killed Brevundimonas produced no measurable biofilm.

Discussion
The experiments reported herein describe the response of recently purified, undomesticated freshwater bacterial isolates to antibiotics and metals during the formation of biofilm. With the exception of P. aeruginosa PA01 and F. columnare, these isolates were purified from the microbiome associated with lake sturgeon eggs. In part, the variability in the amount of biofilm produced by these isolates under our experimental conditions reflects the phylogenetic and metabolic diversity of these bacteria. One isolate, Brevundimonas sp., produced a robust biofilm while the remaining five isolates made only modest amounts, com- pounds in soft agar overlays that were inhibitory to Brevundimonas and Hydrogenophaga, but considerably less so to Acidovorax [24] and had no effect on Massilia. In addition, we also showed that established biofilm of Brevundimonas was significantly attacked by pelagic Pseudomonas C22 [24]. In this series of experiments when both an aggressive antibiotic producing isolate and a sensitive isolate were incubated together, biofilm formation did not necessarily correlate with the results from soft agar overlay. Brevundimonas had reduced biofilm when incubated with either Pseudomonas C22 or D2, consistent with results from a soft agar overlay challenge. In contrast, biofilm in mixed solutions of Hydrogenophaga and Massilia, with either Pseudomonas C22 or D2, was increased. The results with Acidovorax were mixed, showing an increase with Pseudomonas C22 but little change with D2. On testing the ability of Pseudomonas C22 to disrupt established biofilms, we found that it was effective against Brevundimonas, but not other isolates. Thus, even though Brevundimonas was accomplished in establishing a biofilm, that option was not effective against antimicrobials produced by the Pseudomonas isolates.
The results were equally complex when biofilm formation was challenged with tobramycin. In these experiments, biofilm assembly was conducted in the presence of tobramycin and the activity assays were conducted after the assembled biofilms had been washed. Tobramycin was not present during incubation with resazurin. Three isolates, P. aeruginosa PA01, F. columnare and Pseudomonas C22, saw a reduction in the amount of biofilm in the presence of tobramycin, whereas biofilms of Acidovorax, Brevundimonas and Hydrogenophaga increased. However, all six isolates appeared sensitive to tobramycin based on the reduction of metabolic activity. Tobramycin has been shown to inhibit bacterial growth via two mechanisms [32], inhibition of translation and disruption of the outer membrane. These experiments were conducted at a concentration of 5 µg/ml (low-intermediate concentration), at which inhibition is thought to be via interference with translation. At higher concentrations (>8 µg/ml) disruption of the outer membrane is the primary mode of killing.
In mixed biofilms of Brevundimonas-Acidovorax, both biofilm and metabolic activity increased in the presence of tobramycin. Indeed, the biofilm biomass doubled indicating that this two-isolate alliance responded to the tobramycin challenge with increased metabolic activity not seen in the isolates individually.
The nature of this synergism is not known. The opposite was seen with Pseudomonas C22-F. columnare where both biofilm and activity were reduced in the presence of tobramycin, consistent with the results of single species biofilms. The reduction of metabolic activity was substantially greater than the reduction of biofilm in the Brevundimonas-Hydrogenophaga mix but, in the absence of stressor, these two isolates together out-performed each isolate individually. We posit that the isolate combinations of Brevundimonas with either Aciovorax or Hydrogenophaga exhibit attributes that differed significantly from the isolates in isolation. Populations within a biofilm may be noncommunicative, antagonistic or cooperative where the nature of their interactions may stimulate functionality not present in single-species biofilms. This has been termed "emergent properties" of biofilms [46] and is thought to indicate synergisms between species [47].
The best example of this in our data was the interactions between Brevundimonas and Hydrogenophaga. When together, these isolates produced twice as much biofilm as the isolates individually. Mapping the intersections of each species' biofilm physiology will help to determine the essential metabolic features driving these synergisms. As mentioned previously, milk protein was found to stimulate biofilm formation substantially in select species [25]. When 2.5% milk protein was applied as a stressor it elicited a more robust biofilm in Pseudomonas C22, F. columnare, Hydrogenophaga and Brevundimonas as well as an increase in metabolism. Milk protein also stimulated biofilm in Acidovorax but metabolic activity was reduced. Of interest was the response to tobramycin in the presence of milk protein which provided partial (Pseudomonas PA01, Pseudomonas C22, and C22-F. columnare) or complete (Brevundimonas and F. columnare) amelioration of the effects of tobramycin. This seemingly high protein concentration was not selected randomly. In previous work [25] we showed that select bacterial isolates produced abundant biofilm, as measured with the crystal violet assay, in the presence of relatively high concentrations of exogenous protein. We also demonstrated that the number of cells within the biofilm and the amount of protein incorporated into the matrix, increased, in response to exogenous protein.
Relatively high protein concentrations were selected in extended studies [25] to determine possible environmental stimuli of biofilm formation in Serratia marcescens. One potential habitat for this species is the human lung, where the concentration of only the surfactant proteins of alveolar fluid can be as high as 10% of the dry weight [48]. Similar high protein concentration exists in fish gills, targets of F. columnare. One interpretation of this is that in microenvironments with high protein concentration, the efficacy of tobramycin may be limited. Counter to this hypothesis is the observation that enhanced respiration, of the type we see upon addition of 2.5% milk protein, reduces the fraction of persister cells in biofilm of Mycobacterium tuberculosis, rendering it more susceptible to antibiotics [49]. Persister cells are present in biofilm and are thought to account, in part, for the resistance of biofilm to antibiotics [50] [51]. Vilchèze et al. [49] suggest that high metabolic rates and increasing consumption of O 2 lead to fewer persister cells and greater production of reactive oxygen species. While reactive oxygen species have been invoked in the inactivation of antibiotics [52] unstressed conditions similar to those used in the experiments described above.
In a mixed biofilm of Pseudomonas C22 and F. columnare, the mixture was 90% C22. Thus, even in a situation where there was potential for complete dominance by an antimicrobial producing isolate (C22), F. columnare maintained a foothold in the biofilm. We selected magnesium, calcium and nickel to test because these metals enhance rather than inhibit biofilm formation. Both magnesium and calcium are divalent cations commonly found on the outer membrane of bacteria [53] [54] and appear to assist the structuring of biofilm at low concentrations [35] [36] [47]. Nickel has been shown to stimulate biofilm formation in E. coli at 100 µM Ni 2+ [34]. Metals are also toxic to biofilms [15] including nickel [55] [56]. In general, while we could detect statistically relevant differences in biofilm formation with magnesium and calcium, the effects were small. We found no substantial effect of magnesium on biofilm formation at relatively moderate concentrations. Calcium produced a modest stimulation of biofilm at the two highest concentrations for Pseudomonas PA01 and Brevundimonas and a moderate reduction of biofilm for two mixed species biofilms with Brevundimonas. Several hypotheses have been advanced to account for the influence of metals on biofilm including reducing surface charge and improve packing, bridging molecules and contributions to the structuring of the cell surface and conditioning the substratum [36]. Nickel proved to be the most interesting in that Brevundimonas and Pseudomonas PA01 appeared particularly sensitive. The concentrations we employed were based on NiCl 2 , hence the actual Ni 2+ concentrations in the assays were 45 µM, 90 µM, 135 µM and 180 µM. Thus, even at the lowest concentration, strong inhibition of biofilm formation was observed and Brevundimonas was particularly sensitive. While the sensitivity was attenuated in the mixed cultures with Acidovorax and Hydrogenophaga, at the higher concentrations of nickel the biofilm was reduced to barely detectable levels. Brevundimonas may be a critical target for nickel in the sense that our work indicated that Brevundimonas was instrumental in establishing mixed biofilms with emergent properties and higher concentrations of Brevundimonas translated into more robust biofilms. In stream systems, it has been observed that biofilm associated metals influence the microbial composition of the community [57]. All of our isolates were derived from freshwater systems, including Brevundimonas. Given the sensitivity of Brevundimonas to nickel, this group may be a sentinel for metal contamination. Brevundimonas, like the phylogenetically related genus Caulobacter, possesses a holdfast with a sticky terminus, located at one end of the rod-shaped cell [58] [59]. This structure is critical in establishing a connection to the substrata.
Moreover, Brevundimonas biofilm actively recruited both Hydrogenophaga and Acidovorax from broth. We have also observed Brevundimonas induced cell-cell aggregation forming rosettes with Hydrogenophaga and Acidovorax under the light microscope (data not shown), similar to what is seen with Caulobacter [60].
Symbiotic relationships of Brevundimonas with other microbes have been identified [61] [62] and a recent report on nitrogen fixation in this genus [63] may explain in part, its collegiality. These attributes point to a key role for Brevundimonas in establishing biofilms in aquatic settings. Overall, our observations describe a deep complexity in the assembly, function and resistances of multispecies biofilms.

Conclusions
Our investigations into the structure and function of Sturgeon egg microbiomes were designed to assist in the development of hatchery protocols that reduce egg mortality caused by microbial activity. To that end, we have purified over 100 isolates from eggs and in preliminary studies, described antagonism between isolates and characterized biofilm formation [42]. The investigations presented here extend our description of interactions between isolates. By measuring the formation of biofilm comprised of one or two species in the presence and absence of stressors, we have identified several types of isolate interactions that may facilitate the formation of the egg's microbiome. We have identified significant sensitivity of some biofilm-forming isolates to two independently isolated Pseudomonas spp. that produce antimicrobial compounds in situ. In one case, the Pseudomonas isolate (C22) diminished the formation of a robust biofilm of F. columnare, a fish pathogen. While the metabolic activity of all tested isolates was diminished by tobramycin, three produced less biofilm and three produced more biofilm in response to the antibiotic. One of our isolates, Brevundimonas, formed abundant biofilm with increased metabolic activity in the presence of exogenous protein. In several cases, the presence of exogenous protein ameliorated the effects of tobramycin, suggesting that antimicrobial activity is conditional for these strains. Biofilm formation by both Brevundimonas and P. aeruginosa PA01 were exceptionally sensitive to low concentrations of nickel. Finally, we posit that Brevundimonas is a keystone species in the formation of multispecies biofilms based on the ability of established Brevundimonas biofilms to recruit pelagic Hydrogenophaga and Acidovorax and its collaborative interactions with Hydrogenophaga. Advances in Microbiology