Isolation and Identification of Probiotic Bacteria for the Management of Epizootic Shell Disease

I. Abstract Epizootic shell disease (ESD) is an emerging disease in the American lobster (Homarus americanus) characterized by lesions, mainly on the carapace. The diseased appearance of the shell due to the lesions has had significant negative impact upon the lobster fisheries in New York, Connecticut, Rhode Island, and Massachusetts, since fishers are not able to sell affected lobsters to the more lucrative live market. ESD lesions are different from other shell diseases found in the American lobster populations because they are due to a poly-microbial infection that degrades the epicuticle layer, while the pillars of the chitin matrix remain intact in the lesions. ESD was first described in the 1980’s, and has shown the highest prevalence in Southern New England since the late 1990’s. In inshore waters, ESD was estimated to affect 10-40% of the lobsters with 50-80% of the ovigerous females affected by ESD. Additionally, ESD has expanded to lobster populations outside the original geographic range. New or consistent disease observations have been seen in Maine at low levels of disease prevalence. The increase in ESD has generated concern for the health of the lobsters and the economic status of the fishery. It has been shown that probiotics are an effective way to prevent infectious diseases in a variety of animals, including fish and shellfish by inhibition or exclusion of the pathogenic bacteria. The goal of this research was to isolate commensal, potential probiotic, bacteria from the shells of healthy looking lobsters and characterize their ability to reduce or eliminate the ESD-causing organisms. Twentyfour out of 217 isolates from lobsters were characterized as potential probiotic organisms based on their ability to inhibit the growth of putative ESD pathogens Thalassobius sp. I31.1 or Aquimarina macrocephali I32.4 (formerly known as Aquimarina homaria), ability to form strong biofilms, and their effect on Thalassobius sp. I31.1 growth and biofilm formation. While twenty-four isolates exhibited activity against at least one of the target organisms, but only two potential probiotic organisms, Bacillus sp. 06-YP001, and Pseudoalteromonas sp. 10B-YPO11, had inhibitory activity against both pathogens. Biofilm formation on polystyrene, sterilized lobster shell fragments or glass coverslips was variable in strength across isolates. The competition assays demonstrated that four isolates, Loktanella maritima 06-YPC210, Bacillus sp. 06-YP001, Pseudoalteromonas sp. 03-YP014, Pseudoalteromonas sp. 08-YPC21, and Phaeobacter inhibens S4Sm were effective in reducing the growth of Thalassobius sp. I31.1. These results demonstrate that potential probiotic organisms can be isolated from the host (lobsters) and used to reduce growth and biofilm formation of the targeted pathogens (ESD). Looking at the interactions of the pathogens of ESD and the potential probiotics could help elucidate the cause and development of ESD.


II.a. Carapace Structure of H. americanus
The H. americanus cuticle provides structural and protective properties to the organism (18,20). It is composed of two layers: the epicuticle and procuticle. The epicuticle is the diffusion barrier and the procuticle is divided into the exocuticle and the endocuticle (Fig. 1). The procuticle is seen as the mechanical support for the organism (20). The cuticle is composed of a chitin matrix with protein polymers surrounding the matrix.
Chitin, β-(1,4)-2-acetamido-2-deoxy-D-glucopyranose, is a main component of the cuticle. The cuticle is composed of three main minerals: calcite, amorphous calcium carbonate and carbonate-apatite. The chitin is associated with all three major mineral composites including calcium carbonate, calcite, and carbonate-apatite (20). Several polymer sequences have been analyzed and an 18-amino acid residue motif has been identified (x-L/V-I/V-G-P-S-G-I-V-T/S-X-D/N-G-x-N-I/V-Q-V/L), it is hypothesized that the N-terminal amino acid of the 18-residue motif is associated with the chitin matrix and calcium (9).
The chitin matrix has been described as a twisted plywood model ( Figure 2) (19). The layers consist of stacks of helical-like chitin structures that follow a pattern and density depending on examination of the endocuticle and exocuticle layer.
Depending on which layer of the carapace is being observed the density of the chitin stacks will differ. The exocuticle is more compact then the endocuticle layer (20). Along with the chitin matrix the cuticle has a complex pore canal system that aids in the transportation of ions that help when the organism is developing the new exoskeleton (19,20). For the lobster to grow, the old exoskeleton must be replaced by a new exoskeleton through the process of molting. When lobsters molt, the old exoskeleton is shed and the lobsters are unprotected until the new exoskeleton hardens. Once the new exoskeleton is formed it becomes mineralized. Mineralization gives the protective functions to the exoskeleton (19). The severity of ESD has been shown to depend on the molting time of the lobster. The longer the carapace remains infected the more severe the disease is. If the lobster recently acquired ESD and molts relatively soon after the onset of infection, the new developed exoskeleton appears to be initially disease free (3).

II.b. Epizootic Shell Disease Prevalence
ESD has increased in severity and prevalence in the wild lobster population (3). While low prevalence of ESD has been observed since the late 1980s, in 1998 the prevalence increased by ≥20% in inshore populations in Southern New England (3).
New or consistent disease observations have been seen in lobster fisheries in New York, Connecticut, Rhode Island, Maine, and Massachusetts (4). There is also concern that ESD could spread to European lobster populations due to interactions with infected American lobster populations. Due to this concern, some European countries have proposed a ban on the importation of US lobsters (28)

II.c. Epizootic Shell Disease Etiology
The cause(s) responsible for the increase in ESD are not yet known. Originally, three bacterial species, Aquimarina homaria (2) (re-named Aquimarina macrocephali I32.4), Thalassobius sp. (2), and Pseudoalteromonas gracilis (5), were putatively involved in the infection resulting in ESD because they were consistently observed in lesions, even though ESD lesions have a high number of bacterial species colonizing the lesions (2). Pseudoalteromonas gracilis has been described as non-obligatory for the infection and A. homaria and Thalassobius sp.
(2) have been demonstrated to potentially cause lesions, initiation of lesion development is not yet understood.
Lesion formation has been studied in order to better understand the initiation and progression of the disease (7,28). One study showed a shift in the microbial population of the carapace leading to a dysbiosis. Whether the dysbiosis results from or is the cause of lesion formation is unclear (28). In another study, bacterial communities were characterized for transition and affected areas of the lobster carapace (7). It has been suggested that the altered bacterial communities between the transition and lesion areas of the lobster are important in understanding the initiation and progression of ESD (7). Using next generation sequencing, Feinman et al. (7) identified 170 different bacterial species associated with the lobster carapace. They demonstrated a shift in the bacterial composition between the sites of ESD lesions, 5 mm from the sites of lesions (the transition zone), and the sites with no lesion. The bacterial community at the site of the lesion is distinct and is less diverse than the community of the unaffected healthy carapace (7).
Bacteria found in this transition area may be important for initiating disease, while bacteria found in lesions may be secondary or opportunistic colonies (7). Based on differences in microbial community composition between lesions and adjacent areas, the authors hypothesize that lesions of the shell may select for specific taxa, in part due to the host response and melanization. One abundant operational taxonomic unit (OTU) found in the lesions has one hundred percent sequence identity with the genus Aquimarina; this OTU is not found outside of the melanized lesion (7). Another OTU of interest found in the transition microbiome and lesion microbiome is Loktanella.
Loktanella may be affecting the carapace in early colonization and increasing its abundance as the disease progresses (7).
Along with bacterial species linked to ESD, several environmental conditions have been hypothesized to increase ESD prevalence and severity. Environmental factors influence the quality of the shell, which may increase the susceptibility to the likely bacterial effectors of ESD (27). Increase in water temperature has been observed in periods of increased disease prevalence. Warmer water temperatures affect shell composition by affecting the placement and growth of the cuticle (28). The bio-composites of the carapace can become thinner at warmer temperatures (28).
Therefore, the shell becomes less dense and more susceptible to bacterial degradation (28).
Moreover, the marine environment is becoming more acidic and may exacerbate shell disease the proper function of the outside surface cuticle may be affected (12). Ocean acidification is the uptake of anthropogenic carbon dioxide by surface waters (10). Ocean acidification could affect the American lobster in a variety 3 of ways (30). With the absorption of CO2 there is a reduction in seawater pH which reduces the calcification rates; because it depletes carbonate ions needed for the biosynthesis of calcium carbonate (10,30). Ocean acidification could interfere with post molt calcification which is dependent on the uptake of Ca 2+ and HCO -(30).
Ocean acidification could also contribute to increased rates of calcium carbonate dissolving from the existing carapace (12). Kepple et al. (12) performed a study with Homarus americanus larvae examining the effects of ocean acidification on growth.
Larvae exposed to lower pH seawater demonstrates a decreased growth rate and, consequently, a longer amount of time between molting stages. It is hypothesized that the larvae may be redirecting energy to maintain mineralization of the calcified carapace (12).
Genetic differences among lobster populations are suspected to affect disease prevalence. This is observed in the varying disease progressions in the Eastern Long Island Sound and the Western Long Island Sound lobster populations. The western populations were affected by a bottleneck due to higher amounts of pollution and were influenced by selective pressures becoming genetically different when compared to the Eastern populations (2). The Eastern Long Island Sound population is more susceptible to ESD. Contaminants from metals; arsenic, cobalt, copper, and cadmium appear to be linked to ESD prevalence (21). Additionally, diet of the lobster population may influence susceptibility to ESD, as certain foods, such as Atlantic herring, in lobster traps has been connected to observations of increased lesion formation (22). Lobsters in a laboratory setting fed only a herring diet hadincreased shell disease. After testing lobsters in the wild, herring was determined to be a limited food source and demonstrated no increase in shell disease (22).
Although the primary cause of ESD is unknown, colonization by bacteria such as Aquimarina macrocephali I32.4, Thalassobius sp., or Loktanella spp. most probably play a role in disease pathogenesis and lesion progression. I propose that probiotic bacteria may be able to influence the progression of ESD by: 1) prevention of the initial colonization by the pathogen/s and 2) the reduction of polymicrobial community that enhances the ESD lesions.

II.d. Homarus americanus reaction to Epizootic Shell Disease
After initiation of the disease the physical response of the H. americanus There is also a molecular response to disease initiation. Gene expression data demonstrated disruption of molting (based on differential expression of the 8 ecdysteroid response gene), muscular function (arginine kinase) and xenobiotic metabolism (CYP45) in lobsters with ESD (27). Gene expression profiles are consistent with the observation that intermolt duration in lobsters with ESD has been shown to be shortened, suggesting that molting appears to be a defense mechanism against the disease allowing lobsters to shed the infected cuticle before internal damage ensues (27).

II.e. Probiotic use in shellfish
Probiotics have been used to treat or prevent a variety of diseases found in shellfish. Probiotics have been shown to competitively exclude pathogenic bacteria, produce inhibitory compounds, improve water quality and other effects as well (14).
Isolation of probiotic organisms from hosts we are trying to protect have been done with some success in the past, such as oysters (29), crabs, and fish. The probiont P. inhibens S4Sm, previously isolated from the inner shell surface of a healthy oyster, has been demonstrated to provide protection to oysters challenged with the oyster pathogens V. coralliilyticus RE22 and Aliiroseovarius crassostreae (11,29). Mouriño et al. (15) isolated potential probiotic bacteria from the foregut of the South American catfish hybrid, Pseudoplatystoma reticulatum × Pseudoplatystoma corruscans to identify bacteria that were antagonistic to the selected pathogen, Aeromanas hydrophila, and were able to colonize the intestinal tract of the hybrid. The probiotic bacteria were selected through a zone of inhibition test then tested in vivo. The isolates isolated from the foregut did prove able to colonize the intestine and provide protection against A. hydrophila (15). Nogami et al. (16) isolated bacteria from the crustacean culturing pond to isolate potential probiotic bacteria to protect Portunus trituberculatus (Japanese blue crab or horse crab) larvae. They isolated one strain identified as PM-4, which improved larvae growth and reduced the growth of Vibrio anguillarum (16). The results above show that the microbiome of a healthy host may be a source of successful probiotic organisms Larval mollusks are vulnerable to infections in hatcheries. Probiotics can be used to prevent or treat disease outbreaks in aquaculture (1). Phaeobacter inhibens S4 and Bacillus pumilus RI06-95 have been successfully used to prevent larval oyster mortality and give protection to the eastern oyster Crassostrea virginica against challenge with the bacterial pathogens Vibrio coralliilyticus RE22 and Aliiroseovarius crassostreae (11). The probiotic treatment was also effective in reducing mortalities in bay scallops (Argopecten irradians), but not in razor clams (Ensis directus), blue muscles (Mytilus edulis) and northern quahog (Mercenaria mercenaria). The data suggest that the two-probiotic species (Phaeobacter inhibens S4 and Bacillus pumilus) used have a species-specific protective effect for bivalve larvae (25). Since it is likely that this species-specific protective effect is common for all probiotics, specific probiotic organisms will have to be discovered for the treatment of specific infectious diseases in specific host organisms. This is because the host has different pathogen susceptibilities along with different defense mechanisms. The probiotics also benefit the hosts through different mechanisms such as improving water quality, production of antimicrobial compounds, competition with pathogenic bacteria and stimulation of immune response.
Middlemiss et al. (14) studied with the effects of a probiotic Bacillus spp.
against a pathogenic Vibrio spp. in European lobsters. Using a semi-closed recirculating system, they treated the lobsters by adding the probiotic Bacillus spp. to the tank water. They also treated the lobsters separately with UV light and O3 and then compared the effects of the different treatments on the reduction of Vibrio spp. (14).
The bacteriology results were determined for each water treatment. The Bacillus spp.
conferred no health benefits in growth, survival or fitness when compared to the other treatments used. The O3 and UV treatment tanks showed 0.05% of the total CFU to be Vibrio spp. (14), suggesting that the O3 and UV treatment may reduce the normal bacterial flora and create a shift in the bacterial community. This shift in the bacterial community could allow for opportunistic bacteria to take advantage of the change in the community and out compete the normal bacterial flora. This shift in the bacteria community could aid in the successful establishment of the pathogen, generating a more harmful outcome from the treatment. When investigating a treatment for any host it is important to understand the implications the treatment may have on the host and the water quality or habitat.
Talib et al. (26) studied the effect of a multispecies Bacillus culture on the survival of mud crabs (Scyla paramosain) in hatcheries. S. paramosain face large mortalities with bacterial diseases, in hatcheries (26). This study was used to determine if a multispecies Bacillus culture would be effective in increasing the survival rate of mud crabs in hatcheries. The effectiveness of the probiotic treatment was determined based on survival rate (26). Higher concentrations of the Bacillus showed higher survival rates in the hatcheries, demonstrating that culture concentration can influence the success of the probiotic treatment administered (26). probiotic for the treatment of ESD we will look for a species-specific protective effect, but since ESD is a polymicrobial infection it may require more than one probiotic.
With the probiotic treatment, there is a need to understand the implications the treatment may have on the host, water quality, and possibly the habitat. We also must consider the concentration at which we treat the lobsters since it could affect the success of the probiotic treatment.

III. Introduction
Epizootic shell disease (ESD) is a polymicrobial infection that causes lesions in the carapace and claws of the shell of the American lobster, Homarus americanus (2). These lesions are an asymmetrical degradation of the carapace appearing in a black to tan color. ESD varies from other shell diseases found in American lobster populations because it does not degrade the chitin pillars of the cuticle of the shell (15). The unsightly appearance of the carapace due to the lesions has greatly impacted the lobster fisheries in Southern New England, since fishers are not able to sell affected lobsters to the more lucrative live market (4).
Lobster ESD, first observed in the late 1980's, has increased in prevalenceand has expanded its range along the east coast of the United States. Currently, ESD has a high prevalence in eastern Long Island Sound and inshore RI waters (Narragansett Bay) with small outbreaks recorded in Maine and Northern Massachusetts (3,7). In 2012, it was reported that ≥20% of inshore lobsters show evidence of this affliction (3). Further, it was estimated in 2012, that the prevalence of ESD in lobsters found in RI inshore waters was 10-40% (7). Moreover, it is estimated that 50-80% of ovigerous females have ESD (7). Females afflicted with ESD may fail to reproduce because molting frequency increases with the severity of ESD and molting will result in the loss of the entire clutch of eggs. Alternatively, ovigerous females with ESD may they retain the shell and die before the eggs are ready (7). The increase in ESD within the New England lobster population has become a concern for its effects on the health of the lobsters and ultimately the economic health of the fishery.
Probiotics are microorganisms that when administered in certain amounts can confer a health benefit to the host (1). It has been shown that probiotics are an effective way to prevent infectious diseases in a variety of animals, including fish and shellfish, by inhibition or exclusion of the pathogenic bacteria and other mechanisms (1). We suggest that, probiotic organisms are a potential way to prevent or slow the progression of ESD. It has been previously demonstrated that lobsters have a diverse commensal microbial community that colonizes the lobster carapace. We hypothesize that commensal bacterial that colonize the shell of healthy looking lobsters can be a source of probiotic organisms that may be used to reduce or eliminate ESD-causing organisms from the lobster shell, reducing or stopping the progression of ESD.

IV.a. Bacterial strains and growth conditions
Bacterial species routinely grown included Aquimarina macrocephali I32.4, Thalassobius sp. I31.1, and the 24-selected potential probiotic isolates (Table 1). Both P. inhibens S4Sm is a spontaneous streptomycin resistant mutant selected for a previous study (23). The casein and colloidal chitin plates were observed directly for a zone of clearing around the spotted colonies. In order to observe a zone of clearing on the gelatin agar plates a 20% HCl solution was added to cover the agar surface to precipitate unhydrolyzed gelatin (22).

IV.c. Lobster shell and glass coverslip biofilm assays
The glass coverslip biofilm assay developed by Zhao et al. (24) was used and also modified to use lobster shells as an alternative to glass coverslips. To prepare the lobster shell for the assay, lobster molts that were frozen and collected from lobsters kept in aquarium at the Graduate School of Oceanography, were cut into 2 cm × 2 cm squares. The squares were placed individually in 50 mL falcon tubes containing 25 mL of 3% hydrogen peroxide for 24 hours. After 24 hours, the lobster shells were placed into six well plates containing 6 mL of sterile 3% ASW and were placed on a shaker for 2 minutes; this wash step was repeated twice. After the lobster shells were sterilized the biofilm assay was setup as described by Zhao et al. (24).
A competition assay developed by Zhao et al. (24)   isolates are unique or identical species. Additionally, two isolates were identified as L.
maritima. Both were isolated from egg clusters on female lobsters.
The remaining three isolates were identified as Alteromonas, Cobetia, and Bacillus.
Many of these isolates are known in the literature for producing anti-microbial compounds. Pseudoaltermonas are commonly found in association with marine eukaryotes and have been shown to have anti-bacterial, bacteriolytic and algicidal properties (8). Bacillus subtilis are known to produce at least 12 antibiotics (19).

V.b.3. Crystal violet biofilm formation assay:
It was hypothesized that if a potential probiotic organism is a strong biofilm former then it may exhibit a stronger probiotic effect in at least two ways: 1) by colonizing the lobster shell and physically preventing the attachment of potential pathogens to the carapace and 2) producing antibiotic substances on the lobster host, thus preventing colonization by the pathogens. The 24 isolates selected from the zone of inhibition assay were characterized for their ability to form biofilms on polystyrene. A control biofilm former, P. inhibens S4Sm was also examined using this method.

P. inhibens
S4Sm is known to be a strong biofilm former exhibiting an OD580 values of 3.8-4.0 using the crystal violet method (22). Our data measured P. inhibens S4Sm with an OD580 reading of 3.89 matching previously reported data (22). The crystal violet biofilm assay results demonstrate that the twenty-four selected probiotic isolates had a range of OD580 measurements from 1.33-3.71 (Table 4)  that the glass coverslip biofilm formation assay was an accurate, easy, and effective way to measure biofilm formation, and that biofilm formation by candidate probiotic isolates would be tested using this assay.
Biofilm cell densities (CFU/glass coverslip) of the selected candidate probiotic isolates were determined and compared to the biofilm cell density of P. inhibens S4Sm; a probiont known for strong biofilm formation ( Table 4).
The ANOVA analysis showed significant differences between probiotics on biofilm formation on these surfaces [p>0.0001, F(5,12)= 156.54] (Table 4), confirming the results of the crystal violet biofilm assay ( inhibens S4Sm biofilm. Overall, the tested potential probiotics showed a range of ability in forming biofilms on glass cover slips.

V.b.5. Determination of protease and chitinase activities.
Since ESD involves the degradation of protein in the lobster carapace (21) and proteases are frequently associated with virulence, it was important to identify the general proteolytic activities of the candidate probiotic isolates (6). Two different protease assays were used to measure proteolytic activity: one measured casein hydrolysis and the other measured gelatin hydrolysis. Additionally, chitinase activity, while not necessarily associated with ESD based on histological observations, is often associated with pathogenic potential in other lobster diseases (5). These tests were done by zone of proteolysis or zone of chitin degradation.
Twenty candidate probiotic isolates had protease activity against casein.
Nineteen isolates had protease activity against gelatin, and only three isolates exhibited chitinolytic activity. Only one isolate exhibited hydrolytic activity against all 3 substrates. P. inhibens S4Sm, a known probiotic of potential interest (24), had protease activity against casein, but no protease activity against gelatin and no chitinolytic activity ( Table 6).
The two putative pathogens were also tested for their ability to hydrolyze casein, gelatin, and chitin. Thalassobius sp. I31.1 had protease activity against casein and gelatin, but no chitinolytic activity was detected. A. macrocephali I32.4 exhibited protease activity against both casein and gelatin and also had chitinolytic activity (Table 7). P. inhibens S4Sm, a known probiotic of potential interest (24), was used as a control and had protease activity against casein, but no protease activity against gelatin and no chitinolytic activity (Table 6).

VI. Discussion
This study investigated the hypothesis whether bacteria isolated from lobster shells may serve as probiotic organisms that could be used to manage ESD. In this investigation, potential probiotic bacteria active against ESD were isolated from swabs of lobster carapaces.
In this study, lobsters were the source of 217 bacterial isolates that were screened for their ability to act as probiotic organisms to treat ESD. Initial screening for inhibition of growth of two lobster pathogens and for biofilm formation reduced the number of isolates to twenty-four, a ~90% reduction. necessary to determine the effects of these probiotic candidates on the complex microbial communities on the shell.
As noted above, the ability to form a biofilm is an important characteristic for candidate probiotic bacteria. If a candidate probiont can colonize and form a biofilm on the lobster shell, it could prevent the progression of pathogen growth on the lobster shell by producing inhibitory compounds and by physically occupying the lobster shell (1). Two characteristics looked for in a probiotic, production of an inhibitory compound and biofilm formation, both promote probiotic activity. Biomatrix formation results in continuous administration of the inhibitory compound(s) and promotes probiotic activity (23). Additionally, isolation of potential probionts from swabs of lobster carapaces implies that the isolates are adapted to the lobster carapace (1). Previous research in oysters shows that the known probiotic organism, P. inhibens S4Sm, able to protect oysters against challenge with the bacterial pathogen V.
coralliilyticus, is a prolific biofilm former (21), in contrast to the bacterial pathogens V. coralliilyticus and V. anguillarum (OD580 <1) (23). Of the characterized isolates with an OD580 >3, 8 of 11 were Pseudoalteromonas, 2 of 11 were Loktanella and 1 of 11 was Cobetia. The data demonstrate that the lobster isolates are all capable of forming biofilms and some of these isolates form robust biofilms, which suggest that these isolates could be better candidates for use as probionts.
Interestingly, most of the candidate 24 isolates (79%) were members of the genus Pseudoaltermonas. Pseudoaltermonas is found predominantly in the microbiota of a variety of marine fish (26) and has been used as a biocontrol agent in aquaculture (26). Pseudoaltermonas fluorescens has been shown to have inhibitory effect against V. anguillarum when applied to a larval culture of bay scallops (26).
Pseudoaltermonas spp. have been shown to be probiotic organisms in previous studies using various aquaculture systems (26), suggesting that some members of this genus may serve as a probiotic for ESD. One isolate out of the 24 was identified as a member of the genus Bacillus (potentially Bacillus pumilus, had a 98% identity when run through BLASTn), which also has been seen as a successful biocontrol agent (1).
Another example of a probiotic Bacillus is B. pumilus RI06-95, which has been shown to protect larval oysters from mortality after challenge with V. coralliilyticus RE22 (10). Two of the candidate probiotic isolates were identified as Loktanella maritima.
Loktanella spp., however, have been associated with epizootic shell disease lesions in previous ESD studies (5,7,11), and may be considered as a putative pathogen. isolated from the surface biofilm on scleractinian coral and was shown to induce larvae adhesion and metamorphosis (22). Moreover, Loktanella spp. have been identified from tidal flats (17) and from deep floor settlement (14). With the information known about Loktanella spp. it should not be used as a potential probiotic treatment.
The twenty-four candidate probiont isolates were further characterized for their protease and chitinase activity. The H. americanus carapace provides structural and protective properties to the organism (18). The carapace is composed of a chitin matrix with polymers surrounding the matrix (18). Since these candidate probiotic bacteria would be used to treat ESD the determination of the protease and chitinase activities of the isolates would be useful to help assess the potential of these isolates to damage the lobster carapace and should not be included in further testing as probiotic candidates (1). Our data shows many isolates had casein, and or gelatin protease activity ( Table 6). Where only a few isolates had chitinase activity ( Biofilm formation is an important contributor to probiotic activity because it provides mechanisms for probiotic activity, such as, competition for adherence to the surface of the host and preventing contact between the pathogen and the host (23).
Additionally, biofilms of bacteria that secrete antimicrobial compounds (i.e. TDA secreted by P. inhibens S4Sm) contribute to probiotic activity by inhibiting or killing pathogens (23). To determine which substrate would be best to characterize biofilm formation, glass coverslips and lobster shells were used in this assay. Lobster shells were used as another test substrate since the isolates were originally isolated from swabs of lobster carapaces, it was hypothesized that the isolates may form a stronger biofilm on a substrate that it is already known to adhere to (12