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Evaluation of aggregation abilities between commensal fish bacteria and pathogens
Aquaculture 356–357 (2012) 412–414
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Evaluation of aggregation abilities between commensal fish bacteria and pathogens Łukasz Grześkowiak a,⁎, Maria Carmen Collado b, Seppo Salminen a a b
Functional Foods Forum, University of Turku, Turku, Finland Institute of Agrochemistry and Food Science (IATA), Spanish National Research Council (CSIC), Valencia, Spain
a r t i c l e
i n f o
Article history: Received 21 March 2012 Received in revised form 12 April 2012 Accepted 12 April 2012 Available online 21 April 2012 Keywords: Aggregation Commensal bacteria Pathogen Fish Probiotic
a b s t r a c t Bacterial aggregation is related to cell-to-cell adherence between bacteria of the same or different strains. This phenomenon is known to have an important role in microbial interactions and in aquaculture. The aim of the present study was to evaluate the aggregation properties of bacterial candidates for fish probiotics and to assess these properties in viable and non-viable forms with fish bacterial pathogens. The microorganisms Leuconostoc citreum and Enterococcus durans in viable and non-viable forms were evaluated with fish pathogens Aeromonas hydrophila, Aeromonas salmonicida, Edwardsiella tarda and Vibrio anguillarum. L. citreum showed higher auto-aggregation properties in non-viable vs. viable form (P = 0.005). The co-aggregation percentage of viable L. citreum + A. hydrophila differed from those of L. citreum + E. tarda, and L. citreum + V. anguillarum (P = 0.033, P = 0.013, respectively). The co-aggregation abilities with A. salmonicida and E. tarda were higher with non-viable than viable cells of this microorganism (P = 0.023 and P = 0.020, respectively). Non-viable form of E. durans co-aggregated at different percentage with A. hydrophila compared to A. salmonicida and to E. tarda (P = 0.023, P = 0.030, respectively). The results suggest that the ability to auto- and co-aggregate with pathogens can be a useful tool for preliminary screening in order to identify potential probiotic bacteria suitable for use in aquaculture. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Fish species have a variety of commensal bacteria in mucosal surfaces. These regulate the well-being of the fish and any change caused by pathogens or opportunistic microorganisms should reflect the resilience of healthy normal microbiota to outside triggers. Protection of farmed fish from various diseases, especially in early stages of their life, is a prerequisite for increasing production and further development of aquaculture. Thus, an increasing interest in the antimicrobial properties of specific commensal bacteria isolated from fish organisms has been a recent focus of intensive research (Balcázar et al., 2007; Vendrell et al., 2009) to characterise new fish probiotics. The protective role of healthy microbiota may result from the production of inhibitory compounds, competition for nutrients and adhesion sites and protection against pathogens via enhanced natural barrier against their exposure in surfaces such as skin and gills, and gastrointestinal tract. Another target could be facilitating decontamination of water and thereby decreasing exposure to pathogens or harmful chemicals, which might disrupt the mucosal barrier (Brunt et al., 2007; Grześkowiak et al., 2011; Kim and Austin, 2006). This would enhance fish health and also have a positive economic impact. Adhesion to the mucus is often a requirement for colonisation by microorganisms. In case of probiotic bacteria, in order to manifest
beneficial effects they need to achieve adequate numbers through aggregation (Jankovic et al., 2003). Thus the ability to aggregate is a desirable property and an important selection criterion for probiotics. The importance of bacterial aggregation as a first step towards host colonisation has widely been studied for humans (Collado et al., 2007; Ferreira et al., 2011; Palmer et al., 2003). However, only a few studies for microbes of fish origin have been reported (Basson et al., 2008; Jacobs and Chenia, 2009). Therefore, the aim of the present study was to evaluate the aggregation properties of candidate probiotics for fish. For this purpose, we assessed the aggregation properties of Leuconostoc citreum and Enterococcus durans (isolated from the gut of salmon and carp fry, respectively) (Grześkowiak et al., 2011). In particular, the aggregation of species L. citreum and E. durans alone (autoaggregation) and in combination (co-aggregation) and also their co-aggregative properties with fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida, Edwardsiella tarda and Vibrio anguillarum were studied. The impact of viability on aggregation properties of L. citreum and E. durans was also characterised. 2. Materials and methods 2.1. Bacteria and culture conditions
⁎ Corresponding author. Tel.: + 358 2333 6822; fax: + 358 2333 6862. E-mail address: [email protected] (Ł. Grześkowiak). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.04.015
Fish bacterial isolates L. citreum and E. durans and the fish model pathogens A. hydrophila (ATCC 35654), A. salmonicida (ATCC 33658),
Ł. Grześkowiak et al. / Aquaculture 356–357 (2012) 412–414
2.2. Bacterial auto-aggregation assays Auto-aggregation abilities were screened by spectophotometry observation. After incubation, bacterial cells were harvested and washed twice with phosphate-buffered saline (PBS) buffer. The optical density (OD) of bacteria at 600 nm was adjusted to 0.25 ± 0.05 to standardise the bacterial concentration (10 8 cells/ml). The effect of viability on auto-aggregation abilities of tested fish bacterial isolates, L. citreum and E. durans, has also been evaluated. For this purpose, heat-inactivated (95 °C, 10 min) bacterial cells have been used in aggregation assays. Bacterial cell (viable, non-viable forms) suspensions were incubated at 20 °C for 1 h without agitation. Auto-aggregation percentage was measured by A600 nm as: % auto‐aggregation ¼ 1−½At =A0 100 where At represents the absorbance after 1 h of incubation and A0 the absorbance at t = 0. 2.3. Bacterial co-aggregation assays Bacterial suspensions were prepared as described above; A600 nm was adjusted to 0.25 ± 0.05. Equal volumes of cells of different bacteria were mixed and incubated at 20 °C for 1 h without agitation. Percentage of co-aggregation was calculated according to the following formula: % co aggregation ¼ ½ðAmix0 −Amixt Þ=ðAmix0 Þ 100 where Amix0 represents the absorbance of a bacterial mixture at t = 0 and Amixt represents the absorbance of a bacterial mixture after 1 h of incubation. 2.4. Statistical analysis Auto- and co-aggregation assays were determined in five independent experiments. Statistical analyses were done using the SPSS 20.0.0 software (IBM Corp.). Data were subjected to one-way ANOVA and, where appropriate, the Student–Newman–Keuls (S–N–K) test was used for comparison of the means. All results are shown as the mean and the variation is expressed as standard deviation. 3. Results and discussion Aggregation between microorganisms of the same strain (autoaggregation) or between genetically different strains (co-aggregation) is of major importance in several ecological niches. Aggregating bacteria may achieve an adequate mass to form biofilms or adhere to the mucosal surfaces of the host and exert their functions. In addition, the organisms with the ability to aggregate with other bacteria may have an advantage over non-aggregating organisms, which are more easily washed away from the host surfaces. Several studies have focused on the aggregative properties of aquaculture pathogens (Basson et al., 2008; González-Contreras et al., 2011; Jacobs and Chenia, 2009). Our study reports for the first time the aggregative properties of commensal fish bacteria such as L. citreum and E. durans, candidates for fish probiotic bacteria. In the present study, L. citreum showed higher auto-aggregation abilities in non-viable vs. viable form (15.0% ± 6.5 vs. 5.4% ± 2.7, P = 0.005)
25
* 20
% aggregation
E. tarda (ATCC 15947) and V. anguillarum (ATCC 17749) used in previous studies (Grześkowiak et al., 2011) have been evaluated for the aggregation properties. For assays, L. citreum was grown in de Man, Rogosa and Sharpe (MRS) broth (Oxoid, Hampshire, England) and the remaining bacterial strains were grown in Gifu anaerobic medium (GAM; Nissui Pharmaceutical, Tokyo, Japan). All bacteria were grown for 18 h at 20 °C without agitation and under aerobic conditions.
413
15
10
*
5
0
L. citreum
E. durans
L. citreum + E. durans
Fig. 1. Auto- and co-aggregation percentages of L. citreum and E. durans alone and in combination in viable and non-viable forms measured at 1 h of incubation in 20 °C. Results are expressed as mean ± standard deviation (n = 5). Grey bars: viable bacteria, black bars: non-viable bacteria. *Significant difference (P = 0.005) between viable and non-viable forms of bacteria.
(Fig. 1). No such difference was observed between the viability in auto-aggregation of E. durans and co-aggregation of L. citreum with E. durans. This observation would suggest that the cells of L. citreum may be more capable of forming aggregates when used in nonviable forms. This might be due to specific alterations in the bacterial cell surface caused by heating treatment to obtain non-viable cells. In addition, for some probiotic strains, aggregation has been correlated with adhesion (Del Re et al., 2000). Adhesion is known to be a prerequisite for colonisation of the mucosal surfaces in the gastrointestinal tract by many bacteria (Collado et al., 2007). Previously we have reported that viable forms of L. citreum and E. durans adhere to mucus obtained from different sites of carp, salmon and rainbow trout fish (Grześkowiak et al., 2011), thus they are capable of the colonisation. However, studies investigating adhesion properties with non-viable forms of L. citreum and E. durans need further investigation. Aquatic fish pathogens such as Aeromonas spp., Edwardsiella spp. and Vibrio spp. have been shown to form biofilm structures in aquaculture environments and are responsible for numerous infections in fish (Antychowicz, 1996). In our work among the pathogenic strains tested, the most marked ability to aggregate was detected for A. hydrophila (21.0% ± 4.0), followed by E. tarda (4.9% ± 2.6), A. salmonicida (3.8% ± 1.3) and V. anguillarum (3.4% ± 1.7). Co-aggregation has been related to the ability to interact closely with other bacteria (Malik et al., 2003). Both tested commensal fish bacteria, L. citreum and E. durans, showed co-aggregative abilities with specific fish model pathogens but the percentage of aggregation was viability- and pathogen‐dependent. The co-aggregation percentage of viable L. citreum + A. hydrophila differed from that of L. citreum + E. tarda, and L. citreum + V. anguillarum (10.9% ± 6.3 vs. 5.7% ± 1.5, P = 0.033 and 10.9% ± 6.3 vs. 4.8% ± 1.9, P = 0.013, respectively) (Table 1).
Table 1 Co-aggregation percentages of L. citreum (viable and non-viable forms) with pathogens measured at 1 h of incubation in 20 °C. Results are expressed as mean ± standard deviation (n = 5). Pathogen
A. hydrophila A. salmonicida E. tarda V. anguillarum a b c
One-way ANOVA. P = 0.033. P = 0.013.
Pa
L. citreum Viable
Non-viable
10.9 ± 6.3b,c 7.1 ± 0.4 5.7 ± 1.5b 4.8 ± 1.9c
11.1 ± 2.7 7.0 ± 2.3 6.8 ± 2.5 8.5 ± 4.3
0.921 0.947 0.643 0.129
Ł. Grześkowiak et al. / Aquaculture 356–357 (2012) 412–414
414
Table 2 Co-aggregation percentages of E. durans (viable and non-viable forms) with pathogens measured at 1 h of incubation in 20 °C. Results are expressed as mean ± standard deviation (n= 5). Pathogen
A. hydrophila A. salmonicida E. tarda V. anguillarum a b c
Pa
E. durans Viable
Non-viable
5.0 ± 7.3 5.8 ± 0.9 6.2 ± 0.5 3.8 ± 1.5
5.8 ± 1.6b,c 0.3 ± 0.8b 0.6 ± 1.1c 3.4 ± 3.0
0.741 0.023 0.020 0.843
One-way ANOVA. P = 0.023. P = 0.030.
aggregate and co-aggregate is a desirable property for specific microorganisms used as probiotics in health-promoting applications. Further studies should focus on the inhibitory properties of L. citreum and E. durans against fish pathogens, tolerance and survival to gastrointestinal transit and in vivo studies in order to evaluate their selection criteria for probiotics. Taken together, the results suggest that the ability of candidate fish probiotics to auto- and co-aggregate with pathogens is an important selection criterion. The adhesion and pathogen exclusion properties can form a useful tool for screening of host commensal microbiota in order to identify potential probiotic bacteria suitable for use in aquaculture. References
Table 3 Co-aggregation percentages of L. citreum in combination with E. durans (viable and non-viable forms) with pathogens measured at 1 h of incubation in 20 °C. Results are expressed as mean ± standard deviation (n = 5). Pathogen
A. hydrophila A. salmonicida E. tarda V. anguillarum a
Pa
L. citreum + E. durans Viable
Non-viable
7.0 ± 3.6 3.6 ± 3.5 6.5 ± 0.3 5.4 ± 1.7
5.8 ± 1.6 0.3 ± 0.8 0.6 ± 1.1 3.4 ± 3.0
0.817 0.667 0.142 0.921
One-way ANOVA.
When non-viable cells of E. durans were used, the co-aggregation abilities with A. salmonicida and E. tarda were significantly higher than in the presence of viable cells (5.8% ± 0.9 vs. 0.3% ± 0.8, P = 0.023 and 6.2% ± 0.5 vs. 0.6% ± 1.1, P = 0.020, respectively) (Table 2). Non-viable form of E. durans co-aggregated at significantly different percentage with A. hydrophila compared to A. salmonicida and to E. tarda (5.8% ± 1.6 vs. 0.3% ± 0.8, P = 0.023 and 5.8% ± 1.6 vs. 0.6% ± 1.1, P = 0.030, respectively) (Table 3). Co-aggregation ability of L. citreum and E. durans with potential fish pathogens such as those used in the present study could thus contribute to the potential probiotic properties ascribed to specific bacteria (Collado et al., 2007; Gueimonde and Salminen, 2006; Ouwehand and Vesterlund, 2003). Probiotic bacteria through the co-aggregation with pathogens may prevent the development of infection by avoiding the contact of pathogen cells with the host surfaces (prevention of colonisation) but also by inhibiting their metabolic activity (by close contact with pathogen cells) (Collado et al., 2007; Ferreira et al., 2011). The co-aggregation properties of commensal bacteria such as L. citreum and E. durans used in the present study, with fish pathogens, as well as their ability to exclude adhering pathogens from the mucosal surfaces are of importance for therapeutic manipulation of the aberrant intestinal microbiota. Consequently, the ability to auto-
Antychowicz, J., 1996. Choroby i zatrucia ryb. Wydawnictwo SGGW. Balcázar, J.L., Vendrell, D., de Blas, I., Ruiz-Zarzuela, I., Gironés, O., Muzquiz, J.L., 2007. In vitro competitive adhesion and production of antagonistic compounds by lactic acid bacteria against fish pathogens. Veterinary Microbiology 122, 373–380. Basson, A., Flemming, L.A., Chenia, H.Y., 2008. Evaluation of adherence, hydrophobicity, aggregation, and biofilm development of Flavobacterium johnsoniae-like isolates. Microbial Ecology 55, 1–14. Brunt, J., Newaj-Fyzul, A., Austin, B., 2007. The development of probiotics for the control of multiple bacterial diseases of rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 30, 573–579. Collado, M.C., Meriluoto, J., Salminen, S., 2007. Measurement of aggregation properties between probiotics and pathogens: in vitro evaluation of different methods. Journal of Microbiological Methods 71, 71–74. Del Re, B., Sgorbati, B., Miglioli, M., Palenzona, D., 2000. Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Letters in Applied Microbiology 31, 438–442. Ferreira, C.L., Grześkowiak, Ł., Collado, M.C., Salminen, S., 2011. In vitro evaluation of Lactobacillus gasseri strains of infant origin on adhesion and aggregation of specific pathogens. Journal of Food Protection 74, 1482–1487. González-Contreras, A., Magariños, B., Godoy, M., Irgang, R., Toranzo, A.E., AvendañoHerrera, R., 2011. Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L. Journal of Fish Diseases 34, 203–215. Grześkowiak, Ł., Collado, M.C., Vesterlund, S., Mazurkiewicz, J., Salminen, S., 2011. Adhesion abilities of commensal fish bacteria by use of mucus model system: quantitative analysis. Aquaculture 318, 33–36. Gueimonde, M., Salminen, S., 2006. New methods for selecting and evaluating probiotics. Digestive and Liver Disease 38, 242–247. Jacobs, H., Chenia, H.Y., 2009. Biofilm-forming capacity, surface hydrophobicity and aggregation characteristics of Myroides odoratus isolated from South African Oreochromis mossambicus fish. Journal of Applied Microbiology 107, 1957–1966. Jankovic, I., Ventura, M., Meylan, V., Rouvet, M., Elli, M., Zink, R., 2003. Contribution of aggregation-promoting factor to maintenance of cell shape in Lactobacillus gasseri 4B2. Journal of Bacteriology 185, 3288–3296. Kim, D.H., Austin, B., 2006. Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish & Shellfish Immunology 21, 513–524. Malik, A., Sakamoto, M., Hanazaki, S., Osawa, M., Suzuki, T., Tochigi, M., Kakii, K., 2003. Coaggregation among nonflocculating bacteria isolated from activated sludge. Applied and Environmental Microbiology 69, 6056–6063. Ouwehand, A., Vesterlund, S., 2003. Health aspects of probiotics. IDrugs 6, 573–580. Palmer Jr., R.J., Gordon, S.M., Cisar, J.O., Kolenbrander, P.E., 2003. Coaggregation-mediated interactions of streptococci and actinomyces detected in initial human dental plaque. Journal of Bacteriology 185, 3400–3409. Vendrell, D., Balcázar, J.L., Calvo, A.C., de Blas, I., Ruiz-Zarzuela, I., Gironés, O., Múzquiz, J.L., 2009. Quantitative analysis of bacterial adhesion to fish tissue. Colloids and Surfaces. B, Biointerfaces 71, 331–333.
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Evaluation of aggregation abilities between commensal fish bacteria and pathogens Łukasz Grześkowiak a,⁎, Maria Carmen Collado b, Seppo Salminen a a b
Functional Foods Forum, University of Turku, Turku, Finland Institute of Agrochemistry and Food Science (IATA), Spanish National Research Council (CSIC), Valencia, Spain
a r t i c l e
i n f o
Article history: Received 21 March 2012 Received in revised form 12 April 2012 Accepted 12 April 2012 Available online 21 April 2012 Keywords: Aggregation Commensal bacteria Pathogen Fish Probiotic
a b s t r a c t Bacterial aggregation is related to cell-to-cell adherence between bacteria of the same or different strains. This phenomenon is known to have an important role in microbial interactions and in aquaculture. The aim of the present study was to evaluate the aggregation properties of bacterial candidates for fish probiotics and to assess these properties in viable and non-viable forms with fish bacterial pathogens. The microorganisms Leuconostoc citreum and Enterococcus durans in viable and non-viable forms were evaluated with fish pathogens Aeromonas hydrophila, Aeromonas salmonicida, Edwardsiella tarda and Vibrio anguillarum. L. citreum showed higher auto-aggregation properties in non-viable vs. viable form (P = 0.005). The co-aggregation percentage of viable L. citreum + A. hydrophila differed from those of L. citreum + E. tarda, and L. citreum + V. anguillarum (P = 0.033, P = 0.013, respectively). The co-aggregation abilities with A. salmonicida and E. tarda were higher with non-viable than viable cells of this microorganism (P = 0.023 and P = 0.020, respectively). Non-viable form of E. durans co-aggregated at different percentage with A. hydrophila compared to A. salmonicida and to E. tarda (P = 0.023, P = 0.030, respectively). The results suggest that the ability to auto- and co-aggregate with pathogens can be a useful tool for preliminary screening in order to identify potential probiotic bacteria suitable for use in aquaculture. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Fish species have a variety of commensal bacteria in mucosal surfaces. These regulate the well-being of the fish and any change caused by pathogens or opportunistic microorganisms should reflect the resilience of healthy normal microbiota to outside triggers. Protection of farmed fish from various diseases, especially in early stages of their life, is a prerequisite for increasing production and further development of aquaculture. Thus, an increasing interest in the antimicrobial properties of specific commensal bacteria isolated from fish organisms has been a recent focus of intensive research (Balcázar et al., 2007; Vendrell et al., 2009) to characterise new fish probiotics. The protective role of healthy microbiota may result from the production of inhibitory compounds, competition for nutrients and adhesion sites and protection against pathogens via enhanced natural barrier against their exposure in surfaces such as skin and gills, and gastrointestinal tract. Another target could be facilitating decontamination of water and thereby decreasing exposure to pathogens or harmful chemicals, which might disrupt the mucosal barrier (Brunt et al., 2007; Grześkowiak et al., 2011; Kim and Austin, 2006). This would enhance fish health and also have a positive economic impact. Adhesion to the mucus is often a requirement for colonisation by microorganisms. In case of probiotic bacteria, in order to manifest
beneficial effects they need to achieve adequate numbers through aggregation (Jankovic et al., 2003). Thus the ability to aggregate is a desirable property and an important selection criterion for probiotics. The importance of bacterial aggregation as a first step towards host colonisation has widely been studied for humans (Collado et al., 2007; Ferreira et al., 2011; Palmer et al., 2003). However, only a few studies for microbes of fish origin have been reported (Basson et al., 2008; Jacobs and Chenia, 2009). Therefore, the aim of the present study was to evaluate the aggregation properties of candidate probiotics for fish. For this purpose, we assessed the aggregation properties of Leuconostoc citreum and Enterococcus durans (isolated from the gut of salmon and carp fry, respectively) (Grześkowiak et al., 2011). In particular, the aggregation of species L. citreum and E. durans alone (autoaggregation) and in combination (co-aggregation) and also their co-aggregative properties with fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida, Edwardsiella tarda and Vibrio anguillarum were studied. The impact of viability on aggregation properties of L. citreum and E. durans was also characterised. 2. Materials and methods 2.1. Bacteria and culture conditions
⁎ Corresponding author. Tel.: + 358 2333 6822; fax: + 358 2333 6862. E-mail address: [email protected] (Ł. Grześkowiak). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.04.015
Fish bacterial isolates L. citreum and E. durans and the fish model pathogens A. hydrophila (ATCC 35654), A. salmonicida (ATCC 33658),
Ł. Grześkowiak et al. / Aquaculture 356–357 (2012) 412–414
2.2. Bacterial auto-aggregation assays Auto-aggregation abilities were screened by spectophotometry observation. After incubation, bacterial cells were harvested and washed twice with phosphate-buffered saline (PBS) buffer. The optical density (OD) of bacteria at 600 nm was adjusted to 0.25 ± 0.05 to standardise the bacterial concentration (10 8 cells/ml). The effect of viability on auto-aggregation abilities of tested fish bacterial isolates, L. citreum and E. durans, has also been evaluated. For this purpose, heat-inactivated (95 °C, 10 min) bacterial cells have been used in aggregation assays. Bacterial cell (viable, non-viable forms) suspensions were incubated at 20 °C for 1 h without agitation. Auto-aggregation percentage was measured by A600 nm as: % auto‐aggregation ¼ 1−½At =A0 100 where At represents the absorbance after 1 h of incubation and A0 the absorbance at t = 0. 2.3. Bacterial co-aggregation assays Bacterial suspensions were prepared as described above; A600 nm was adjusted to 0.25 ± 0.05. Equal volumes of cells of different bacteria were mixed and incubated at 20 °C for 1 h without agitation. Percentage of co-aggregation was calculated according to the following formula: % co aggregation ¼ ½ðAmix0 −Amixt Þ=ðAmix0 Þ 100 where Amix0 represents the absorbance of a bacterial mixture at t = 0 and Amixt represents the absorbance of a bacterial mixture after 1 h of incubation. 2.4. Statistical analysis Auto- and co-aggregation assays were determined in five independent experiments. Statistical analyses were done using the SPSS 20.0.0 software (IBM Corp.). Data were subjected to one-way ANOVA and, where appropriate, the Student–Newman–Keuls (S–N–K) test was used for comparison of the means. All results are shown as the mean and the variation is expressed as standard deviation. 3. Results and discussion Aggregation between microorganisms of the same strain (autoaggregation) or between genetically different strains (co-aggregation) is of major importance in several ecological niches. Aggregating bacteria may achieve an adequate mass to form biofilms or adhere to the mucosal surfaces of the host and exert their functions. In addition, the organisms with the ability to aggregate with other bacteria may have an advantage over non-aggregating organisms, which are more easily washed away from the host surfaces. Several studies have focused on the aggregative properties of aquaculture pathogens (Basson et al., 2008; González-Contreras et al., 2011; Jacobs and Chenia, 2009). Our study reports for the first time the aggregative properties of commensal fish bacteria such as L. citreum and E. durans, candidates for fish probiotic bacteria. In the present study, L. citreum showed higher auto-aggregation abilities in non-viable vs. viable form (15.0% ± 6.5 vs. 5.4% ± 2.7, P = 0.005)
25
* 20
% aggregation
E. tarda (ATCC 15947) and V. anguillarum (ATCC 17749) used in previous studies (Grześkowiak et al., 2011) have been evaluated for the aggregation properties. For assays, L. citreum was grown in de Man, Rogosa and Sharpe (MRS) broth (Oxoid, Hampshire, England) and the remaining bacterial strains were grown in Gifu anaerobic medium (GAM; Nissui Pharmaceutical, Tokyo, Japan). All bacteria were grown for 18 h at 20 °C without agitation and under aerobic conditions.
413
15
10
*
5
0
L. citreum
E. durans
L. citreum + E. durans
Fig. 1. Auto- and co-aggregation percentages of L. citreum and E. durans alone and in combination in viable and non-viable forms measured at 1 h of incubation in 20 °C. Results are expressed as mean ± standard deviation (n = 5). Grey bars: viable bacteria, black bars: non-viable bacteria. *Significant difference (P = 0.005) between viable and non-viable forms of bacteria.
(Fig. 1). No such difference was observed between the viability in auto-aggregation of E. durans and co-aggregation of L. citreum with E. durans. This observation would suggest that the cells of L. citreum may be more capable of forming aggregates when used in nonviable forms. This might be due to specific alterations in the bacterial cell surface caused by heating treatment to obtain non-viable cells. In addition, for some probiotic strains, aggregation has been correlated with adhesion (Del Re et al., 2000). Adhesion is known to be a prerequisite for colonisation of the mucosal surfaces in the gastrointestinal tract by many bacteria (Collado et al., 2007). Previously we have reported that viable forms of L. citreum and E. durans adhere to mucus obtained from different sites of carp, salmon and rainbow trout fish (Grześkowiak et al., 2011), thus they are capable of the colonisation. However, studies investigating adhesion properties with non-viable forms of L. citreum and E. durans need further investigation. Aquatic fish pathogens such as Aeromonas spp., Edwardsiella spp. and Vibrio spp. have been shown to form biofilm structures in aquaculture environments and are responsible for numerous infections in fish (Antychowicz, 1996). In our work among the pathogenic strains tested, the most marked ability to aggregate was detected for A. hydrophila (21.0% ± 4.0), followed by E. tarda (4.9% ± 2.6), A. salmonicida (3.8% ± 1.3) and V. anguillarum (3.4% ± 1.7). Co-aggregation has been related to the ability to interact closely with other bacteria (Malik et al., 2003). Both tested commensal fish bacteria, L. citreum and E. durans, showed co-aggregative abilities with specific fish model pathogens but the percentage of aggregation was viability- and pathogen‐dependent. The co-aggregation percentage of viable L. citreum + A. hydrophila differed from that of L. citreum + E. tarda, and L. citreum + V. anguillarum (10.9% ± 6.3 vs. 5.7% ± 1.5, P = 0.033 and 10.9% ± 6.3 vs. 4.8% ± 1.9, P = 0.013, respectively) (Table 1).
Table 1 Co-aggregation percentages of L. citreum (viable and non-viable forms) with pathogens measured at 1 h of incubation in 20 °C. Results are expressed as mean ± standard deviation (n = 5). Pathogen
A. hydrophila A. salmonicida E. tarda V. anguillarum a b c
One-way ANOVA. P = 0.033. P = 0.013.
Pa
L. citreum Viable
Non-viable
10.9 ± 6.3b,c 7.1 ± 0.4 5.7 ± 1.5b 4.8 ± 1.9c
11.1 ± 2.7 7.0 ± 2.3 6.8 ± 2.5 8.5 ± 4.3
0.921 0.947 0.643 0.129
Ł. Grześkowiak et al. / Aquaculture 356–357 (2012) 412–414
414
Table 2 Co-aggregation percentages of E. durans (viable and non-viable forms) with pathogens measured at 1 h of incubation in 20 °C. Results are expressed as mean ± standard deviation (n= 5). Pathogen
A. hydrophila A. salmonicida E. tarda V. anguillarum a b c
Pa
E. durans Viable
Non-viable
5.0 ± 7.3 5.8 ± 0.9 6.2 ± 0.5 3.8 ± 1.5
5.8 ± 1.6b,c 0.3 ± 0.8b 0.6 ± 1.1c 3.4 ± 3.0
0.741 0.023 0.020 0.843
One-way ANOVA. P = 0.023. P = 0.030.
aggregate and co-aggregate is a desirable property for specific microorganisms used as probiotics in health-promoting applications. Further studies should focus on the inhibitory properties of L. citreum and E. durans against fish pathogens, tolerance and survival to gastrointestinal transit and in vivo studies in order to evaluate their selection criteria for probiotics. Taken together, the results suggest that the ability of candidate fish probiotics to auto- and co-aggregate with pathogens is an important selection criterion. The adhesion and pathogen exclusion properties can form a useful tool for screening of host commensal microbiota in order to identify potential probiotic bacteria suitable for use in aquaculture. References
Table 3 Co-aggregation percentages of L. citreum in combination with E. durans (viable and non-viable forms) with pathogens measured at 1 h of incubation in 20 °C. Results are expressed as mean ± standard deviation (n = 5). Pathogen
A. hydrophila A. salmonicida E. tarda V. anguillarum a
Pa
L. citreum + E. durans Viable
Non-viable
7.0 ± 3.6 3.6 ± 3.5 6.5 ± 0.3 5.4 ± 1.7
5.8 ± 1.6 0.3 ± 0.8 0.6 ± 1.1 3.4 ± 3.0
0.817 0.667 0.142 0.921
One-way ANOVA.
When non-viable cells of E. durans were used, the co-aggregation abilities with A. salmonicida and E. tarda were significantly higher than in the presence of viable cells (5.8% ± 0.9 vs. 0.3% ± 0.8, P = 0.023 and 6.2% ± 0.5 vs. 0.6% ± 1.1, P = 0.020, respectively) (Table 2). Non-viable form of E. durans co-aggregated at significantly different percentage with A. hydrophila compared to A. salmonicida and to E. tarda (5.8% ± 1.6 vs. 0.3% ± 0.8, P = 0.023 and 5.8% ± 1.6 vs. 0.6% ± 1.1, P = 0.030, respectively) (Table 3). Co-aggregation ability of L. citreum and E. durans with potential fish pathogens such as those used in the present study could thus contribute to the potential probiotic properties ascribed to specific bacteria (Collado et al., 2007; Gueimonde and Salminen, 2006; Ouwehand and Vesterlund, 2003). Probiotic bacteria through the co-aggregation with pathogens may prevent the development of infection by avoiding the contact of pathogen cells with the host surfaces (prevention of colonisation) but also by inhibiting their metabolic activity (by close contact with pathogen cells) (Collado et al., 2007; Ferreira et al., 2011). The co-aggregation properties of commensal bacteria such as L. citreum and E. durans used in the present study, with fish pathogens, as well as their ability to exclude adhering pathogens from the mucosal surfaces are of importance for therapeutic manipulation of the aberrant intestinal microbiota. Consequently, the ability to auto-
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