- Email: [email protected]
Comparative study of cytotoxicity of Aeromonas spp. on four different cell lines
ARTICLE IN PRESS
Comparative Immunology, Microbiology & Infectious Diseases 29 (2006) 232–240 www.elsevier.com/locate/cimid
Comparative study of cytotoxicity of Aeromonas spp. on four different cell lines S. Ghataka, R.K. Agarwalb,, K.N. Bhilegaonkara a
Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar 243 122, India b National Salmonella Centre, Bacteriology and Mycology Division, Indian Veterinary Research Institute, Izatnagar 243 122, India Accepted 17 June 2006
Abstract In vitro cytotoxicity is an important virulence property of motile mesophilic Aeromonas species. Cell-free supernatant prepared from 55 Aeromonas isolates including one A. hydrophila type strain (MTCC 646) were examined for their cytotoxic potential on four different cell lines (Vero, BHK-21, MDBK, B 95a). Results of the study revealed cytotoxic potential in 92.72% of the isolates. Analysis of data exposed significant variation among isolates in respect of their cytotoxicity. Vero cells proved to be most sensitive t o aeromonal toxins and B 95a cells showed significantly (Po0:01) lower response compared to other cell lines. Sensitivities of BHK-21 and MDBK cell lines were in between Vero and B 95a. r 2006 Elsevier Ltd. All rights reserved. Keywords: Aeromonas; Cytotoxicity; Vero; MDBK; BHK; B 95a
Re´sume´ Cytotoxicity in vitro est une proprie´te´ de virulence importante d’espe`ce de Aeromonas de mesophilic mobile. La cellule libe`re supernatant a pre´pare´ de 55 Aeromonas isole inclut un la Corresponding author.
E-mail address: [email protected] (R.K. Agarwal). 0147-9571/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2006.06.005
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tension de type de A. hydrophila (MTCC 646) ont e´te´ examine´ pour leur potentiel de cytotoxic sur quatre lignes de cellule diffe´rentes (Vero, BHK-21, MDBK, LE B 95a). Les re´sultats de l’e´tude ont re´ve´le´ le potentiel de cytotoxic dans 92,72% de l’isole. L’analyse de donne´es a expose´ la variation significative parmi isole dans le respect de leur cytotoxicity. Les cellules de Vero se sont re´ve´le´es le plus sensible aux toxines de aeromonal et au B 95a cellules montre´es significativement (Po0:01) abaisse la re´ponse en comparaison des autres lignes de cellule. Les sensibilite´s de BHK-21 et lignes de cellule de MDBK e´taient entre Vero et le B 95a. r 2006 Elsevier Ltd. All rights reserved. Mots-cle´: Aeromonas, Cytotoxicity, Vero, MDBK, BHK, B 95a
1. Introduction Motile and mesophilic members of the genus Aeromonas are pathogens of humans and animals [1]. Besides their role in gastrointestinal infections they have been implicated in a multitude of infections including septicaemia, wound infections, urinary tract infections, myocarditis ocular complications, osteomyelitis, meningitis, peritonitis cholangitis, etc. [2]. The ability of the organisms to inflict diseases in animals and humans is attributable to an impressive array of virulence factors [3]. The virulence factors of Aeromonas may be broadly classified into extracellular factors, structural features and cell-associated factors [2]. Among the most discussed of these are the extracellular factors, which include haemolysins, enterotoxin, protease, amylase and chitinase [2]. Apart from the specific roles played by these factors in Aeromonas-induced disease processes, one of the most important virulence property of haemolysins and enterotoxin of aeromonads involves exhibition of cytotoxicity in vitro. To assess the cytotoxic potential, of the organisms different cell lines have been used by the researchers [3]. The cell lines that have been used for the purpose ranges from mammalian cell lines viz. Vero, Int 407, Chimp liver, Chinese hamster ovary, HeLa, HEp-2, Y1 adrenal to fish cell line, epithelioma papulosum of carp (Cyprinus carpio) [4–10]. However, different cell lines vary in their sensitivities to different toxins thereby making the comparison of different studies difficult. Nevertheless, reports about comparative efficacy of different cell lines to aeromonal toxins are either very rare or absent. In addition, a majority of researchers employed only one kidney derived cell line (Vero) for studying cytotoxicity of Aeromonas spp. leaving other cell lines of similar origin uninvestigated [11–18]. And, relatively newly established cell lines of lymphoid origin e.g. B 95a [19] were even less explored in the context of cytotxic behaviour of Aeromonas spp. Therefore, the present study was undertaken to assess and compare sensitivities of different cell lines to toxins of Aeromonas spp. and to investigate the effect of aeromonal toxins on a lymphoid cell line.
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2. Materials and methods 2.1. Bacterial strains Included in the study was a set of 55 strains of Aeromonas spp. of diverse origin (Table 1). A. hydrophila type strain, MTCC 646 was obtained from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. Rest of the strains were from Veterinary Public Health Division, Indian Veterinary Research Institute, Izatnagar, India. All strains of Aeromonas were grown on Trypticase soy agar (Difco) at 37 1C for 18–24 h. A known verocytotoxic Escherichia coli strain maintained at our laboratory was used as positive control for cytotoxicity assay and was grown on Eosine methylene blue agar. For long-term storage15% (v/v) glycerol–Trypticase soy broth was used and stored at 70 1C. 2.2. Preparation of cell-free supernatant (CFS) Cell-free supernatants (CFSs) of the isolates were prepared as per the method described by Gray et al. [20] with necessary modifications. The organisms were grown in 5 ml of brain heart infusion (BHI) broth (Hi-Media) fortified with 0.6% (w/v) yeast extract and 3% (w/v) cassamino acid. Following an incubation of 12 h at 37 1C, 50 ml of supplemented BHI broth was inoculated with the initial broth culture and incubated in a shaker set at 200 rpm at 37 1C. After incubation of 14–16 h, while the growth was in mid-log phase the broth cultures were taken to 50 ml sterilized tubes and centrifuged at 10000 g for 30 min at 4 1C. The supernatants were transferred to fresh tubes and filtered through 0.22 mm filters (Sartorius, Germany). The sterility of each preparation was checked by streaking onto nutrient agar and incubating at 37 1C for 24 h. Similarly, the pH of the filtrates were checked and stored at 20 1C till further use.
Table 1 Aeromonas strains used in the study Strains
A. A. A. A. A. A.
hydrophila caviae veronii bt sobria veronii bt veronii jandaei trota a
Origin Clinical
Chicken
Fish
Unknowna
2 5 1 — — 2
5 6 — — — —
5 3 19 1 — —
1 3 1 — 1 —
Strains of unknown origin were Aeromonas strains maintained in the Division of VPH, IVRI, India.
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2.3. Assay for cytotoxicity Cytotoxic potential of the isolates were tested on four different cell lines viz. African green monkey kidney cells (Vero), Madin darby bovine kidney (MDBK) cells, Baby hamster kidney cells (BHK-21) and Marmoset B lymphoblastoid cells (B 95a) [19] as per the method of Barer et al. [6] with suitable adaptations. The cell lines except B 95a were propagated in Eagle’s minimum essential medium (EMEM, Sigma) with 10% and 2% foetal bovine serum (FBS, Sigma) during growth and maintenance, respectively. The B 95a cells were propagated in Rosewell Parker Memorial Institute (RPMI) 1640 medium (Sigma, USA) with 10% and 5% FBS during growth and maintenance, respectively. The toxin activity of the cell-free preparations was assayed in 96 well tissue culture plates (Greiner, Germany). In each well cells were grown to confluent monolayers. In a 96 chamber deep well plate serial two-fold dilutions of each CFS were prepared in the same medium in which cells were grown. The medium from cell culture plate was discarded and 100 ml of each dilution of each CFS was added in the wells of the cell culture plates in triplicate. In each plate CFS of verocytotoxic E. coli and a preparation of BHI broth (Hi Media) with 0.6% (w/v) yeast extract and 3% (w/v) cassamino acid, processed similarly as with other CFS preparation, were included as positive and negative controls, respectively. The plates were incubated at 37 1C in 5% CO2 atmosphere and examined six hourly in an inverted microscope (Nikon, Japan) for up to 72 h for characteristic cytopathic effect (CPE). CFS preparation inducing CPE at least up to 1:8 dilution or more in 50% or more cells were recorded as positive and further titrated if necessary. For any individual CFS preparation, the reciprocal of the highest dilution inducing CPE in 50% or more cells was recorded as its cytotoxicity titre. Following full incubation, the plates were stained with 1% crystal violet after fixing with 10% (v/v) formaldehyde solution. After staining, they were observed macroscopically and microscopically and the results were compared with previous microscopic observation. 2.4. Analysis of results Data generated from cytotoxicity assays were subjected to statistical analysis as per Snedecor and Cochran [21]. Since values of cytotoxicity titres were not normally distributed, the data were transformed to logarithmic scale with base 2 following addition of unity (1) to individual data. Log-transformed data were statistically analysed employing the following statistical model: Y ijk ¼ m þ Si þ C j þ ðs cÞij þ eijk , where Yijk is the log2 titre of ith isolate, jth cell line, kth observation, m the overall mean, Si the effect of ith isolate, Cj the effect of jth cell line, (s c)ij the interaction between ith isolate and jth cell line, eijk the experimental error (eijk is independently, randomly and normally distributed with mean 0 and variance s2).
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To make multiple comparisons of mean titres, least significant difference test was conducted [21].
3. Results Cytotoxic potential of Aeromonas isolates was studied on four different cell lines including BHK-21, Vero, MDBK and B 95a cell lines. Microscopic examination of different cells following exposure to and incubation with CFS prepared from cytotoxic isolates revealed number of changes including rounding and shrivelling of cells, loss of cytoplasmic extensions, disorganization of cell sheets, shrinking of cytoplasms. Cytotoxicity was induced by 92.72% of the isolates in all four cell culture systems. Except for one isolate of chicken origin and three of unknown origin, all other isolates from all sources were found to be cytotoxic (Table 2). Species differentiation of cytotoxic isolates indicated 82.35% of A. caviae, 95.24% of A. veronii bt sobria and all isolates of other species were cytotoxic in all four cell lines tested. Statistical analysis to differentiate between sensitivity among cell culture systems as well as, between cytotoxic potentials of isolates indicated significant difference Table 2 Cytotoxic potential of Aeromonas isolates Source/species
No. of isolates examined
No. of cytotoxic isolates (%)
Canine A. caviae A. veronii bt sobria
4 3 1
4 (100.00) 3 (100.00) 1 (100.00)
Human A. hydrophila A. caviae A. trota
6 2 2 2
6 2 2 2
Chicken A. hydrophila A. caviae
11 5 6
10 (90.91) 5 (100.00) 5 (83.33)
Fish A. hydrophila A. caviae A. veronii bt sobria A. veronii bt veronii
28 5 3 19 1
28 5 3 19 1
(100.00) (100.00) (100.00) (100.00) (100.00)
6 1 3 1 1
3 1 1 1 0
(50.00) (100.00) (33.33) (100.00) (0.00)
Unknowna A. hydrophila(MTCC646) A. caviae A. jandaei veronii bt sobria Total
55
(100.00) (100.00) (100.00) (100.00)
51 (92.72)
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Table 3 Analysis of variance of cytotoxicity titres (log2 titre) for different Aeromonas isolates under various cell culture systems Source of variation
Degree of freedom
Mean square
Isolates Cell lines Isolates cell lines Error
54 3 162 440
6.603 2.12 0.974 0.278
Po0:01.
Table 4 Mean and standard error (SE) of cytotoxicity titres (log2 titre) for different cell culture systems Cell line (s)
Mean7SE
BHK-21 Vero MDBK B 95a
4.1570.04b 4.2570.04b 4.1370.04b 3.9870.04a
Means having same superscripts do not differ significantly (Po0:01).
12
Isolates
10 8 6
C
4
5.05
4.95
4.85
4.75
4.65
4.55
4.45
4.35
4.25
4.15
4.05
3.95
3.85
3.75
3.65
3.55
3.45
3.35
3.25
3.15
3.05
2.95
2.85
0
2.75
2
Titre (s) Fig. 1. Frequency distribution of cytotoxicity titres (log2 titre) of Aeromonas isolates.
(Po0:01) between isolates and between cell lines (Table 3). Individual Aeromonas isolates differed significantly (Po0:01) among themselves in inducing cytotoxicity on four cell culture systems. Also four cell lines showed variation (significant at Po0:01) in their response to cytotoxins liberated by isolates. Interestingly, the interaction between isolates and cell lines was also significantly variable (Po0:01). This indicated that the relative ability of individual isolates to induce cytotoxicity was not uniform across four different cell culture systems. Mean cytotoxicity titres individual cell lines taking all isolates into account were in the range of 3.98–4.2570.04 (Table 4). Of the four cell lines tested, Vero cells expressed highest
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sensitivity with mean titre being 4.2570.04 and the least sensitive cell line as apparent from the results was B 95a (mean titre ¼ 3.9870.04). Other two cell lines (BHK-21 and MDBK) were of almost similar sensitivity against the CFS prepared from the isolates under study. From frequency distribution of mean cytotoxicity titres of isolates the wide variation in titres among isolates was evident. Highest titre (log2 titre) was 5.04, whereas lowest was 2.75 (Fig. 1). As much as 10 isolates induced high degree of cytotoxicity (log2 titre ¼ 5.04) over all the four cell culture systems, while low titred (log2 titre o3) isolates were four in number. Following these were two major groups of isolates with mean titres of about 4.05 and 4.75, respectively. Thirty-six percent of the isolates were highly cytotoxic across all four cell lines showing cytotoxicity titres of 44.00 (log2 titre).
4. Discussion In the present study, we have presented the results of testing of Aeromonas CFS on four different cell lines. Three of these cell lines were basically kidney derived and B 95a was a B lymphocyte-derived cell line [19]. Most (92.72%) of the isolates studied were cytotoxic. Similar high prevalence of cytotoxic potentials as observed in the present study among aeromonads of diverse origin has been reported by others too [11,22,23]. Analysis of results of cytotoxicity studies revealed significant (Po0:01) differences among individual isolates as well as among different cell lines. Also the cell line–isolate interaction varied significantly (Po0:01) implying non-uniform response of individual isolates across four different cell lines tested. This substantiates our previous assumption about incomparability of data generated from cytotoxicity testing on different cell lines. Our results showed that Vero cells were most sensitive to CFS preparations from isolates, followed this were BHK-21 and MDBK with no significant difference between them. But B 95a cells were significantly (Po0:01) less responsive. Perhaps this is why a large number of researchers preferred Vero cell for cytotoxicity testing [11–18]. Interestingly, low response of B 95a cells, which are lymphocytic cells, has a deeper implication. B lymphocytes are known to be responsive to bacterial lipopolysaccharides [24]. In our study during preparation of CFS, cells were centrifuged out during the mid-log growth phase to keep the cell lysis and thereby endotoxin release in the culture broth to a minimum. The low response of B 95a cells indicates that CFS preparation was indeed relatively low in endotoxin content. This served as an internal control over the entire cell culture study implicating that response in other cell lines was actually due to aeromonal cytotoxins. However, there are also chances of cell lysis during growth of bacterial cells in broth culture. Since polymyxin B is known to inhibit activities of lipopolysaccharide in other cell cultures [25,26] treatment of CFS preparations with polymyxin B before testing them on cell lines will be an interesting approach for further studies.
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Finally, response of B 95a cells to CFS from Aeromonas isolates indicates possible presence of some lymphotoxic factor(s) yet to be characterized. And to our knowledge this study documents the first report of cytotoxicity testing of aeromonads on a lymphocytic cell line. Frequency distribution of cytotoxicity titres of the isolates indicated that this behaviour was widely prevalent and highlights possible public health risks lurking among Aeromonas spp. studied. In conclusion, we suggest use of Vero cells in preference to other cell lines examined for testing of cytotoxic potential of Aeromonas spp.
Acknowledgement Authors are thankful to Director, IVRI, Dr. RK. Singh and Dr. V. Bhanuprakash for providing necessary facilities and Dr. M. Nath for his help in data analysis.
References [1] Janda JM, Abott SL. Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations and unanswered questions. Clin Infect Dis 1998;27:332–44. [2] Janda JM. Recent advances in the study of the taxonomy, pathogenicity and infectious syndromes associated with the genus Aeromonas. Clin Microbiol Rev 1991;4:397–410. [3] Cahill MM. Virulence factors in motile Aeromonas species. J Appl Bacteriol 1990;69:1–6. [4] Boulanger Y, Lallier R, Consineau G. Isolation of enterotoxigenic Aeromonas from fish. Can J Microbiol 1977;23:1161–4. [5] Ljungh A, Eneroth P, Wadstrom T. Steroid secretion in adrenal Y1 cells exposed to Aeromonas hydrophila enterotoxin. FEMS Microbiol Lett 1982;15:141–4. [6] Barer MR, Millership SE, Tabaqchali S. Relationship of toxin production to species in the genus Aeromonas. J Med Microbiol 1986;22:302–9. [7] Kindschuh M, Pickering LK, Cleary TG, Ruiz-Palacios G. Clinical and biochemical significance of toxin production by Aeromonas hydrophila. J Clin Microbiol 1987;25:916–21. [8] Thornley JP. Aeromonas caviae exhibits aggregative adherence to HEp-2 cells. J Clin Microbiol 1994;32:2631–2. [9] Rahman M, Navarro PC, Kuhn I, Huys G, Swings J, Mollby R. Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh. Appl Environ Microbiol 2002;68:650–5. [10] Balaji V, Jesudason MV, Sridharan G. Cytotoxin testing of environmental Aeromonas spp. in vero cell culture. Indian J Med Res 2004;119:189–98. [11] Ormen O, Ostensvik O. The occurrence of aerolysin positive Aeromonas spp. and their cytotoxicity in Norwegian water sources. J Appl Microbiol 2001;90:797–802. [12] Asao T, Kozaki S, Kato K, Kinoshita Y, Otsu K, Uemura T, et al. Purification and characterization of an Aeromonas hydrophila hemolysin. J Clin Microbiol 1986;24:228–32. [13] Paniagua C, Rivero O, Anguita J, Naharro G. Pathogenicity factors and virulence for Rainbow Trout (Salmo gairdneri) of motile Aeromonas spp. isolated from a river. J Clin Microbiol 1990;28:350–5. [14] Pollard DR, Johnson WM, Lior H, Tyler SD, Rozee KR. Detection of aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction. J Clin Microbiol 1990;28:2477–81. [15] Wang G, Tyler KD, Munro CK, Johnson WM. Characterization of cytotoxic, hemolytic Aeromonas caviae clinical isolates and their identification by determining presence of a unique hemolysin gene. J Clin Microbiol 1996;34:3203–5.
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[16] Heuzenroeder MW, Wong CYF, Flower RLP. Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model. FEMS Microbiol Lett 1999;174:131–6. [17] Wang G, Clark CG, Liu C, Pucknell C, Munro CK, Kruk TMAC, et al. Detection and characterization of the hemolysin gene in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR. J Clin Microbiol 2003;41:1048–54. [18] Ullmann D, Krause G, Knabner D, Weber H, Beutin L. Isolation and characterization of potentially human pathogenic, cytotoxin-producing Aeromonas strains from retailed seafood in Berlin, Germany. J Vet Med B 2005;52:82–7. [19] Kobune F, Sakata H, Sugiyama M, Sugiura A. B 95a, a marmoset lymphoblastoid cell line, as a sensitive host for rinderpest virus. J Gen Virol 1991;72:687–92. [20] Gray SJ, Stickler DJ, Bryant TN. The incidence of virulence factors in mesophilic Aeromonas species isolated from farm animals and their environment. Epidemiol Infect 1990;105:277–94. [21] Snedecor GW, Cochran WG. Statistical methods. Ames, IA: Iowa State University Press; 1989. [22] Jesudason MV, John M, Koshi G. Comparison of three assays to detect enterotoxigenicity of Aeromonas isolates from patients with diarrhoea in Vellore, south India. J Diarrh Dis Res 1989;7:77–80. [23] Martins LM, Marquez RF, Yano T. Incidence of toxic Aeromonas isolated from food and human infections. FEMS Immunol Med Microbiol 2002;32:237–42. [24] Goldsby RA, Kindt TJ, Osborne BA. Kuby immunology. USA: W.H. Freeman & Company; 2000. [25] Hu ZQ, Asano K, Yamazaki T, Shimamura T. Effect of lipopolysaccharide on mouse mast cell induction by a splenic cell culture system. Infect Immun 1994;62:3844–9. [26] Cuellar A, Fonseca A, Gomez A. Effect of lipopolysaccharides on human dendritic cell cultures and its inhibition by polymyxin B. Biomedica 2004;24:413–22.
Comparative Immunology, Microbiology & Infectious Diseases 29 (2006) 232–240 www.elsevier.com/locate/cimid
Comparative study of cytotoxicity of Aeromonas spp. on four different cell lines S. Ghataka, R.K. Agarwalb,, K.N. Bhilegaonkara a
Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar 243 122, India b National Salmonella Centre, Bacteriology and Mycology Division, Indian Veterinary Research Institute, Izatnagar 243 122, India Accepted 17 June 2006
Abstract In vitro cytotoxicity is an important virulence property of motile mesophilic Aeromonas species. Cell-free supernatant prepared from 55 Aeromonas isolates including one A. hydrophila type strain (MTCC 646) were examined for their cytotoxic potential on four different cell lines (Vero, BHK-21, MDBK, B 95a). Results of the study revealed cytotoxic potential in 92.72% of the isolates. Analysis of data exposed significant variation among isolates in respect of their cytotoxicity. Vero cells proved to be most sensitive t o aeromonal toxins and B 95a cells showed significantly (Po0:01) lower response compared to other cell lines. Sensitivities of BHK-21 and MDBK cell lines were in between Vero and B 95a. r 2006 Elsevier Ltd. All rights reserved. Keywords: Aeromonas; Cytotoxicity; Vero; MDBK; BHK; B 95a
Re´sume´ Cytotoxicity in vitro est une proprie´te´ de virulence importante d’espe`ce de Aeromonas de mesophilic mobile. La cellule libe`re supernatant a pre´pare´ de 55 Aeromonas isole inclut un la Corresponding author.
E-mail address: [email protected] (R.K. Agarwal). 0147-9571/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2006.06.005
ARTICLE IN PRESS S. Ghatak et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 232–240
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tension de type de A. hydrophila (MTCC 646) ont e´te´ examine´ pour leur potentiel de cytotoxic sur quatre lignes de cellule diffe´rentes (Vero, BHK-21, MDBK, LE B 95a). Les re´sultats de l’e´tude ont re´ve´le´ le potentiel de cytotoxic dans 92,72% de l’isole. L’analyse de donne´es a expose´ la variation significative parmi isole dans le respect de leur cytotoxicity. Les cellules de Vero se sont re´ve´le´es le plus sensible aux toxines de aeromonal et au B 95a cellules montre´es significativement (Po0:01) abaisse la re´ponse en comparaison des autres lignes de cellule. Les sensibilite´s de BHK-21 et lignes de cellule de MDBK e´taient entre Vero et le B 95a. r 2006 Elsevier Ltd. All rights reserved. Mots-cle´: Aeromonas, Cytotoxicity, Vero, MDBK, BHK, B 95a
1. Introduction Motile and mesophilic members of the genus Aeromonas are pathogens of humans and animals [1]. Besides their role in gastrointestinal infections they have been implicated in a multitude of infections including septicaemia, wound infections, urinary tract infections, myocarditis ocular complications, osteomyelitis, meningitis, peritonitis cholangitis, etc. [2]. The ability of the organisms to inflict diseases in animals and humans is attributable to an impressive array of virulence factors [3]. The virulence factors of Aeromonas may be broadly classified into extracellular factors, structural features and cell-associated factors [2]. Among the most discussed of these are the extracellular factors, which include haemolysins, enterotoxin, protease, amylase and chitinase [2]. Apart from the specific roles played by these factors in Aeromonas-induced disease processes, one of the most important virulence property of haemolysins and enterotoxin of aeromonads involves exhibition of cytotoxicity in vitro. To assess the cytotoxic potential, of the organisms different cell lines have been used by the researchers [3]. The cell lines that have been used for the purpose ranges from mammalian cell lines viz. Vero, Int 407, Chimp liver, Chinese hamster ovary, HeLa, HEp-2, Y1 adrenal to fish cell line, epithelioma papulosum of carp (Cyprinus carpio) [4–10]. However, different cell lines vary in their sensitivities to different toxins thereby making the comparison of different studies difficult. Nevertheless, reports about comparative efficacy of different cell lines to aeromonal toxins are either very rare or absent. In addition, a majority of researchers employed only one kidney derived cell line (Vero) for studying cytotoxicity of Aeromonas spp. leaving other cell lines of similar origin uninvestigated [11–18]. And, relatively newly established cell lines of lymphoid origin e.g. B 95a [19] were even less explored in the context of cytotxic behaviour of Aeromonas spp. Therefore, the present study was undertaken to assess and compare sensitivities of different cell lines to toxins of Aeromonas spp. and to investigate the effect of aeromonal toxins on a lymphoid cell line.
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2. Materials and methods 2.1. Bacterial strains Included in the study was a set of 55 strains of Aeromonas spp. of diverse origin (Table 1). A. hydrophila type strain, MTCC 646 was obtained from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. Rest of the strains were from Veterinary Public Health Division, Indian Veterinary Research Institute, Izatnagar, India. All strains of Aeromonas were grown on Trypticase soy agar (Difco) at 37 1C for 18–24 h. A known verocytotoxic Escherichia coli strain maintained at our laboratory was used as positive control for cytotoxicity assay and was grown on Eosine methylene blue agar. For long-term storage15% (v/v) glycerol–Trypticase soy broth was used and stored at 70 1C. 2.2. Preparation of cell-free supernatant (CFS) Cell-free supernatants (CFSs) of the isolates were prepared as per the method described by Gray et al. [20] with necessary modifications. The organisms were grown in 5 ml of brain heart infusion (BHI) broth (Hi-Media) fortified with 0.6% (w/v) yeast extract and 3% (w/v) cassamino acid. Following an incubation of 12 h at 37 1C, 50 ml of supplemented BHI broth was inoculated with the initial broth culture and incubated in a shaker set at 200 rpm at 37 1C. After incubation of 14–16 h, while the growth was in mid-log phase the broth cultures were taken to 50 ml sterilized tubes and centrifuged at 10000 g for 30 min at 4 1C. The supernatants were transferred to fresh tubes and filtered through 0.22 mm filters (Sartorius, Germany). The sterility of each preparation was checked by streaking onto nutrient agar and incubating at 37 1C for 24 h. Similarly, the pH of the filtrates were checked and stored at 20 1C till further use.
Table 1 Aeromonas strains used in the study Strains
A. A. A. A. A. A.
hydrophila caviae veronii bt sobria veronii bt veronii jandaei trota a
Origin Clinical
Chicken
Fish
Unknowna
2 5 1 — — 2
5 6 — — — —
5 3 19 1 — —
1 3 1 — 1 —
Strains of unknown origin were Aeromonas strains maintained in the Division of VPH, IVRI, India.
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2.3. Assay for cytotoxicity Cytotoxic potential of the isolates were tested on four different cell lines viz. African green monkey kidney cells (Vero), Madin darby bovine kidney (MDBK) cells, Baby hamster kidney cells (BHK-21) and Marmoset B lymphoblastoid cells (B 95a) [19] as per the method of Barer et al. [6] with suitable adaptations. The cell lines except B 95a were propagated in Eagle’s minimum essential medium (EMEM, Sigma) with 10% and 2% foetal bovine serum (FBS, Sigma) during growth and maintenance, respectively. The B 95a cells were propagated in Rosewell Parker Memorial Institute (RPMI) 1640 medium (Sigma, USA) with 10% and 5% FBS during growth and maintenance, respectively. The toxin activity of the cell-free preparations was assayed in 96 well tissue culture plates (Greiner, Germany). In each well cells were grown to confluent monolayers. In a 96 chamber deep well plate serial two-fold dilutions of each CFS were prepared in the same medium in which cells were grown. The medium from cell culture plate was discarded and 100 ml of each dilution of each CFS was added in the wells of the cell culture plates in triplicate. In each plate CFS of verocytotoxic E. coli and a preparation of BHI broth (Hi Media) with 0.6% (w/v) yeast extract and 3% (w/v) cassamino acid, processed similarly as with other CFS preparation, were included as positive and negative controls, respectively. The plates were incubated at 37 1C in 5% CO2 atmosphere and examined six hourly in an inverted microscope (Nikon, Japan) for up to 72 h for characteristic cytopathic effect (CPE). CFS preparation inducing CPE at least up to 1:8 dilution or more in 50% or more cells were recorded as positive and further titrated if necessary. For any individual CFS preparation, the reciprocal of the highest dilution inducing CPE in 50% or more cells was recorded as its cytotoxicity titre. Following full incubation, the plates were stained with 1% crystal violet after fixing with 10% (v/v) formaldehyde solution. After staining, they were observed macroscopically and microscopically and the results were compared with previous microscopic observation. 2.4. Analysis of results Data generated from cytotoxicity assays were subjected to statistical analysis as per Snedecor and Cochran [21]. Since values of cytotoxicity titres were not normally distributed, the data were transformed to logarithmic scale with base 2 following addition of unity (1) to individual data. Log-transformed data were statistically analysed employing the following statistical model: Y ijk ¼ m þ Si þ C j þ ðs cÞij þ eijk , where Yijk is the log2 titre of ith isolate, jth cell line, kth observation, m the overall mean, Si the effect of ith isolate, Cj the effect of jth cell line, (s c)ij the interaction between ith isolate and jth cell line, eijk the experimental error (eijk is independently, randomly and normally distributed with mean 0 and variance s2).
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To make multiple comparisons of mean titres, least significant difference test was conducted [21].
3. Results Cytotoxic potential of Aeromonas isolates was studied on four different cell lines including BHK-21, Vero, MDBK and B 95a cell lines. Microscopic examination of different cells following exposure to and incubation with CFS prepared from cytotoxic isolates revealed number of changes including rounding and shrivelling of cells, loss of cytoplasmic extensions, disorganization of cell sheets, shrinking of cytoplasms. Cytotoxicity was induced by 92.72% of the isolates in all four cell culture systems. Except for one isolate of chicken origin and three of unknown origin, all other isolates from all sources were found to be cytotoxic (Table 2). Species differentiation of cytotoxic isolates indicated 82.35% of A. caviae, 95.24% of A. veronii bt sobria and all isolates of other species were cytotoxic in all four cell lines tested. Statistical analysis to differentiate between sensitivity among cell culture systems as well as, between cytotoxic potentials of isolates indicated significant difference Table 2 Cytotoxic potential of Aeromonas isolates Source/species
No. of isolates examined
No. of cytotoxic isolates (%)
Canine A. caviae A. veronii bt sobria
4 3 1
4 (100.00) 3 (100.00) 1 (100.00)
Human A. hydrophila A. caviae A. trota
6 2 2 2
6 2 2 2
Chicken A. hydrophila A. caviae
11 5 6
10 (90.91) 5 (100.00) 5 (83.33)
Fish A. hydrophila A. caviae A. veronii bt sobria A. veronii bt veronii
28 5 3 19 1
28 5 3 19 1
(100.00) (100.00) (100.00) (100.00) (100.00)
6 1 3 1 1
3 1 1 1 0
(50.00) (100.00) (33.33) (100.00) (0.00)
Unknowna A. hydrophila(MTCC646) A. caviae A. jandaei veronii bt sobria Total
55
(100.00) (100.00) (100.00) (100.00)
51 (92.72)
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Table 3 Analysis of variance of cytotoxicity titres (log2 titre) for different Aeromonas isolates under various cell culture systems Source of variation
Degree of freedom
Mean square
Isolates Cell lines Isolates cell lines Error
54 3 162 440
6.603 2.12 0.974 0.278
Po0:01.
Table 4 Mean and standard error (SE) of cytotoxicity titres (log2 titre) for different cell culture systems Cell line (s)
Mean7SE
BHK-21 Vero MDBK B 95a
4.1570.04b 4.2570.04b 4.1370.04b 3.9870.04a
Means having same superscripts do not differ significantly (Po0:01).
12
Isolates
10 8 6
C
4
5.05
4.95
4.85
4.75
4.65
4.55
4.45
4.35
4.25
4.15
4.05
3.95
3.85
3.75
3.65
3.55
3.45
3.35
3.25
3.15
3.05
2.95
2.85
0
2.75
2
Titre (s) Fig. 1. Frequency distribution of cytotoxicity titres (log2 titre) of Aeromonas isolates.
(Po0:01) between isolates and between cell lines (Table 3). Individual Aeromonas isolates differed significantly (Po0:01) among themselves in inducing cytotoxicity on four cell culture systems. Also four cell lines showed variation (significant at Po0:01) in their response to cytotoxins liberated by isolates. Interestingly, the interaction between isolates and cell lines was also significantly variable (Po0:01). This indicated that the relative ability of individual isolates to induce cytotoxicity was not uniform across four different cell culture systems. Mean cytotoxicity titres individual cell lines taking all isolates into account were in the range of 3.98–4.2570.04 (Table 4). Of the four cell lines tested, Vero cells expressed highest
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sensitivity with mean titre being 4.2570.04 and the least sensitive cell line as apparent from the results was B 95a (mean titre ¼ 3.9870.04). Other two cell lines (BHK-21 and MDBK) were of almost similar sensitivity against the CFS prepared from the isolates under study. From frequency distribution of mean cytotoxicity titres of isolates the wide variation in titres among isolates was evident. Highest titre (log2 titre) was 5.04, whereas lowest was 2.75 (Fig. 1). As much as 10 isolates induced high degree of cytotoxicity (log2 titre ¼ 5.04) over all the four cell culture systems, while low titred (log2 titre o3) isolates were four in number. Following these were two major groups of isolates with mean titres of about 4.05 and 4.75, respectively. Thirty-six percent of the isolates were highly cytotoxic across all four cell lines showing cytotoxicity titres of 44.00 (log2 titre).
4. Discussion In the present study, we have presented the results of testing of Aeromonas CFS on four different cell lines. Three of these cell lines were basically kidney derived and B 95a was a B lymphocyte-derived cell line [19]. Most (92.72%) of the isolates studied were cytotoxic. Similar high prevalence of cytotoxic potentials as observed in the present study among aeromonads of diverse origin has been reported by others too [11,22,23]. Analysis of results of cytotoxicity studies revealed significant (Po0:01) differences among individual isolates as well as among different cell lines. Also the cell line–isolate interaction varied significantly (Po0:01) implying non-uniform response of individual isolates across four different cell lines tested. This substantiates our previous assumption about incomparability of data generated from cytotoxicity testing on different cell lines. Our results showed that Vero cells were most sensitive to CFS preparations from isolates, followed this were BHK-21 and MDBK with no significant difference between them. But B 95a cells were significantly (Po0:01) less responsive. Perhaps this is why a large number of researchers preferred Vero cell for cytotoxicity testing [11–18]. Interestingly, low response of B 95a cells, which are lymphocytic cells, has a deeper implication. B lymphocytes are known to be responsive to bacterial lipopolysaccharides [24]. In our study during preparation of CFS, cells were centrifuged out during the mid-log growth phase to keep the cell lysis and thereby endotoxin release in the culture broth to a minimum. The low response of B 95a cells indicates that CFS preparation was indeed relatively low in endotoxin content. This served as an internal control over the entire cell culture study implicating that response in other cell lines was actually due to aeromonal cytotoxins. However, there are also chances of cell lysis during growth of bacterial cells in broth culture. Since polymyxin B is known to inhibit activities of lipopolysaccharide in other cell cultures [25,26] treatment of CFS preparations with polymyxin B before testing them on cell lines will be an interesting approach for further studies.
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Finally, response of B 95a cells to CFS from Aeromonas isolates indicates possible presence of some lymphotoxic factor(s) yet to be characterized. And to our knowledge this study documents the first report of cytotoxicity testing of aeromonads on a lymphocytic cell line. Frequency distribution of cytotoxicity titres of the isolates indicated that this behaviour was widely prevalent and highlights possible public health risks lurking among Aeromonas spp. studied. In conclusion, we suggest use of Vero cells in preference to other cell lines examined for testing of cytotoxic potential of Aeromonas spp.
Acknowledgement Authors are thankful to Director, IVRI, Dr. RK. Singh and Dr. V. Bhanuprakash for providing necessary facilities and Dr. M. Nath for his help in data analysis.
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