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|Year : 2003 | Volume
| Issue : 4 | Page : 233--238
Biochemical characteristics, serogroups, and virulence factors of aeromonas species isolated from cases of diarrhoea and domestic water samples in Chennai
SV Alavandi, S Ananthan
Department of Microbiology, Dr. ALM Post-Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, India
Department of Microbiology, Dr. ALM Post-Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113
PURPOSE: The objective of the present study was to delineate the differences between the clinical and environmental Aeromonas species with respect to their biochemical characteristics, serogrouping and virulence factors, in order to find a phenotypic marker of enteropathogenicity. METHODS: A total of 55 Aeromonas spp. inclusive of 19 isolates from cases of diarrhoea, and 36 from water samples comprising, 10 isolates of A. hydrophila, 21 isolates each of A. sobria, and A. caviae, two isolates of A. jandaei and one isolate of A. veronii were subjected to analysis of their biochemical characteristics, serogrouping, and virulence factors. RESULTS: Among the differences recorded in the biochemical characteristics in the three major species, the most striking characteristic was fermentation of lactose, which was observed in all the 11 A. caviae isolates recovered from water samples. None of the 10 clinical isolates of A. caviae tested fermented lactose. The clinical Aeromonas isolates belonged to seven typable serogroups, O:13, O:14, O:16, O:21, O:27, O:32 and O:35. The environmental isolates belonged to eight different serogroups, such as, O:3, O:11, O:14, O:16, O:18, O:28, O:64 and O:78 and were predominated by serotypes O:18 and O:64. Among the virulence factors tested, 89% of the environmental isolates produced b haemolysin, while only 62.3% of clinical isolates were able to do so. There was no significant difference between the clinical and environmental aeromonads with respect to their enterotoxigenicity in suckling mice in vivo, cytotoxicity in vitro in Vero cell monolayers, and ability to produce siderophores. CONCLUSION: Efforts to delineate the differences between the clinical and environmental Aeromonas spp. did not reveal significant difference between them. However, difference was observed with respect to their ability to produce b haemolysin, wherein, higher percentage of environmental isolates was haemolytic. The results also suggest that all the haemolytic environmental isolates need not be enteropathogenic. Further, serogroups O:18 and O:64 may not be involved in aeromonal diarrhoea in children in this geographic region.
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Alavandi S V, Ananthan S. Biochemical characteristics, serogroups, and virulence factors of aeromonas species isolated from cases of diarrhoea and domestic water samples in Chennai.Indian J Med Microbiol 2003;21:233-238
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Alavandi S V, Ananthan S. Biochemical characteristics, serogroups, and virulence factors of aeromonas species isolated from cases of diarrhoea and domestic water samples in Chennai. Indian J Med Microbiol [serial online] 2003 [cited 2019 Dec 11 ];21:233-238
Available from: http://www.ijmm.org/text.asp?2003/21/4/233/8034
The motile mesophilic Aeromonas species are primarily organisms of aquatic environment and are present in fresh water, estuarine and coastal water bodies and even in chlorinated water., Aeromonads have been also reported to be associated with human disease, especially in diarrhoea in children, aged individuals and immunocompromised patients., Incidence of Aeromonas associated gastroenteritis has been reported from world over and its incidence in the developed countries has been reported to be relatively low compared to that in the developing countries. In India, Aeromonas associated diarrhoea has been reported from Bombay, Calcutta, Goa, Vellore, Pondicherry and Chennai,, and the incidence in these areas was reported to range between less than 1% to about 13%.
Several molecular typing techniques have been employed for typing of Aeromonas spp. and these studies have indicated that the clinical and environmental aeromonads are different. Aeromonas strains from human diarrhoeal stools and drinking water samples were reported to be dissimilar by biotyping in conjunction with gas liquid chromatography of cell wall fatty acid methyl esters. In a similar study, aeromonads isolated from public water supply in Iowa city were reported to be unrelated to those isolated from patients with gastroenteritis by ribotyping. Our studies using RAPD fingerprinting also indicated that there were differences between the clinical and environmental strains of Aeromonas spp., and that only some strains occurring in water are potentially enteropathogenic. What makes some of these strains pathogenic is not known. in order to find out the differences between clinical and environmental Aeromonas spp. we analysed the biochemical characteristics, serogroups, and virulence factors.
Materials and Methods
Fifty-two isolates were subjected to biochemical characterization using standard protocols. Production of b haemolysin was determined by conventional blood agar inoculation. Presence of large zone of b haemolysis around the colonies and titres were determined according to protocols described earlier. Aeromonas isolates were tested for enterotoxigenicity in vivo in 3-4 day old suckling mice. Cytotoxic activity of the culture filtrates of Aeromonas isolates to Vero cell monolayers was tested as per the protocols described elsewhere. Production of siderophores was determined by protocols described earlier using Chrome azurol S (CAS) agar and in cell free culture filtrates using CAS assay solution. Aeromonas isolates were serotyped by tube agglutination method using polyvalent antisera at National Institute of Infectious Diseases (Tokyo, Japan) by Dr. Toshio Shimada.
All the A. hydrophila isolates and 63.6% of A. sobria were aerogenic. All the A. caviae isolates were anaerogenic [Table:1] and 39.4% of the isolates fermented lactose.
None of the Aeromonas isolates from clinical sources fermented lactose. A. caviae were the only species which were isolated from domestic water samples and which fermented lactose. Fermentation of arabinose was observed in 42.1% of the isolates from each of the sources, and this feature was also associated with A. caviae. Production of acetyl methyl carbinol was observed in 15.8% of the clinical samples and 27.3% of the isolates from water, and was usually associated with A. hydrophila and A. sobria, while none of the A. caviae isolates produced acetyl methyl carbinol. Aesculin hydrolysis was observed in 57.9% of the clinical isolates and 60.6% of the isolates from domestic water samples, and this trait was found to be associated with A. hydrophila and A. caviae.
A total of 38 isolates comprising 16 from cases of diarrhoea and 22 from domestic water samples were serotyped. Aeromonas isolates obtained from clinical samples could be placed in seven typable groups, viz., O:13, O:14, O:16, O:21, O:27, O:32, and O:35. Being rough strains, two isolates of A. caviae and one isolate of A. sobria could not be serotyped [Table:2].
Aeromonads isolated from water samples belonged to O:3, O:11, O:14, O:16, O:18, O:28, O:64 and O:78 serogroups. Two serogroups (O:18 and O:64) occurred frequently in the water samples. Two isolates of A.sobria belonged to unknown 'O' serogroup and one isolate of A. hydrophila was rough type.
Majority of the clinical isolates (63.2%) produced haemolysin, while a relatively higher percentage i.e., 88.9% of aeromonads recovered from the water samples produced b - haemolysin [Table:3].
Figures in parenthesis are number of isolates tested
There appeared to be no significant difference between the clinical and environmental Aeromonas isolates with regard to their ability to produce cytotoxicity in Vero cells in vitro. The clinical isolates (63.2%) were cytotoxic to Vero cells, while a relatively less percentage (55.6%) of those of environmental origin were able to do so. The clinical isolates (68.4%) were also enterotoxigenic in the suckling mice. It is important to note here that 60% of the Aeromonas isolates of environmental origin were found to be enterotoxigenic by the suckling mouse assay.
Nineteen of the 21 clinical isolates and 26 of the 27 isolates from water produced siderophores indicating their ability to survive and grow in the iron deficient conditions [Table:4].
Among the 52 Aeromonas isolates subjected to biochemical characterization, A. hydrophila and A. sobria were aerogenic while the A. caviae isolates were anaerogenic in nature. Similarly, production of acetoin was confined to A. hydrophila and A. sobria only. However, several other biochemical reactions, such as fermentation of arabinose, lactose, aesculin hydrolysis, and lack of acid production from salicin were found to deviate from the ideal phenotype for each isolate. Janda et al have earlier reported similar deviation in the biochemical characteristics from ideal phenotypes. Thirty nine per cent of the Aeromonas isolates obtained from domestic water samples, mainly comprising A. caviae, fermented lactose. This fact needs to be considered when selective isolation media are not employed for isolation of aeromonads from clinical and environmental sources. In the present study 42.1% of the Aeromonas isolates from each of the sources fermented arabinose, majority of which were A. caviae. In an earlier study from Australia, a relatively higher percentage (58.5%) of aeromonads were reported to be able to ferment arabinose. Decarboxylation of lysine was associated with A. hydrophila (82.6%) and A. sobria (90.5%), while 33% of A. caviae were also associated with this trait in the present study. These results corroborate with those reported earlier. Production of CAMP-like factor was usually found in haemolytic strains of A. hydrophila and A. sobria isolates. A large number of A. caviae isolates were also able to produce CAMP-like factor. Hence, the proposal of Figura and Guglielmetti, that the motile mesophilic Aeromonas strains can be presumptively differentiated based on this trait do not appear to be applicable. The results obtained in the present study do not support their observation.
The genus Aeromonas is antigenically diverse, being composed of more than 96 distinct serogroups on the basis of presence of unique somatic antigens. These serogroups are not species specific. A recent study on serotyping of Aeromonas isolates from diverse clinical and environmental sources along with reference strains indicated that serogroups O:11, O:16 and O:34 predominate in about 48% of the clinical samples. They also reported that individual serogroups could be found in more than one species. High incidence of O:11, O:16 and O:34 serogroups has been reported by several investigators., ,  However in the present study, these serogroups do not seem to occur with as much frequency. While serotype O:16 was found in only one clinical isolate O:11 and O:34 were not found in the clinical samples. Three out of 16 clinical isolates of A. caviae and one isolate of A. sobria were untypable (rough types). A similar observation was made by Havelaar and co-workers, who reported that about 45% of their strains, predominantly A. caviae, were untypable. Among the 22 aeromonads serotyped from environmental source, two isolates belonged to O:11 and two others to O:16. The environmental aeromonads recovered in the present study were predominated by O:64 and O:18 [5 each of the 22 (22.7%) isolates serotyped]. It may be construed that the serogroups O:11, O:16 and O:34 reported from other geographic regions are uncommon in this region.
Among the Aeromonas isolates obtained in the present study, 63% of the clinical isolates were haemolytic, while, more than 88% of those obtained from environment were able to haemolyse the erythrocytes. Some studies indicated haemolysin production in a relatively lesser percentage of isolates compared to the results obtained in this study. As reported in an earlier study, a majority of A. caviae isolated in the present study were also haemolytic. Out of 10 isolates from water samples tested for enteropathogenicity by suckling mouse assay, 6 (60%) of them showed their ability to induce enterotoxigenic response in the suckling mice. It has been reported that enteropathogenicity of environmental isolates was low (15%) compared to those from clinical source (42%) by suckling mouse assay. In the present study, there appeared to be no significant difference in the ability to produce cytotoxins by aeromonads from the two sources. Cumberbatch et al reported higher (69%) incidence of cytotoxic strains of A. hydrophila from diarrhoeal source. They also reported that cytotoxin was a stable property and could not be associated with any plasmid. Some authors had suggested that cytotoxin production was species specific and that only A. hydrophila and A. sobria produced enterotoxin.,
Majority of the isolates obtained in the present study produced siderophores. In spite of the iron limiting conditions in the culture medium, Aeromonas isolates readily grew well, indicating their ability to survive in the iron deficient conditions prevalent in the host environment. It has been reported that amonabactin is possibly an important virulence factor of Aeromonas species. Earlier studies have indicated that ability to elaborate siderophores correlated with higher virulence in Aeromonas species. Further, iron deficiency in the medium induces microbes to activate iron acquisition mechanisms and in turn, constitutes an important signal, which regulates expression of a number of virulence factors unrelated to iron metabolism.
Analysis of Aeromonas isolates could not reveal any striking differences between those isolated from clinical and environmental sources. The conventional notion that the Aeromonas species are always non-lactose fermentors may prove wrong and hence, selective media such as Inositol Brilliant green Bile salts (IBB) agar or Cefsulodin Irgasan Novobiocin (CIN) agar must be incorporated along with the battery of enteric media for the recvovery of Aeromonas spp. Further, serogroups other than O:11, O: 16 and O: 34 could play a role in diarrhoea in this geographical region, and that, at least 60% of the environmental Aeromonas isolates could be enteropathogenic.
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