Isolation And Characterization Of Vibrio Parahaemolyticus And Aeromonas Hydrophila From Fish In Kenyan Waters And Their Possible Role In Causing Infection To Man
Damiano, Wale Andrari , B.V.M.
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With the increase in utilization of fish resources by man, there has been a concomitant demand for quality control of fish and fish products. An important aspect of this has been the need for the study of microbiological contribution to spoilage changes. Both the inherent bacterial flora of fish and those acquired through contamination after catch, together with other factors like the enzymatic activities, are important in causing changes which lead to lowering of quality. Of major public health importance, however, is the fact that some of these microorganisms cause infection in man. Amongst this group are Vibrio parahaemolvticus and motile Aeromonas species. lihrlQ has long been known to be one of the aetiologlc agents in food-poisoning causing acute gastroenteritis, particularly common in areas where marine fish is eaten raw in large quantities. Aeromonas species, together with Campylobacter and Yersinia species, on the other hand, are considered to be in the group of emerging enteropathogens, which cause acute diarrhoeal diseases particularly in infants. This study was aimed at assessing the hygienic standards of fish from some of the fishing areas in Kenya by*determining the total bacterial * counts, isolation and characterization of Y. parahaemolvticus and motile Aeromonas species. (V) Total bacterial counts were performed on fish and water samples obtained directly from landing sites of Lake Naivasha, River Sombeli (Magadi), and Hasinga Dam. A total of 30 samples were collected. For isolation of the two bacteria, a total ox 763 samples were collected from all the major fishing sites in Kenya. Of these, 723 were fish and 40 were water samples. Primary isolation was done by the use of selective media and the isolates were confirmed by means of biochemical tests. In the case of Aeromonas isolates, two methods, that is, biochemical and CAHP- like factor detection, were used to further identify to species level as A fiaYiaa. A hY.drfiEhila and A- flfittcia- The ability of these isolates to cause infection was determined by assessing their pathogenic pobential in terms of toxin production. For V.parahaemolvticus. this was done by detection of Kariagawa haemolysis. In case of AarGmenaa species, toxigenicity was tested for by detection of enterotoxin and cytotoxin production using suckling mice and vero cell lines respectively. The antibiograms of the isolates of the two genera were studied by determining the zones of inhibition of bacterial growth on Mueller-Hinton agar by various antimicrobial agents. Analysis of the plasmid contents was performed by lysis of the cell-walls of the * isolates and then separating them by means of electrophoresis. (Vi) Slide coagglutination technique using antisera raised against the somatic antigens of the two genera coated on Staphylococcus aureus strain Cowan I, was used to identify isolates having homologous antigens. Results obtained for total bacterial counts on fish sampled from the three areas ranged from 105 to 10* and Id to 105 for water samples ' • Out of 666 fish and water samples from both freshwater and marine environs screened for £. parahemolyticus. 29 isolates were made, representing an isolation rate of only 4%. However, a high isolation rate of 46% (27/62) was found amongst fish samples obtained from the coastal waters. The prevalence in fish from this area was 44% (26/57) and in water was 40% (2/6). Only 1% (1/99) and 0.3% (1/326) of the samples from Lake Naivasha and Lake Victoria respectively yielded the organism. The two isolates in both cases were from fish. The organism was not isolated from lake Turkana, River Sombeli (Magadi; and Masinga Dam samples. All the 763 fish and water samples collected were screened for motile Aeromonas species. An isolation rate of 47% (366/763) was made. The two methods used for species differentiation were observed to give a slight variation of different rates of ♦ occurrence of the three species. The rates obtained by using biochemical tests were A- CfiYiflfi 42% (vii) (150/356); hxd£OEhila 37% (132/356) and A. aobria 21% (74/356) while that of CAMP-like factor detection were AsayIaa 44% (165/356), A. hydrophila 36% (123/366) and A- aobria 22% (78/356). The 29 V.parahaemolvticua isolates were all Kanagawa negative. The general optimum salt requirement for growth was 3%, although one isolate from Lake Naivasha had a low salt requirement because it grew very well in a medium containing i% salt concentration. Of the typable twenty isolates serotyped, only five were typable, representing 20% typability. Ail the typable isolates were from the coastal waters and were serotypes 02:K28; 03*. K29 and 04:K42. A total of 166 Aeromonas were tested for their enterotoxigenicities. Fourty seven (28%) isolates were found to produce enterotoxins. The occurrence of enterotoxin producing strains amongst the isolates varied considerably with A- sobrla having the highest rate of 51% (20/39), followed by A- hydrophijg 35% (23/65) and then A- GAYiAA 6% (4/62). In the cell culture model, 53% (42/80) of the isolates were found to be cytotoxic to vero cells. The ¥.. parahaemolvticua strains were generally sensitive to chloramphenicol, sulphonamides, while ♦ being resistant to penicillin and fucidin. Aeromonag isolates ,on the other hand, showed resistance to more (vlli) agents like ampicillin, bacitracin, neomycin, penicillin, oxacillin and nitrofuran. Moat of them, however, reaponded to chloramphenicol, tetracyclinea, nalidixic acid, fucidin and aulphonamidea. Among the Aeromonaa isolates, 32 of them were found to carry plasmids of varying sizes and numbers. Most of them were light, although some isolates carried heavy plasmids as well. Coagglutination test using y. parahaemolyticuq isolates showed that only four of the 29 isolates were serologically identical to the somatic antigen of the reference strains to which the antiserum was raised. Among the Aeromonaa isolates, only 29 out of 356 gave positive reaction. Two of these were A.sobria while the rest were A- hydrophila. The total bacterial counts for fish were found to be generally higher than those for water, because apart from serving as a substrate for attachment of microorganisms, some fish can actively concentrate bacteria during feeding as they filter water through their gills. The prevalence of y. parahaemolvticus in Kenyan marine fish was higher than that of fresh water fish. A i high percentage of motile Aeromonaa species was isolated from both freshwater and marine environs. ♦ Although the results of differentiation into the three species using the two methods differed slightly the (lx) general trend was that A- cavi^e had the highest frequency of occurrence followed by A- hvdrophila and A. aabriii Toxigenicity assay for both enterotoxins and cytotoxins revealed that higher toxigenic strains occur amongst A- sebrifi followed by A- hydrophilfl and then A- cavlae. Since pathogenicity is associated with toxin production, it can be deduced that although A* sobria has low occurrence among environmental isolates, they are more pathogenic than the other two species. Antimicrobial susceptibilities of J. parahaemolyticus showed that the isolates were susceptible to most broad spectrum antibiotics. The plasmid carriage amongst the Isolates was low which might imply that plasmid-induced resistance is not present in these isolates. The few light plasmids detected were assumed to be cryptic as no specific functions could be assigned to them. Aeromonas species, on the other hand, showed multiple resistance to a number of agents. One isolate was found to be resistant to all the tested agents. It was found that Aeromonas species showed greater variation in antibiotic susceptibility patterns than earahafim^iyticqs. Although coagglutination technique enhanced sensitivity of sli<ie agglutination test, its use for identification of the two organisms was limited because (X) of the heterogenicity of the antigens of the organisms. In conclusion, this study reports a high prevalence of V. parahaemolytlcus amongst marine fish but a low prevalence in fresh water fish in Kenya. The high prevalence of the organism amongst marine fish might be a potential health hazard. There is also a high occurrence of Aeromonas species in Kenyan waters and fish, and the isolation of toxigenic strains of &. aabrifi and A. hydrophila among them indicates that they may present a potential health problem