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 ~  Abstract
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 ~  Results and Disc...
 ~  Acknowledgement
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ORIGINAL ARTICLE
Year : 2001  |  Volume : 19  |  Issue : 4  |  Page : 190-192
 

Isolation and identification of aeromonas from patients with acute diarrhoea in Kolkata, India


Department of Microbiology, All India Institute of Hygiene and Public Health, Kolkata - 700 073, India

Correspondence Address:
Department of Microbiology, All India Institute of Hygiene and Public Health, Kolkata - 700 073, India

 ~ Abstract 

Isolation of diarrhoea causing Aeromonas was carried out in the division of Active Surveillance, National Institute of Cholera and Enteric Diseases (NICED), Kolkata for a period of 12 months from January 1999 to December 1999. Out of 602 stool samples collected from patients with acute diarrhoea admitted in Infectious Diseases (ID) Hospital, Kolkata, 64 (10.6%) samples were identified positive for Aeromonas as the pathogen. The different isolated and identified species from patients with acute diarrhoea were A. hydrophila (60%), A. caviae (20%), A. veronii (10%), A. schubertii (4%), A. jandaei (3%), and A. trota (3%). Most of the isolated Aeromonas strains showed resistance to commonly employed antibiotics. All the clinical isolates of Aeromonas possessed virulence genes encoding for aerolysin and cytotonic enterotoxin genes. Except A. schubertii and A. jandaei, all the other species possessed the gene for haemolysis.

How to cite this article:
Kannan S, Chattopadhyay U K, Pal D, Shimada T, Takeda Y, Bhattacharya S K, Ananthanarayanan P H. Isolation and identification of aeromonas from patients with acute diarrhoea in Kolkata, India. Indian J Med Microbiol 2001;19:190-2


How to cite this URL:
Kannan S, Chattopadhyay U K, Pal D, Shimada T, Takeda Y, Bhattacharya S K, Ananthanarayanan P H. Isolation and identification of aeromonas from patients with acute diarrhoea in Kolkata, India. Indian J Med Microbiol [serial online] 2001 [cited 2020 Jul 16];19:190-2. Available from: http://www.ijmm.org/text.asp?2001/19/4/190/8187


Aeromonas species are gram-negative, motile, facultative anaerobic, rod shaped, oxidase positive bacteria of the recently assigned family Aeromonadaceae[1]. The significance of Aeromonas species as causative agent of human diarrhoea has recently been established[2]. Aeromonads have been found to cause a variety of primary infections in the normal host as well as severe infections in immuno-compromised patients.[3] The aeromonads from the heterogenous environment possessed several virulence factors such as enterotoxins, haemolysins, cytotoxins, proteases, adhesins etc.[4] Six different species of Aeromonas viz., A. hydrophila, A. caviae, A. veronii (biovar sobria and veronii), A. schubertii, A. jandaei and A. trota are associated with various human infections.[5] Till now, the association of A. schubertii and A. jandaei has not been established with diarrhoeal cases.[6] Interestingly, our study revealed that these two species were also causing acute diarrhoea.

 ~ Materials and Methods Top

Isolation of Aeromonas
A total of 602 stool samples from the diarrhoeal patients with age group ranging between 1 and 62 years were tested for the presence of Aeromonas. The stool samples were transported to the laboratory in Cary-Blair transport medium. All the samples were enriched in alkaline peptone water (APW) at 37C for 18 hours. The enriched inoculum from APW was streaked on Ampicillin sheep blood agar (ASBA) and xylose deoxycholate citrate agar (XDCA) followed by incubation at 370C for 24 hours.[7] Both haemolytic, nonhaemolytic and non-xylose fermenting colonies were tested for oxidase test.[8] Strains were stocked on nutrient agar stabs at room temperature for further studies.
Identification and antibiogram studies on Aeromonas
The oxidase positive colonies were further confirmed by the biochemical tests and species differentiation was performed by the method described by Aerokey II group of tests for the identification of Aeromonas.[9] The serotyping of Aeromonas was carried out at the NIID, Tokyo, Japan using Aeromonas specific O antiserum.[8] The antibiotic susceptibility testing was conducted with commercially available antibiotic discs (HiMedia, Mumbai) by the method described by Kirby and Bauer.[10] The drugs such as ampicillin, cefamandole, chloramphenicol, novobiocin, tetracycline, erythromycin, trimethoprim-sulfamethoxazole, vancomycin, colistin, gentamycin, streptomycin, neomycin, polymyxin, ciprofloxacin and nalidixic acid were tested for sensitivity.
PCR-analysis of virulence genes
Aeromonas specific virulence genes such as cytotonic enterotoxin, aerolysin and hemolysin were detected using specific oligonucleotide primers by PCR analysis.[11] The strains were grown in Luria broth (Difco) and incubated for 24 hours at 370C in shake culture. The denatured DNA templates for PCR was made available by boiling the bacterial culture for 10 minutes and immediately keeping in ice. The reaction mixture for PCR analysis consisted of 2.5 l of 10X amplification buffer (500mM KCl, 100mM Tris HCl, 15mM MgCl2 pH 8.3), 8l of 25mM MgCl2, 2.5l each of 2mM dATP, dTTP, dGTP and dCTP, 10 nM each of the primers and 0.625 Unit of Taq DNA Polymerase (Takara Shuzo, Otsu, Japan) and 3 l of the denatured DNA template. The reaction conditions for PCR assay were the same mentioned elsewhere,[12],[13] using automated thermal cycler (Perkin Elmer, CA, USA). The PCR amplicons were visualized by ethidium bromide staining after electrophoresis using 1.5% agarose gel.

 ~ Results and Discussion Top

Six different species of Aeromonas causing diarrhoea were isolated and identified from patients with acute diarrhoea. Isolation of Aeromonas with reference to patient's age and sex are given in the [Table - 1]. The rates of isolation of these species were A. hydrophila (60%), A. caviae (20%), A. veronii (10%), A. schubertii (4%), A. jandaei (3%) and A. trota (3%). Ampicillin sheep blood agar failed to select the colonies of A. trota from the stool samples.
This property was attributed to the ampicillin susceptibility of this particular species as shown in the antibiogram [Table - 2]. Most of the Aeromonas strains exhibited resistance to many commonly used antibiotics. The serotyping of the isolated Aeromonas strains are given in [Table - 3]. A. schubertii and A. jandaei produced non-hemolytic colonies on ASBA. PCR analysis also indicated that except A. schubertii and A. jandaei, other species possessed the hemolysin gene. The non-hemolytic property may be correlated to the less virulent nature of A. schubertii and A. jandaei in causing diarrhoea.
All the strains of Aeromonas isolated showed the presence of cytotonic enterotoxin [Figure]and aerolysin genes. This study also reveals that possession of virulence factors such as cytotonic enterotoxins and aerolysins are vital for diarrhoegenicity of Aeromonas. The infection might have occurred after consumption of contaminated drinking water or foodstuffs with aeromonads. Considering the prevalence of aeromonads in water and foods, there is a necessity to include this organism while assessing the microbiological quality of drinking water and foods.

 ~ Acknowledgement Top

This work was supported, in part, by the Japan International Co-operative Agency (JICA/NICED Project 054-1061-E-O). 

 ~ References Top

1.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.  Back to cited text no. 1    
2.Farmer III JJ, Arduino MJ, Hickman-Brenner FW. The genera Aeromonas and Plesiomonas In: Balows A, Truper HG, Dworkin M, Harder W, Schleifer KH, eds. The prokaryotes: a handbook on the biology of bacteria, ecophysiology, isolation, identification, applications. (New York: Springer-Verlog) 1992;3013-45.  Back to cited text no. 2    
3.Kirov SM. The public health significance of Aeromonas spp. in foods. Int J Food Microbiol 1993; 20:179-98.  Back to cited text no. 3    
4.Janda JM, Duffey PS. Mesophilic aeromonads in human disease: current taxonomy, laboratory identification and infectious disease spectrum. Rev Infect Dis 1998; 10:980-97.  Back to cited text no. 4    
5.Carnahan AM. Aeromonas taxonomy: a sea of change. Med Microbiol (Lett) 1993; 2:206-11.  Back to cited text no. 5    
6.Janda JM, Abbott SL. Evolving concepts regarding the genus Aeromonas: An expanding panorama of species, disease presentations and unanswered questions. Clin Infect Dis 1998; 27:332-44.  Back to cited text no. 6    
7.Misra SK, Nair GB, Bhadra RK, Sikdar SN, Pal SC. Comparison of selective media for primary isolation of Aeromonas species from human and animal feces. J Clin Micro 1987; 25:2040.  Back to cited text no. 7    
8.Misra SK, Shimada T, Bhadra RK, Pal SC, Nair GB. Serogroups of Aeromonas from clinical and environmental sources in Calcutta. J Diarrhoeal Dis 1989; 7:8-12.  Back to cited text no. 8    
9.Carnaham AM, Behram S, Joseph SW. Aerokey II: A flexible key for identifying clinical Aeromonas species. Clin Microbiol 1991;12:2843-49.  Back to cited text no. 9    
10.Bauer AW, Kirby WM, Sherris KC, Turck M. Antibiotic susceptibility testing by a standardised single disc method. Am J Clin Pathol 1966; 45: 493.  Back to cited text no. 10    
11.Janda JM, Abbott SL. Human pathogens. In: Austin B, Altwigg M, Gosling PJ, Joseph S, eds. The genus Aeromonas. (Chichester, England: John Wiley & Sons) 1996;151-73.  Back to cited text no. 11    
12.Granum PE, Sullivan KO, Tomas JM, Ormen O. Possible virulence factors of Aeromonas spp. from food and water. (1998) 21: 131-137. FEMS Immun and Med Microbiol 1998; 21: 131-7.  Back to cited text no. 12    
13.Pollard DR, Johnson WM, Lior H, Tyler SD, Rozee KR. Detection of the Aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction. J Clin Microbiol 1990;11:2477-81.  Back to cited text no. 13    
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