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December 1997, Volume 47, Issue 12

Original Article

Isolation and Identification of Aeromonas Species from Human Stools

Altaf Ahmed  ( Ziauddin Medical University Hospital, North Nazimabad, Karachi. )
Salim HaIiz  ( Ziauddin Medical University Hospital, North Nazimabad, Karachi. )
Afia Zafar  ( Ziauddin Medical University Hospital, North Nazimabad, Karachi. )
Tahir Shamsi  ( Ziauddin Medical University Hospital, North Nazimabad, Karachi. )
Jamiluddin Rizvi  ( Ziauddin Medical University Hospital, North Nazimabad, Karachi. )
Serajuddaula Syed  ( Ziauddin Medical University Hospital, North Nazimabad, Karachi. )

Abstract

One thousand and three diarrhoeal stool samples were processed in our laboratory during the period 1996/1997 for the presence of enteric pathogens especially Aeromonas spp., which has emçrged as a new agent causing diarrhoea. Ampicillin sheep blood agar was found to be the best medium for the isolation of Aeromonas spp. from stool specimens. Enteric pathogens were found in 200 (20%) stools, of which Aeromonas spp, was the second commonest pathogen isolated amounting to 21% of isolates. This study clearly incidicates that Aeromonas spp. must be looked for in every diarrhoea! stool samples, specially in children below 10 years of age. Isolation and identification is cost effective and easy, if the given protocol is observed (JPMA 47:305, 1997).

Introduction

The Aemmonas, an aquatic bacterium usually found in fresh waters e.g., rivers, lakes etc. is a gram negative straight rod with rounded ends and not a nonnal flora of the intestine. Aeromonas spp. have been established as an important gastrointestinal entenc pathogen1-4. The low level of isolation presently reported in most clinical laboratories may not be a true reflection of prevalence of the pathogen especially in our country where diarrhoeal illnesses are common. This is supported by the fact that isolation rate of Aeromonas spp. frequently increases in laboratories which have taken specific measures to isolate it. Isolation of Aeromonas spp. from stool samples requires the use of culture media which fulfills two criteria: an inhibiting effect on the growth of saprophvtes, and a discriminating capacity which allows it to be recognised among other species which are capable of growth on the medium. A variety of media are available in the market, the efficacy ofwhichare not well established. The confirmation of Aeromonas species is usually based on the production of oxidase, inhibition of growth in 6.5% NaCI and on TCBS and being resistant to Ampicillin (except for few strains)5. However identification upto species level is costly and a cumbersome affair.
The purpose of this study was to develop a cost effective method for the isolation and identification of Aeromonas from stool specimen for a mutine clinical laboratory. In all, seven media were evaluated to test the efficacy which included four routine culture media, MacConkey’s agar (MAC), Thioglycollate bile salt sucrose agar (TCBS) and Xylose lysine desoxycholate agar(XLD) and four special media, Ampicillin sheep blood agar with 30mg/litre of ampicillin (ASBA30), Aeromonas agar(Aer), Cefusulodin Irgasan and Novobiocin agar (CIN) and Glutamate starch phenol red agar(GSP).

Material and Methods

One thousand three stool samples were collected from patients with complaint of diarrhoea submitted to the clinical laboratory, during the year 1996-1997. Dianthoea was defined as 3 or more loose stools per day. The stool specimens were provided by patients both in the outdoor clinic and admitted in the wards of the hospital.
Stool samples were plated within one hourof collection, directly onto MacConkeys agar No.3 (Oxoid-CM 115), XLD (Oxoid-CM469), ASBA30  (Mueller-hinton agar-Oxoid-CM337 +5% Sheep blood + Ampicillin selective supplement SR136)6-8. CIN agar (Oxoid- CM653 + Yersinia selective supplement - Oxoid, SR 109) Aeromonas agar(Oxoid-CM833), TCBS agar (Oxoid-CM333) and GSP agar(Merck- 10230). The inoculated plates were incubated for 24-48 hours at 36°C + 1°C aerobically. Each plate was examined after 24 hours and 48 hours. Oxidase test was performed from blood agar plate by pouring oxidase reagent on the plate.Oxidase positive colonies were picked up within 10-12 seconds after the appearance of purple colour. All Oxidase positive colonies were isolated and reactions were set up which included , Triple sugar iron agar (TSI), Urea,Indole, H2S, motility, Xylose, Inositol, Mannitol. Dulcitol and 6.5%NaCI. The results were confirmed by API 10S(Analytab Products International,S.A. ,Vercieu,France).
Statistical Analysis: Data on planing media were analyzed statistically by McNemar’s test9

Results

The basis of recognition of Aeromonas spp. on different media is given in Table I.

Beta-hemolysis was observed only in 36%of isolates on ASBA30. Forty-one cases of Aerornonas were isolated, of which 75% were lactose fermenters and 25% were non-lactose fermenters as seen on MacConkeys agar.

Table II summanses the optimum biochemical tests for the identification and confirmation of Aeromonas species. The screening reactions were found to be reliable and reproducible. The ideal and cost effective tests were positive reaction with Oxidase,inhibition of growth in 6.5% NaCI and TCBS, no H2S production, motile, no pigment production on nutrient agar. negative reactions with urease, xylose.inositol.dulcitol and fermentation of mannitol.

Table III shows the sensitivities of the seven media separately. The rate of isolation on ASBA (4%), GSP (3.5%), MAC (3.5%) was significantly higher than that on XLD (0.3%), CIN (1.5%), TCBS (0.1%) and Aeromonas agar(0.5%)(P<0.001). Sensitivity of MAC and GSP was 78% as compared to ASBA. Recovery was poorest from XLD. All Aeromonas which grew on different media other than ASBA, also grew on ASBA. The effective combination of two media with which maximum number of all isolates were detected was ASBA30. MacConkey and GSP agar.

Discussion

The study illustrated that the isolation of Aeromonas spp. was not difficult and certaini not very costly. However, while choosing a selective media containing an antibiotic, the high bacterial resistance in this region should be kept in view. We found Ampicillin blood agar with 30img/l Ampicillin the most convenient, productive and cost-effective media for the isolation of Aerornonas. It was observed that haemolysis and oxidase test could be done without any interference. ASBA30 was not inhibitory to competing faecal flora as mentioned by some workers10.
As certain Aeromonas spp. are anipicillin sensitive, they may be missed on ASBA3O. To isolate these , an additional rnedia,i.e., MacConkey agar, or GSP should be used10. Yersinia and Aeromonas spp. can be isolated on a single medium CIN agar when incubated at 25°C, though for the latter it is not as sensitive as ASBA3O. The growth of Aeromonas spp. can be increased if modified CIN with 4 mg/i of Cefsulodin instead of 15mg/i is used. This is because higher concentrations of Cefsulodin inhibits nornial flora of gut and Aeromonas as well11. GSPwas a good medium forthe primary isolation of ampicillin sensitive strains .It gave prominent yellow coloured colonies surrounded by a yellow zone. Colonies were easy to find among the growth of other normal flora of the gut. These colonies were then isolated on a nutrient agar plate for oxidase testing because this important test should not be performed on a media with sugar content to avoid false results. TCBS does not help the growth of Aeromonas spp. at all. XLD agar and Aerornonas agar were also not so productive as compared to ASBA,GSP and MAC. Alkaline peptone waterenrichmentteclmique was not applied in this study because of its doubtful significance in isolation of Aeromonas spp. from clinical cases. It has been found that the use of enrichment culture frequcently detected Aeromonas spp. in asvmptomatic individuals12.
We recommend the use of two media, ASBA3O and GSP agar for the primaiy isolation of aeromonas ,spp. because the study clearly indicated that the best medium was ASBA3O with maximum isolation followed by GSP or MAC medium. with an added advantage of growing those Aeromonas which are ampicillin sensitive. Although MacConkeys medium was also sensitive enough, but it does not differentiate the colonies of Aeromonas from other coliforms and it grows Aeromonas both as non-lactose fermenters and lactose fermenters.A large number of biochemical tests have been evaluated for the final identification ofAeromonas spp.13,14 which have proved tobe specific and reproducible. Based on the results of this study, we propose a simple scheme for the identification of suspected Aeromonas isolates in a routine laboratoiy (Figure).

The scheme was cost effective since the oxidase test excluded members of Enterobacteriaceae at an early stage of investigation, while growth in 6.5%NaCI broth and greenish pigmentation on nutrient agar eliminated the halophilic vibrios and some Pseudomonads respectively. H2S production and positive indole reaction also helped in eliminating any Salmonella orvibrios. Some laboratories may slill find it costly to use ASBA3O as an additional media, then Aeromonas can easily be picked up from MacConkeys media if the recommended isolation protocol is adhered. In clinical laboratories of our counily, the need for isolation and identification of Aeromonas spp. is important. The suggested scheme should prove useful.

References

1.Burke, V ,Gracey, M.,Robinson,J. et al. The microbiology of childhood gastroenteritis: Aeromonas species and other infective agents. J. Infect.Dis., 1983; 148:68-73.
2. Gracey,M., Burke, V. and Robinson. J. Aeromonas associated gastroenteri­tis.Lancet, 1982;ii: 1304-1306.
3. Baman,S. I. Aeromonas hydrophila as the etiologic agent in severe gastroenteri­tis:report ofa case. AmJ.Med. Technol., 1980;46: 179- 181.
4. Rahman,A.F.M.S. and Willoughby, J.M.T. Dysentry like syn drome associated with Aeromonas hydrophila.Br.Med.J. 1980;28 1976.
5. Carnahan,A.M., Chakraborty,T., Fanning,G.R. et al.Aeromonas trots sp. nov., an Ampicillin-susceptible species isolated from clinical specimens. J.Clin. Microbiol., 1991;29: 1206-1210.
6. Kay,B.A., Guerrero,C.E. and Sack, RB. Media for the isolation of Aeromonas hydrophila. J.Clin, Microbiol., 1985 ;22 :888-890.
7. Kelly, M.T., Stroh E.M.D, and Jessop, J Comparison of blood agar, ampicillin blood agar, MacConkey-ampicillin-Tween agar and modified cefsulodin-ir­gasan-novobiocin agarfor isolation ofAeromonas spp. from stool specimens. J. Clin. Microbiol., 1988;26:1738.1740.
8. Robinson, J., Burke,V., Worthy, P.J. et al. Media for isolation of Aeromonas spp. from faeces. J.Med. Microbiol, 1984;18:405-411.
9. Knapp, G.R. and Miller, MC. Clinical epidemiology and biostatistics. NHS Williams and Wilkins, Baltimore, Maryland, Harward Publishing Company, Malvem, Pennsylvania. 1992, pp. 221- 225.
10. Mishra, S., Balakrish G.N., Bhadra,R.K et al. Comparison ofselectivemedia for primary isolation of Aeromonas species from human and animal faeces. J.Clin. Microbiol., 1987; 25: 2040-2043.
11. Altorfer, R., Altwegg, M., Zollinger-Iten,J. et al. Growth of Aeromonas spp. on Cefsulodin-irgasan-novobiocin agarselective for Yersinia enterocolitica. J.Clin. Microbiol., 1985;22:478-480.
12. Robinson, J., Beaman, J., Wagner,L. et al. Comparision ofdirect plating with the use of enrichment culture for isolation of Aeromonas species from faeces. J.Med.Microbiol., 1986; 22:315-317.
13. Carnahan, AM., Bebram, S., Joseph. SW. Aerokey II: A flexible key for identifying clinical aeromonas species.J.ClinMicrobiol., 1991 ;29:2843­2849.
14. Janda, J.M., Abbott, S.L., Khashe, S. ct al. Further studies on biochemical characteristics and serologic properties of the genus aeromonas. J.Clin.Micro­biol., 1996; 34: 1930-1933.

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