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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 96(2): 169-173, February 2001 Prevalence, Species Differentiation, Haemolytic Activity, and Antibiotic Susceptibility of Aeromonads in Untreated Khalifa Sifaw Ghenghesh/+, Abdelmula El-Ghodban*, Rabia Dkakni, Salaheddin Abeid, Abdurazzaq Altomi, Abdussalam Tarhuni**, Department of Medical Microbiology, Faculty of Medicine, P.O. Box 80013, Tripoli, Libya *Faculty of Science, Eotvos Lorand University, Budapest, Hungary **Arab Company of Soft Drinks and Mineral Water, The use of untreated water for drinking and other activities have been associated with intestinal and extraintestinal infections in humans due to Aeromonas species. In the present study aeromonadswere isolated from 48.7% of 1,000 water samples obtained from wells and other miscellaneous sources.
Aeromonas species were detected in 45% of samples tested in spring, 34.5% in summer, 48% in autumnand 60% of samples tested in winter. Speciation of 382 strains resulted in 225 (59%) being A. hydrophila,103 (27%) A. caviae, 42 (11%) A. sobria and 11 (3%) atypical aeromonads. Of 171 Aeromonas strainstested for their haemolytic activity, 53%, 49%, 40% and 37% were positive in this assay using human,horse, sheep and camel erythrocytes respectively. The results obtained indicate that potentiallyenteropathogenic Aeromonas species are commonly present in untreated drinking water obtainedfrom wells in Libya (this may also apply to other neighbouring countries) which may pose a healthproblem to users of such water supplies. In addition, ceftriaxone and ciprofloxacin are suitable drugsthat can be used in the treatment of Aeromonas-associated infections, particularly in theimmunocompromised, resulting from contact with untreated sources of water. Key words: Aeromonas - water - haemolysin - erythrocytes - antibiotics Members of the genus Aeromonas are gram- al. 1983, Janda et al. 1983, George et al. 1985, San- negative, oxidase-positive, facultative anaerobic, Joaquin & Picket 1988). Several studies have rod-shaped bacteria of the family Vibrionaceae.
reported that the drinking of untreated water is the They occur naturally in fresh water sources and most probable manner of acquiring these organisms are established pathogens of fish and amphibians (Holmberg et al. 1986, Moyer 1987). At least 13 (Hazen et al. 1978, Hazen & Fleirmans 1979, species are included in the genus Aeromonas at this time (Janda 1991). Three of these, namely aeromonads have for some time been recognized hydrophila, caviae and sobria, are most commonly as opportunistic pathogens in the immuno- associated with disease in humans. A number of compromised (von Graevenitz & Mensch 1968, virulence factors have been associated with these Washington 1972). They have been isolated from organisms and may be responsible for their skin and soft tissue infections of patients without enteropathogenicity, these include the production underlying conditions, but who suffered a trauma of cytotoxins, enterotoxins and haemolysins followed by exposure to water (Gold & Salit 1993).
(Gracey et al. 1982, Singh & Sanyal 1992, Majeed & Furthermore, Aeromonas species have been Macrae 1994). Burke et al. (1983b) have shown that implicated as causative agents of diarrhoea in haemolytic aeromonads are also enterotoxigenic and children and adults (Burke et al. 1983a, Goodwin et suggested that the detection of the haemolyticactivity is sufficient to discriminate enterotoxigenicAeromonas species. Although, Aeromonas-associated disease in the very young, the old andthe immunocompromised often requires antimicro-bial therapy, reports on the susceptibility of these +Corresponding author. Fax: 218-21-333.4474. E-mail: organisms to antimicrobial agent are rare in our re- gion. Furthermore, information concerning species distribution of aeromonads in well water is lacking.
The present study was carried out to determine the Aeromonas in Well Water• Khalifa Sifaw Ghenghesh et al.
prevalence, species differentiation, haemolytic ac- (Bauer et al. 1966). The following antibiotics were tivity (using four different types of erythrocytes), tested (Oxoid, UK): ampicillin, ceftriaxone, and antibiotic susceptibility of Aeromonas strains ciprofloxacin, cephaloridine, chloramphenicol, gen- in untreated well water in Tripoli area (population ~ tamicin, kanamycin, nalidixic acid, tetracycline and MATERIALS AND METHODS
Aeromonas species were isolated from 487 From March, 1993 to December, 1994, 1,000 (48.7%) water samples. Speciation of 382 strains drinking water samples obtained from wells (98% resulted in 59% being A. hydrophila, 27% A. caviae, of samples) and other miscellaneous sources of un- 11% A. sobria and 3% atypical aeromonads (Table treated water were examined. Water samples were I). Information on the depth of 481 wells was collected in sterile containers and processed within available. The wells were divided into three groups 3 h of collection. When it was known, the depth of according to their depth and aeromonads were isolated form 56% of 178 wells less than 20 m, 53%of 238 wells 20-30 m and 49% of 65 wells more than 30 m deep. The isolation rates of Aeromonas species Isolation - For the isolation of aeromonads, 2.5 from the three groups of wells are not statistically ml of the water samples were added to 25 ml of significant (P > 0.05). Aeromonads were detected in 45% of samples tested in spring, 34.5% in summer, enrichment. After an overnight incubation at 37oC, 48% in autumn and 60% of samples tested in winter a loopful from the APW was plated onto blood (Table II). Haemolytic activity of 171 Aeromonas agar supplemented with 15 mg/l ampicillin and the strains tested against human, horse, sheep and plates were incubated at 37oC overnight.
camel erythrocytes is shown in Table III. Regardless Identification - Identification of Aeromonas of the erythrocytes used, the results obtained show species was carried out using API 20E System (Bio- a statistically significant difference between Merieux, France) and the following tests: haemolysin production with A. sobria and A. production of oxidase, resistance to agent O/129 hydrophila compared with A. caviae strains (Oxoid, UK), gas production from glucose and (P<0.001, Chi-square test). Susceptibility testing of aesculin hydrolysis. Isolates identified as 40 Aeromonas strains (24 A. hydrophila, 12 A. Aeromonas by the API 20E, and which did not caviae and 4 A. sobria) against antimicrobial agents produce gas from glucose nor hydrolyse aesculin resulted in 100% being resistant to ampicillin, 95% were considered as atypical aeromonads.
to cephaloridine, and 5% to tetracycline. All (100%) Haemolysin assay - Bacterial strains were inoculated into 10 ml of brain heart infusion brothsupplemented with 0.3% (wt/vol) yeast extract(Oxoid), and incubated at 37oC with agitation (200rpm) in a waterbath shaker (Karl Kolb, West Germany) for 48 h. Cell-free supernatants were Results of speciation of 381 Aeromonas prepared by centrifugation (4000×g, 30 min) at 4oC and filtration (0.45 µm membrane filters, Sartorius, West Germany). Doubling dilutions of the cell-free supernatant test solutions in phosphate buffered saline (pH 7.4) were made in microtiter trays. Equal volumes (100 µl) of a 1% suspension of fresh, washed (three times) human, horse, sheep or camelerythrocytes was added. Phosphate buffered saline and broth blanks were included in each tray. Trayswere sealed and incubated for 1 h at 37oC and then for 1 h at 4oC. Haemolytic activity of cell-free Seasonal distribution of Aeromonas supernatants was considered positive if dilutions of >1:4 of each supernatant yielded 50% haemolysisof the erythrocytes. A total 171 Aeromonas strains Antibiotic susceptibility tests - The sus- ceptibility of 40 Aeromonas strains to antimicrobial agent was determined by the disc diffusion method Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 96(2), February 2001 Haemolytic activity of Aeromonas species isolated from well water in Tripoli area % of Aeromonas species showing haemolysis a: mean value for the sum of all assays is the predominant species in freshwater and mu- ciprofloxacin, chloramphenicol, gentamicin, kana- Using tissue culture and horse erythrocytes, a statistically significant correlation betweenproduction of cytotoxic haemolysin and the DISCUSSION
presence of diarrhoea has been reported by Brauer In the present study, nearly 50% of the untreated et al. (1985). Although in the present study the cytotoxic activity of the Aeromonas strains was Aeromonas species and nearly all of these samples not determined, the results obtained show that were obtained from wells. In a study, on the role of nearly half of our isolates were haemolytic and Aeromonas species in intestinal infections in the United States, Holmberg et al. (1986) reported that, Erythrocytes from small laboratory animals of 20 patients who could specify their water supply (mouse, rabbit, guinea pig) have been reported to in the week before their gastrointestinal illness, 18 be more sensitive than human and sheep erythro- had obtained their water from private wells and two cytes in the Aeromonas haemolysis assay had been drinking untreated spring water.
(Handfield et al. 1996). However, if the haemolysin Aeromonas species were reported to be isolated assay is to be used routinely in clinical laboratories in higher numbers during the summer (Ljungh & to detect enteropathogenic aeromonads, small ani- Wadstrom 1985). Also Aeromonas-associated mals are not a practical source of erythrocytes to diarrhoea was found to be high during the summer be used in such an assay. Our findings and those months (Burke et al. 1984, Agger et al. 1985). In the of others (Monfort & Baleux 1991) support the use present work, isolation rates of aeromonads were of human or horse erythrocytes in the haemolysin highest in the months of winter and lowest in assay and show that this simple assay can be eas- summer. Although there is no clear explanation to ily used to assist in the detection of pathogenic our findings, we can speculate this is may be due strains of Aeromonas species isolated from un- to the relatively mild winter and hot summer seasons treated water sources. Although camel erythrocytes in our region. Pathak et al. (1988), studying the have not been reported previously to detect seasonal distribution of aeromonads in river water, haemolytic aeromonads, they are not recommended for use in Aeromonas haemolysis assay.
We found no differences in the isolation rates In addition to drinking untreated water that con- of Aeromonas species in the water samples tains Aeromonas species, the taking of antibiotics obtained from wells with different depths. However, such as ampicillin to which these organisms are it is worth mentioning that one strain (A. caviae) resistant, may be a predisposing factor for the de- was isolated from a water sample obtained from a velopment of gastroenteritis (Holmberg et al. 1986, Moyer 1987). Antibiotic-resistant strains of There is little information available about Aeromonas have been isolated from aquatic envi- species distribution in aquatic environment (Araujo ronments and this resistance is principally plasmid Boira 1996). In the present study, A. hydrophila mediated (Hedges et al. 1985, Borrego et al. 1991).
was the most common species representing nearly Similar to other reports (Altwegg & Geiss 1989) all 60% of the aeromonads identified to the species our isolates were resistant to ampicillin and 95% to level. These results are consistent with the findings cephaloridine. All were susceptible to ceftriaxone, of others (Krovacek et al. 1992, Hanninen et al. 1997, cholramphenicol, ciprofloxacin and gentamicin.
Kuhn et al. 1997) who reported that A. hydrophila Although the isolates were also susceptible to Aeromonas in Well Water• Khalifa Sifaw Ghenghesh et al.
trimethoprim-sulphamethoxazole and nalidixic acid, Burke V, Gracey M, Robinson J, Peck D, Beaman J, recently we isolated aeromonads from children with Bundell C 1983a. The microbiology of children diarrhoea and from chicken carcases that were gastroenteritis: Aeromonas species and other highly resistant to trimethoprim-sulphamethoxazole infective agents. J Infect Dis 148: 68-74.
Burke V, Robinson J, Beaman J, Gracey M, Lesmana M, and nalidixic acid respectively (Ghenghesh et al.
Rockhill R, Echeverria P, Janda JM 1983b. Correla- 1998). Because of the isolation of multiple-resis- tion of enterotoxicity with biotype in Aeromonas tant Aeromonas species (including to trimethoprim- spp. J Clin Microbiol 18: 1196-1200.
sulphamethoxazole and nalidixic acid) from Burke V, Robinson J, Gracey M, Peterson D, Patridge freshwater in other parts of the world (Borrego et K 1984. Isolation of Aeromonas hydrophila from a al. 1991), our findings warrant the need to take metropolitan water supply: seasonal correlation with proper measures to prevent the introduction of clinical isolates. Appl Environ Microbiol 48: 361- aeromonads, that are resistant to these drugs, to George WL, Nakata MM, Thompson J, White ML 1985. Aeromonas related diarrhoea in adults. Arch In Libya, as it is in other developing countries, Intern Med 145: 2207-2211.
it is common to use water obtained from wells and Ghenghesh KS, Abeid S, Dkakni R, Tawil A, Elkot R other untreated sources, in addition to drinking, 1998. Susceptibility of Aeromonas species isolated for bathing and other purposes and this may be in Libya to antimicrobial agents. Second International hazardous to individuals with wounds, lacerations Meetings on the Therapy of Infections, Florence, or abrasions (Janda & Duffey 1988, Gold & Salit 1993, Kelly et al. 1993, Newton & Kennedy 1993).
Gold WL, Salit IE 1993. Aeromonas hydrophila Patients at risk to infections with Aeromonas infections of skin and soft tissue: report of 11 cases species also include those with underlying and review. Clin Infect Dis 16: 69-70.
Goodwin CS, William ES, Stewart JK, Gracey M, Burke malignancies and hepatobiliary disease (Goodwin V, Robison J 1983. Enterotoxigenic Aeromonas hydrophila and diarrhoea in adults. Med J Australia We conclude that potentially enteropathogenic Aeromonas species are commonly present in Gracey M, Burke V, Robinson J 1982. Aeromonas- untreated drinking water obtained from wells in associated gastroenteritis. Lancet ii: 1304-1306.
Libya (this may also apply to other neighbouring Handfield M, Smard P, Cuillard M, Letarte R 1996.
countries) which may pose a health problem to users Aeromonas hydrophila isolated from food and of such water supplies. In addition, ceftriaxone and drinking water: hemagglutination, hemolysis, and ciprofloxacin are suitable drugs that can be used in cytotoxicity for a human intestinal cell line (HT-29).
Appl Environ Microbiol 62: 3459-3461.
the treatment of Aeromonas-associated infections, Hanninen ML, Oivanen P, Hirvela-Koski V 1997.
particularly in the immunocompromised, resulting Aeromonas species in fish, fish-eggs, shrimp and from contact with untreated sources of water.
fresh water. Inter J Food Microbiol 34: 17-26.
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