versión impresa ISSN 0301-732X
Arch. med. vet. v.35 n.1 Valdivia ene. 2003
Isolation of thermotolerant species of Campylobacter from river water
using two collection methods*
Aislamiento de especies termotolerantes de Campylobacter de aguas fluviales
utilizando dos métodos de colecta.
H. FERNANDEZ1 , TM, D.Sc.; L. OTTH, TM; M. WILSON, TM.
Instituto de Microbiología Clínica, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.
* This work received financial support from Grants FONDECYT 1980920 and DID-UACh S-2001-33.
1 Corresponding author.e-mail: firstname.lastname@example.org
Campylobacter jejuni y C. coli son agentes zoonóticos de infecciones intestinales que pueden contaminar cuerpos de agua. Varios brotes de gastroenteritis por Campylobacter asociados al consumo de agua han sido descritos. En este estudio se compara el rendimiento de los métodos de la tórula de Moore (MSM) y de filtración por membrana para el aislamiento de Campylobacter en 181 muestras de agua obtenidas del curso urbano del río Calle-Calle. En el 24.3% de las muestras obtenidas por MSM y en el 7.2% de las obtenidas por MFM fue posible aislar especies termotolerantes de Campylobacter, siendo C. coli aislado en mayor proporción que C. jejuni. Los resultados sugieren que MSM es un método eficiente para el aislamiento de estas bacterias a partir de agua.
Palabras claves: Campylobacter jejuni, C. coli, agua, tórula de Moore,técnica de filtración por membranas.
Key words: Campylobacter jejuni, C. coli, water, Moore swab, membrane filtration technique.
The thermotolerant species of Campylobacter, C. jejuni and C. coli are considered as important agents of intestinal infection all over the world (Allos and Blaser, 1995; Fernández, 1992; Friedman et al., 2000). Many domestic and wild animals are known as natural reservoirs that, together with sewage, could contaminate water bodies. Both species can survive in water from several days to several weeks, depending on water temperature (Anderson et al., 1997; Pickert and Bozenhart, 1985).
Several outbreaks of Campylobacter gastroenteritis associated with the consumption of contaminated water have been reported (Anderson et al., 1997; Sacks et al., 1986; Skerjve and Brennhovd, 1992; Jacobs-Reitsma, 2000).
Various methods have been described in order to determine the presence of enteropathogenic bacteria in water, being enrichment procedures and filtration through membranes the most frequently used (Bolton et al., 1987; Mathewson et al., 1983). The Moore swab is a concentration method described in 1948 to isolate Salmonella and Vibrio cholerae from water (Barret et al., 1980). However, little information is available in relation to the isolation of Campylobacter species from water bodies using this method. The Moore swab was used previously in a pilot study in our laboratory (Fernández et al., 1990).
In this study we compared the performance of the Moore swab (MSM) and the filtration through membrane (MFM) methods for the isolation of thermotolerant species of Campylobacter from water samples obtained from the Calle-Calle river, Valdivia, Chile (39º 47 Southern latitude, 73º 15 Western latitude).
MATERIAL AND METHODS
We obtained duplicated water samples (181 for MSM and 181 for MFM) from different points of the urban course of the Calle-Calle river (26 Km long, 297 m average wide), according to the following methods:
Moore Swab Method (MSM) (Fernández et al., 1990): consists of a piece of gauze of 70x22 cm, folded several times to form cylindrical rolls which were wrapped in thick paper and autoclaved. Each swab, attached to a 5 m long nylon string, was left for 24 hrs in the river. When removed, each swab was placed into a flask containing 125 ml of an enrichment broth with a formulation twice higher than the normal one, to compensate the dilution produced by the water absorbed by the gauze. This medium consisted of Brucella broth, FBP supplement (ferrous sulphate, sodium metabissulfite and sodium pyruvate 0.5 g/L of each), vancomycine (20 mg/ L), polimyxin B (5.000 I.U./L), trimethoprim (10 mg/L), cephalotine (20 mg/L) and anphotericin (4 mg/L). The flasks were incubated at 42ºC for 48 hrs under microaerobic atmosphere, and 200 µl were seeded on Butzler selective medium incubated as described above.
Membrane Filtration Method (MFM) (Mathewson et al., 1983): Four litre of water were passed through a sterile 47 mm membrane filter (Millipore) with 0.45 µm pore size, using a vacuum pump. The filter was aseptically placed into a flask containing 100 ml of the enrichment broth described above. The rest of the procedure was similar to that used with the MSM.
Suspect Campylobacter colonies were seeded on blood agar in order to obtain pure culture and then, they were identified and biotyped by their morphological characteristics, susceptibility to cephalotine and nalidixic acid and by their capacity to produce hypuricase, desulfhydrase and DNAse (Fernández et al., 1990).
RESULTS AND DISCUSSION
As shown in table I, 24.3% of the samples were positive for Campylobacter using the MSM method whereas, with the MFM, only in 7.2% these micro-organisms were isolated, being this difference statistically significative (chi-square test). The higher isolation rate obtained with the MSM could be due to the fact that the swabs remain during 24 hrs into the water, thus allowing filtration of a great quantity of water and the capture of Campylobacter cells, as well as other micro-organisms, suspended in the water. The water sample volume used for the MFM was 4 L, an amount that compared with the MSM, could be insufficient to allow a higher isolation rate.
Table I. Isolation rates of Campylobacter species using the Moore Swab and the Membrane Filtration Methods.
Aislamiento de especies de Campylobacter utilizando la tórula de Moore y el Método de Filtración por Membrana.
|N= number of positive samples; n= total number of samples; *p<0.01|
Contrariously to earlier reports (Bolton et al., 1987; Skerjve and Brennhovd, 1992), C. coli was most frequently isolated (77.3%) than C. jejuni (22.7%). Similar results were obtained with both methods (MASM and MFM). A higher isolation rate of C. coli from wastewater samples was observed by Höller (1988) and in river water samples by Fernández et al. (1990). Some authors suggested that C. coli could be more resistant than C. jejuni to environmental conditions (Tresierra-Ayala et al., 1999). That could explain the differences observed in the isolation rates between both species. Currently, experimental studies are carried out in our laboratory in order to establish if C. coli is more resistant than C. jejuni to environmental conditions. Despite this difference, it is necessary to bear in mind that both species are considered as important agents of diarrheal disease, and contaminated water are recognised as an epidemiological source of outbreaks as well as sporadic cases of intestinal campylobacteriosis.
Our results show that the MSM is an efficient method to isolate Campylobacter species. Recently we start to use the MSM to isolate Arcobacter species from the same type of samples with good results (data not showed). Based on these results we propose the use of MSM as an adequate, easy and inexpensive method for the isolation of Campylobacter species from river water samples.
Campylobacter jejuni and C. coli are zoonotic agents of intestinal infection that could contaminate water bodies. Several outbreaks of Campylobacter gastroenteritis associated with the consumption of contaminated water have been reported. Various methods have been described to determine the presence of these bacteria in water bodies. In this study the performance of the Moore swab (MSM) and the filtration through membrane methods (MFM) for the isolation of Campylobacter from 181 water samples obtained from the urban course of the Calle-Calle river water samples was compared. Campylobacter species were isolated in 24.3% of the samples obtained with MSM and in 7.2% of those processed by MFM thus, appearing MSM as an efficient method to isolate Campylobacter from river water samples.
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