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The occurrence of Aeromonas spp. in natural mineral water and well water
International Journal of Food Microbiology 63 (2001) 169–173 www.elsevier.nl / locate / ijfoodmicro
Short communication
The occurrence of Aeromonas spp. in natural mineral water and well water a, a b Salvatore Massa *, Clelia Altieri , Antonio D’Angela a
Istituto di Produzioni e Preparazioni Alimentari, Facolta` di Agraria, via Napoli n. 25, 71100 Foggia, Italy b Presidio Multizonale di Prevenzione, via Maglie n. 5, 73100 Lecce, Italy Accepted 7 August 2000
Abstract The presence of motile Aeromonas spp. in natural mineral water (NMW) and drinking well water in the area of Lecce (Italy) was investigated. Aeromonas spp. were not detected in any of the 60 NMW samples either by the direct and enrichment method. From a total of 20 wells, five were found to contain Aeromonas species with cell number ranging from 26 to 1 609 250 ml 21 . In two wells the presence of Aeromonas spp. was not associated to the presence of faecal indicators, i.e. coliforms and faecal coliforms. Sixty-five Aeromonas spp. strains isolated in this survey were identified at species level and some were examined for haemolysis, Voges Proskauer reaction, lysine decarboxylase and sorbitol fermentation, tests which had been previously shown to correlate with production of enterotoxin / cytotoxin. Isolates identified as A. hydrophila and A. sobria showed potentially virulent properties. 2001 Elsevier Science B.V. All rights reserved. Keywords: A. hydrophila; Natural mineral water; Well drinking water
1. Introduction The Aeromonas genus is taxonomically complex and 14 DNA hybridization groups (HGs) have now been identified (Kirow, 1993; 1997). The species A. hydrophila, A. caviae and A. sobria (Janda, 1991) are motile and have been linked to two major group of human diseases: septicemia and gastroenteritis (Merino et al., 1995). Recent studies on the prevalence of motile Aeromonas spp. in the stools of 618 *Corresponding author. Tel.: 1 39-0881-714-544; fax: 1 390881-740-211. E-mail address: [email protected] (S. Massa).
children with diarrhoea in Northen, Central and Southern Italy, and in stool samples from 6403 gastroenteritis cases in Tuscany area, have reported a percentage of isolation of 2.2 and 1.4%, respectively (Caprioli et al., 1996; Figura et al., 1997) The motile species are known to occurr widely in the environment, especially in fresh water (Hazen et al., 1978), and they have been recovered from both unchlorinated (Burke et al., 1984a) and chlorinated water supplies (Le Chevallier et al., 1982; Burke et al., 1984b; Massa et al., 1999). The presence in drinking water is receiving increasing attention in recent years, because they have been associated with enteric infections, in particular
0168-1605 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00410-4
S. Massa et al. / International Journal of Food Microbiology 63 (2001) 169 – 173
170
A. hydrophila and A. sobria (Champsaur et al., 1982; Burke et al., 1984a; Krovacek et al., 1989). Drinking untreated water has been identified as a significant risk factor for Aeromonas-associated diseases (Kirow, 1997). Although the mechanism of their enteropathogenicity is not fully elucidated and understood, these organisms produce enterotoxins, haemolysins, cytotoxins, potentially acting as virulence factors (Cahill, 1990). A correlation seems to exist between enterotoxin and cytotoxin production and certain biochemical tests, i.e. haemolysis of rabbit erythrocytes, production of acetoin (Voges– Proskauer (VP) reaction), lysine decarboxylation and sorbitol fermentation (Cahill, 1990). The aims of the present study were: (i) to determine the presence of motile Aeromonas spp. in drinking water not treated with chlorine, such as natural mineral water and well water; (ii) to identify the isolated strains to species level; and (iii) to investigate if they were correlated with virulent factors production.
2. Materials and methods
2.1. Samples Sixty natural mineral water (NMW) samples representative of 15 water brands sold all over Italy, were tested. All samples were purchased at retail outlets in Lecce (southern Italy). Water samples were classified on the basis of the fixed residual (FR) (indicated on the label) as follows: (a) minimal mineralized water (FR , 50 mg l 21 ); (b) oligomineral water (FR 5 50–500 mg l 21 ); and (c) mineral water (FR . 500 mg l 21 ) (Table 1). All the NMWs
were tested for Aeromonas spp. and heterothrophic plate count (HPC). In the same period (May– November, 1998), water samples from 20 private wells (depth 15–80 m) were tested for coliforms and faecal coliforms, faecal enterococci and Aeromonas spp. The water samples, collected asceptically in 1 l sterile plastic bottles, were carried to the laboratory immediately and processed within 4 h of collection.
2.2. Microbiological analysis Heterotrophic plate counts were determined in Plate Count Agar (Oxoid) inoculated in duplicate with 1 ml volume of undiluted samples and 1 ml of decimal dilution in sterile saline and incubated at 228C for 72 h and at 378C for 24 h, respectively. Coliforms and faecal coliforms (TC and FC), and faecal enterococci (FS) were determined according to Massa et al. (1998). Aeromonas spp. were detected by membrane filtration (0.45 mm, Millipore) of 250 ml water and incubation of the filter in 100 ml of Alcaline Peptone Water (APW, Oxoid) at 288C for 24 h. After incubating, the enriched cultures in APW were streaked onto Starch Ampicillin Agar (SAA, Biolife, Milan, Italy) and incubated at 308C for 24 h. For the direct method Aeromonas spp. were isolated and enumerated by filtering suitable volumes through 0.45 mm membrane filters, which were cultured on SAA. Suspected colonies were streaked on Nutrient Agar (Oxoid) to ensure purity. Isolates were Gram stained and identified as presumptive Aeromonas spp. if they were Gram-negative, oxidase-positive and glucose-fermenting (O / F test). The strains were further identified with API 20 NE (Biomerieux, France), according to the manufacturer’s instructions. The following tests were then performed, all at 308C:
Table 1 Heterotrophic plate counts (HPC) at different temperatures in natural mineral water samples, subdivided according to fixed residual Fixed residual a
, 50 50–500 . 500
Number of samples
15 30 15
HPC (8C)
Aeromonas spp.
22 Mn b
Mx
M
Mn
37 Mx
M
Direct cultured
Enrichment cultured
54 6 4
663 1480 106
358 743 55
3320 89 77
3840 9160 3800
3580 4624 228
ND c ND ND
ND ND ND
Units in mg l 21 . Mn, minimum; Mx, maximum; M, median. c ND, not detected. a
b
S. Massa et al. / International Journal of Food Microbiology 63 (2001) 169 – 173
motility, lysine decarboxylase (LDC), VP reaction, ability to produce gas from glucose, aesculin hydrolysis and fermentation of arabinose, salicin and sorbitol and differentiated at species level (A. hydrophila, A. caviae, A. sobria) using the criteria proposed by Popoff (1984). Haemolysis was studied on 7% (v / v) blood agar plates containing washed rabbit erythrocytes.
Table 2 Microbiological analysis of drinking water samples from 20 wells Wells
A B C D E a
3. Results and discussion Recently, Italian law requires that A. hydrophila should not exceed 10 and 100 cfu ml 21 , at source and during marketing, respectively (GUDRI, 1997). Aeromonas spp. were not detected in any of the natural mineral water samples either by the direct or enrichment method. Not much data are available in literature about the presence of Aeromonas spp. in natural mineral water. Schwaller and Schmidt-Lorenz (1981) specified that no Aeromonas spp. was identified among the isolates recovered. Similar results were reported by other authors: Hunter and Burge (1987) examined 58 bottles of mineral water from European countries (29 still and 29 carbonated) by a specific enrichment procedure and did not demonstrate the presence of Aeromonas spp. in any of the 100 ml samples. Similarly, Havelaar et al. (1990) did not observe growth of aeromonads in any of 64 samples of still mineral water examined using the membrane filtration method. Only two papers from Spain positively report the isolation of Aeromonas from mineral waters, Gonzales et al. (1987) detected this organism in one of three sources investigated, while Quevedo-Sarmiento et al. (1986) showed that ca. 10% of all colonies recovered on nutrient agar at 228C belonged to Aeromonas spp. Table 1 shows a large variation of HPC from 6 to 1480 and from 89 to 9160 cfu ml 21 , respectively at 22 and 378C, in the samples with 50–500 mg l 21 fixed residual. These findings are comparable to that reported in other studies (Manaia et al., 1990; Massa et al., 1998; Warburton et al., 1998). The concern over high bacterial counts in bottled water is due to the masking of potential pathogenic bacteria (Massa et al., 1998). Aeromonas spp. were isolated from five of 20 examined wells, with cell numbers ranging from 26
171
b
Coliforms a Total
Faecal
500 10 ND ND 5
200 3 ND ND 4
Faecal enterococci
Aeromonas spp.a
ND b ND ND ND ND
1609 918 26 94 49
cfu 250 ml 21 . ND, not detected.
to 1 609 250 ml 21 (Table 2). In two wells the presence of Aeromonas spp. was not associated to the presence of faecal indicators, i.e. coliforms and faecal coliforms. Other authors have found that coliform counts did not correlate with Aeromonas spp. count. Some reports from Australia (Burke et al., 1984a) or from Northen America (Le Chevallier et al., 1982), question the suitability of coliforms as index of water quality. Similarly, an investigation about the occurrence of Aeromonas spp. in drinking water supplies in a mountain area in Northeast Italy (Legnani et al., 1998), showed that no correlation was demonstrated between the concentration of Aeromonas spp. and faecal indicator organisms. In the present study the high number of Aeromonas spp. indicated that they may be good indicators of hygienic quality of well water. Consequently their search should be used to indicate unsatisfying conditions, especially in private well water not undergoing systematic chlorine treatment. All the 65 strains selected for identification were found to be motile Aeromonas spp. Based on their reaction in biochemical tests, i.e. acid from arabinose and salicin, aesculin hydrolysis and gas from glucose (Popoff, 1984) the strains were identified as A. hydrophila (20, 20 and five isolates from well A, B and C, respectively), A. sobria (10 from well D) and A. caviae (10 from well E) (Table 3). Ten Aeromonas isolates were further investigated. Although Janda et al. (1983) found that only 42% of 12 cytotoxic A. hydrophila strains from pediatric patients were VP-positive, a strong correlation between cytotoxicity and positive VP reaction has been reported by Cumberbatch et al. (1979), Kaper et al. (1981) and Burke et al. (1983). Other investigators have correlated Aeromonas spp. toxigenicity with a
172
S. Massa et al. / International Journal of Food Microbiology 63 (2001) 169 – 173
Table 3 Species of Aeromonas isolated from drinking well water Species
Number of strains
Acid from Arabinose
Salicin
A. hydrophila A. hydrophila A. hydrophila A. sobria A. caviae
20 20 5 10 10
1 1 1 2 1
1 1 1 2 1
positive LDC reaction and sorbitol fermentation (Cumberbatch et al., 1979; Janda et al., 1983; Callister and Agger, 1987). Finally, according to Burke et al. (1983), 97% of cytotoxic Aeromonas spp. strains could be correctly classified by haemolysin assay, and the correlation was stronger for drinking water isolates than for food (raw meat, ready to eat products, milk) isolates (Handfield et al., 1996). On the basis of these references, seven strains among those examined in the present study showed putative virulence properties, in particular the A. hydrophila strains and one A. sobria strain (results not shown). Both A. hydrophila and A. sobria appear to be inherently more virulent than those organisms classified as A. caviae. The public health significance of these findings is unknown since further research is nedeed to identify more specifically the properties responsible for enteropathogenicity in the aeromonads (Kirow, 1997).
References Burke, V., Robinson, J., Beaman, J., Gracey, M., Lesmana, M., Rockhill, R., Echeverria, P., Janda, J.M., 1983. Correlation of enterotoxicity with biotype in Aeromonas spp. J. Clin. Microbiol. 18, 1196–1200. Burke, V., Robinson, J., Gracey, M., Petersen, D., Meyer, N., Haley, V., 1984a. Isolation of Aeromonas spp. from an unchlorinated domestic water supply. Appl. Environ. Microbiol. 48, 367–370. Burke, V., Robinson, J., Gracey, M., Petersen, D., Partridge, P., 1984b. Isolation of Aeromonas spp. from a metropolitan water supply: seasonal correlation with clinical isolates. Appl. Environ. Microbiol. 48, 361–366. Cahill, M., 1990. A review: virulence factors in Aeromonas species. J. Appl. Bacteriol. 69, 1–16. Callister, S., Agger, W., 1987. Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae from grocery store produce. Appl. Environ. Microbiol. 53, 249–253.
Esculin hydrolysis
Gas from glucose
Well
1 1 1 2 1
1 1 1 1 2
A B C D E
Caprioli, A., Pezzella, C., Morelli, R., Giammanco, A., Arista, S., Crotti, D., Facchini, M., Guglielmetti, P., Piersimoni, C., Luzzi, I., 1996. Enteropathogens associated with childhood diarrhea in Italy. Pediatr. Infect. Dis. J. 15, 876–883. Champsaur, H., Andremont, A., Mathieu, D., Rottman, E., Auzepy, P., 1982. Cholera-like illness due to Aeromonas sobria. J. Infect. Dis. 145, 248–254. Cumberbatch, N., Gurwith, M.J., Langston, C., Sacks, R.B., Brunton, J.L., 1979. Cytotoxix enterotoxin produced by Aeromonas hydrophila: relationship of toxigenic isolates to diarrheal disease. Infect. Immun. 23, 829–837. Figura, N., Guglielmetti, P., Zanchi, A., Signori, R., Rossolini, A., Lior, H., Russi, M., Musmanno, R., 1997. Species, biotype and serogroup of Campylobacter spp. isolated from children with diarrhoea over a 10-year period. New Microbiol. 20, 303–310. Gonzales, C., Gutierrez, C., Grande, T., 1987. Bacterial flora in bottled uncarbonated mineral drinking water. Can. J. Microbiol. 33, 1120–1125. GUDRI 1997 (Gazzetta Ufficiale della Repubblica Italiana 23 / 7 / 1997). Decreto 8 luglio 1997. Integrazioni dei criteri di valutazione della caratteristiche microbiologiche delle acque minerali naturali. Handfield, M., Simard, P., Couillard, M., Letarte, R., 1996. Aeromonas hydrophila isolated from food and drinking water: haemoagglutination, haemolysis and cytotoxicity for a human intestinal cell-line (HT-29). Appl. Environ. Microbiol. 62, 3459–3461. Havelaar, A., Toorop-Bouma, A., Medema, G., 1990. The occurrence and significance of Aeromonas in water with special reference to natural mineral water. Rivista Italiana di Igiene 50, 349–356. Hazen, T.C., Fliermans, C.B., Hirsch, R.P., Esch, P.T., 1978. Prevalence and distribution of Aeromonas hydrophila in the United States. Appl. Environ. Microbiol. 36, 731–738. Hunter, P.R., Burge, S.H., 1987. The bacteriological quality of bottled mineral waters. Epidemiol. Infect. 99, 439–443. Janda, J.M., Bottone, E.J., Skinner, C.V., Calcaterra, D., 1983. Phenotypic markers associated with gastrointestinal Aeromonas hydrophila isolates from symptomatic children. J. Clin. Microbiol. 17, 588–591. Janda, J.M., 1991. Recent advances in the study of the taxonomy, pathogenicity and infections syndromes associated with the genus Aeromonas. Clin. Microbiol. Rev. 4, 397–410. Kaper, J.B., Lockman, H., Colwell, R.R., 1981. Aeromonas hydrophila: ecology and toxigenic of isolates from an estuary. J. Appl. Bacteriol. 50, 359–377.
S. Massa et al. / International Journal of Food Microbiology 63 (2001) 169 – 173 Kirow, S.M., 1993. The public health significance of Aeromonas spp. in foods. Int. J. Food Microbiol. 20, 179–198. Kirow, S.M., 1997. Aeromonas and Plesiomonas species. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology. Fundamentals and Frontiers. ASM Press, Washington DC, USA, pp. 265–287. Krovacek, K., Peterz, M., Faris, A., Mansson, I., 1989. Enterotoxigenicity and drug sensitivity of Aeromonas hydrophila isolated from well water in Sweden: a case study. Int. J. Food Microbiol. 8, 149–154. Le Chevallier, M.W., Evans, T.M., Seidler, R.J., Daily, O.P., Merrel, B.R., Rollins, D.M., Joseph, S.W., 1982. Aeromonas sobria in chlorinated drinking water supplies. Microb. Ecol. 8, 325–333. Legnani, P., Leoni, E., Soppelsa, F., Burigo, R., 1998. The occurrence of Aeromonas spp. in drinking water supplies of an area of Dolomite mountains, Italy. J. Appl. Microbiol. 85, 271–276. Manaia, C.M., Nunes, O.C., Morais, P.V., Da Costa, M.S., 1990. Heterotrophic plate counts and isolation of bacteria from mineral waters on selective and enrichment media. J. Appl. Bacteriol. 69, 871–876. Massa, S., Fanelli, M., Brienza, M.T., Sinigaglia, M., 1998. The bacterial flora in bottled natural mineral water sold in Italy. J. Food Qual. 21, 175–185.
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Massa, S., Armuzzi, R., Canganella, F., Tosques, M., Trovatelli, L.D., 1999. Susceptibility to chlorine of Aeromonas hydrophila in water. J. Appl. Microbiol. 86, 169–173. ` J.M., 1995. EmergMerino, S., Rubires, X., Knøchel, S., Tomas, ing pathogens: Aeromonas spp. Int. J. Food Microbiol. 28, 157–168. Popoff, M., 1984. Genus III. Aeromonas Kluyver and van Niel 1936, 1938. In: Krieg, N.R., Holt, J.G. (Eds.). Bergey’s Manual of Systematic Bacteriology, Vol. 1. Williams and Wilkins, Baltimore, pp. 545–548. Quevedo-Sarmiento, J., Ramos-Cormenzana, A., Gonzales-Lopez, J., 1986. Isolation and characterization of aerobic and heterotrophic bacteria from natural springs in the Lanjaron aree (Spain). J. Appl. Bacteriol. 61, 365–372. Schwaller, P., Schmidt-Lorenz, W., 1981. La flore microbienne de ´ ´ quatre eaux mineralel non Gazeifies et mises en bouteilles. ` ´ 2eme communication. Les Pseudomonas et autres bacteries gram-negatif. Composition fine de la flore. Zentralblatt fur Bakteriologie und Hygiene I abt. Orig. C 2, 179–196. Warburton, D., Harrison, B., Crawford, C., Foster, R., Fox, C., Gour, L., Krol, P., 1998. A further review of the microbiological quality of bottled water sold in Canada: 1992–1997 survey results. Int. J. Food Microbiol. 39, 221–226.
Short communication
The occurrence of Aeromonas spp. in natural mineral water and well water a, a b Salvatore Massa *, Clelia Altieri , Antonio D’Angela a
Istituto di Produzioni e Preparazioni Alimentari, Facolta` di Agraria, via Napoli n. 25, 71100 Foggia, Italy b Presidio Multizonale di Prevenzione, via Maglie n. 5, 73100 Lecce, Italy Accepted 7 August 2000
Abstract The presence of motile Aeromonas spp. in natural mineral water (NMW) and drinking well water in the area of Lecce (Italy) was investigated. Aeromonas spp. were not detected in any of the 60 NMW samples either by the direct and enrichment method. From a total of 20 wells, five were found to contain Aeromonas species with cell number ranging from 26 to 1 609 250 ml 21 . In two wells the presence of Aeromonas spp. was not associated to the presence of faecal indicators, i.e. coliforms and faecal coliforms. Sixty-five Aeromonas spp. strains isolated in this survey were identified at species level and some were examined for haemolysis, Voges Proskauer reaction, lysine decarboxylase and sorbitol fermentation, tests which had been previously shown to correlate with production of enterotoxin / cytotoxin. Isolates identified as A. hydrophila and A. sobria showed potentially virulent properties. 2001 Elsevier Science B.V. All rights reserved. Keywords: A. hydrophila; Natural mineral water; Well drinking water
1. Introduction The Aeromonas genus is taxonomically complex and 14 DNA hybridization groups (HGs) have now been identified (Kirow, 1993; 1997). The species A. hydrophila, A. caviae and A. sobria (Janda, 1991) are motile and have been linked to two major group of human diseases: septicemia and gastroenteritis (Merino et al., 1995). Recent studies on the prevalence of motile Aeromonas spp. in the stools of 618 *Corresponding author. Tel.: 1 39-0881-714-544; fax: 1 390881-740-211. E-mail address: [email protected] (S. Massa).
children with diarrhoea in Northen, Central and Southern Italy, and in stool samples from 6403 gastroenteritis cases in Tuscany area, have reported a percentage of isolation of 2.2 and 1.4%, respectively (Caprioli et al., 1996; Figura et al., 1997) The motile species are known to occurr widely in the environment, especially in fresh water (Hazen et al., 1978), and they have been recovered from both unchlorinated (Burke et al., 1984a) and chlorinated water supplies (Le Chevallier et al., 1982; Burke et al., 1984b; Massa et al., 1999). The presence in drinking water is receiving increasing attention in recent years, because they have been associated with enteric infections, in particular
0168-1605 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00410-4
S. Massa et al. / International Journal of Food Microbiology 63 (2001) 169 – 173
170
A. hydrophila and A. sobria (Champsaur et al., 1982; Burke et al., 1984a; Krovacek et al., 1989). Drinking untreated water has been identified as a significant risk factor for Aeromonas-associated diseases (Kirow, 1997). Although the mechanism of their enteropathogenicity is not fully elucidated and understood, these organisms produce enterotoxins, haemolysins, cytotoxins, potentially acting as virulence factors (Cahill, 1990). A correlation seems to exist between enterotoxin and cytotoxin production and certain biochemical tests, i.e. haemolysis of rabbit erythrocytes, production of acetoin (Voges– Proskauer (VP) reaction), lysine decarboxylation and sorbitol fermentation (Cahill, 1990). The aims of the present study were: (i) to determine the presence of motile Aeromonas spp. in drinking water not treated with chlorine, such as natural mineral water and well water; (ii) to identify the isolated strains to species level; and (iii) to investigate if they were correlated with virulent factors production.
2. Materials and methods
2.1. Samples Sixty natural mineral water (NMW) samples representative of 15 water brands sold all over Italy, were tested. All samples were purchased at retail outlets in Lecce (southern Italy). Water samples were classified on the basis of the fixed residual (FR) (indicated on the label) as follows: (a) minimal mineralized water (FR , 50 mg l 21 ); (b) oligomineral water (FR 5 50–500 mg l 21 ); and (c) mineral water (FR . 500 mg l 21 ) (Table 1). All the NMWs
were tested for Aeromonas spp. and heterothrophic plate count (HPC). In the same period (May– November, 1998), water samples from 20 private wells (depth 15–80 m) were tested for coliforms and faecal coliforms, faecal enterococci and Aeromonas spp. The water samples, collected asceptically in 1 l sterile plastic bottles, were carried to the laboratory immediately and processed within 4 h of collection.
2.2. Microbiological analysis Heterotrophic plate counts were determined in Plate Count Agar (Oxoid) inoculated in duplicate with 1 ml volume of undiluted samples and 1 ml of decimal dilution in sterile saline and incubated at 228C for 72 h and at 378C for 24 h, respectively. Coliforms and faecal coliforms (TC and FC), and faecal enterococci (FS) were determined according to Massa et al. (1998). Aeromonas spp. were detected by membrane filtration (0.45 mm, Millipore) of 250 ml water and incubation of the filter in 100 ml of Alcaline Peptone Water (APW, Oxoid) at 288C for 24 h. After incubating, the enriched cultures in APW were streaked onto Starch Ampicillin Agar (SAA, Biolife, Milan, Italy) and incubated at 308C for 24 h. For the direct method Aeromonas spp. were isolated and enumerated by filtering suitable volumes through 0.45 mm membrane filters, which were cultured on SAA. Suspected colonies were streaked on Nutrient Agar (Oxoid) to ensure purity. Isolates were Gram stained and identified as presumptive Aeromonas spp. if they were Gram-negative, oxidase-positive and glucose-fermenting (O / F test). The strains were further identified with API 20 NE (Biomerieux, France), according to the manufacturer’s instructions. The following tests were then performed, all at 308C:
Table 1 Heterotrophic plate counts (HPC) at different temperatures in natural mineral water samples, subdivided according to fixed residual Fixed residual a
, 50 50–500 . 500
Number of samples
15 30 15
HPC (8C)
Aeromonas spp.
22 Mn b
Mx
M
Mn
37 Mx
M
Direct cultured
Enrichment cultured
54 6 4
663 1480 106
358 743 55
3320 89 77
3840 9160 3800
3580 4624 228
ND c ND ND
ND ND ND
Units in mg l 21 . Mn, minimum; Mx, maximum; M, median. c ND, not detected. a
b
S. Massa et al. / International Journal of Food Microbiology 63 (2001) 169 – 173
motility, lysine decarboxylase (LDC), VP reaction, ability to produce gas from glucose, aesculin hydrolysis and fermentation of arabinose, salicin and sorbitol and differentiated at species level (A. hydrophila, A. caviae, A. sobria) using the criteria proposed by Popoff (1984). Haemolysis was studied on 7% (v / v) blood agar plates containing washed rabbit erythrocytes.
Table 2 Microbiological analysis of drinking water samples from 20 wells Wells
A B C D E a
3. Results and discussion Recently, Italian law requires that A. hydrophila should not exceed 10 and 100 cfu ml 21 , at source and during marketing, respectively (GUDRI, 1997). Aeromonas spp. were not detected in any of the natural mineral water samples either by the direct or enrichment method. Not much data are available in literature about the presence of Aeromonas spp. in natural mineral water. Schwaller and Schmidt-Lorenz (1981) specified that no Aeromonas spp. was identified among the isolates recovered. Similar results were reported by other authors: Hunter and Burge (1987) examined 58 bottles of mineral water from European countries (29 still and 29 carbonated) by a specific enrichment procedure and did not demonstrate the presence of Aeromonas spp. in any of the 100 ml samples. Similarly, Havelaar et al. (1990) did not observe growth of aeromonads in any of 64 samples of still mineral water examined using the membrane filtration method. Only two papers from Spain positively report the isolation of Aeromonas from mineral waters, Gonzales et al. (1987) detected this organism in one of three sources investigated, while Quevedo-Sarmiento et al. (1986) showed that ca. 10% of all colonies recovered on nutrient agar at 228C belonged to Aeromonas spp. Table 1 shows a large variation of HPC from 6 to 1480 and from 89 to 9160 cfu ml 21 , respectively at 22 and 378C, in the samples with 50–500 mg l 21 fixed residual. These findings are comparable to that reported in other studies (Manaia et al., 1990; Massa et al., 1998; Warburton et al., 1998). The concern over high bacterial counts in bottled water is due to the masking of potential pathogenic bacteria (Massa et al., 1998). Aeromonas spp. were isolated from five of 20 examined wells, with cell numbers ranging from 26
171
b
Coliforms a Total
Faecal
500 10 ND ND 5
200 3 ND ND 4
Faecal enterococci
Aeromonas spp.a
ND b ND ND ND ND
1609 918 26 94 49
cfu 250 ml 21 . ND, not detected.
to 1 609 250 ml 21 (Table 2). In two wells the presence of Aeromonas spp. was not associated to the presence of faecal indicators, i.e. coliforms and faecal coliforms. Other authors have found that coliform counts did not correlate with Aeromonas spp. count. Some reports from Australia (Burke et al., 1984a) or from Northen America (Le Chevallier et al., 1982), question the suitability of coliforms as index of water quality. Similarly, an investigation about the occurrence of Aeromonas spp. in drinking water supplies in a mountain area in Northeast Italy (Legnani et al., 1998), showed that no correlation was demonstrated between the concentration of Aeromonas spp. and faecal indicator organisms. In the present study the high number of Aeromonas spp. indicated that they may be good indicators of hygienic quality of well water. Consequently their search should be used to indicate unsatisfying conditions, especially in private well water not undergoing systematic chlorine treatment. All the 65 strains selected for identification were found to be motile Aeromonas spp. Based on their reaction in biochemical tests, i.e. acid from arabinose and salicin, aesculin hydrolysis and gas from glucose (Popoff, 1984) the strains were identified as A. hydrophila (20, 20 and five isolates from well A, B and C, respectively), A. sobria (10 from well D) and A. caviae (10 from well E) (Table 3). Ten Aeromonas isolates were further investigated. Although Janda et al. (1983) found that only 42% of 12 cytotoxic A. hydrophila strains from pediatric patients were VP-positive, a strong correlation between cytotoxicity and positive VP reaction has been reported by Cumberbatch et al. (1979), Kaper et al. (1981) and Burke et al. (1983). Other investigators have correlated Aeromonas spp. toxigenicity with a
172
S. Massa et al. / International Journal of Food Microbiology 63 (2001) 169 – 173
Table 3 Species of Aeromonas isolated from drinking well water Species
Number of strains
Acid from Arabinose
Salicin
A. hydrophila A. hydrophila A. hydrophila A. sobria A. caviae
20 20 5 10 10
1 1 1 2 1
1 1 1 2 1
positive LDC reaction and sorbitol fermentation (Cumberbatch et al., 1979; Janda et al., 1983; Callister and Agger, 1987). Finally, according to Burke et al. (1983), 97% of cytotoxic Aeromonas spp. strains could be correctly classified by haemolysin assay, and the correlation was stronger for drinking water isolates than for food (raw meat, ready to eat products, milk) isolates (Handfield et al., 1996). On the basis of these references, seven strains among those examined in the present study showed putative virulence properties, in particular the A. hydrophila strains and one A. sobria strain (results not shown). Both A. hydrophila and A. sobria appear to be inherently more virulent than those organisms classified as A. caviae. The public health significance of these findings is unknown since further research is nedeed to identify more specifically the properties responsible for enteropathogenicity in the aeromonads (Kirow, 1997).
References Burke, V., Robinson, J., Beaman, J., Gracey, M., Lesmana, M., Rockhill, R., Echeverria, P., Janda, J.M., 1983. Correlation of enterotoxicity with biotype in Aeromonas spp. J. Clin. Microbiol. 18, 1196–1200. Burke, V., Robinson, J., Gracey, M., Petersen, D., Meyer, N., Haley, V., 1984a. Isolation of Aeromonas spp. from an unchlorinated domestic water supply. Appl. Environ. Microbiol. 48, 367–370. Burke, V., Robinson, J., Gracey, M., Petersen, D., Partridge, P., 1984b. Isolation of Aeromonas spp. from a metropolitan water supply: seasonal correlation with clinical isolates. Appl. Environ. Microbiol. 48, 361–366. Cahill, M., 1990. A review: virulence factors in Aeromonas species. J. Appl. Bacteriol. 69, 1–16. Callister, S., Agger, W., 1987. Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae from grocery store produce. Appl. Environ. Microbiol. 53, 249–253.
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Caprioli, A., Pezzella, C., Morelli, R., Giammanco, A., Arista, S., Crotti, D., Facchini, M., Guglielmetti, P., Piersimoni, C., Luzzi, I., 1996. Enteropathogens associated with childhood diarrhea in Italy. Pediatr. Infect. Dis. J. 15, 876–883. Champsaur, H., Andremont, A., Mathieu, D., Rottman, E., Auzepy, P., 1982. Cholera-like illness due to Aeromonas sobria. J. Infect. Dis. 145, 248–254. Cumberbatch, N., Gurwith, M.J., Langston, C., Sacks, R.B., Brunton, J.L., 1979. Cytotoxix enterotoxin produced by Aeromonas hydrophila: relationship of toxigenic isolates to diarrheal disease. Infect. Immun. 23, 829–837. Figura, N., Guglielmetti, P., Zanchi, A., Signori, R., Rossolini, A., Lior, H., Russi, M., Musmanno, R., 1997. Species, biotype and serogroup of Campylobacter spp. isolated from children with diarrhoea over a 10-year period. New Microbiol. 20, 303–310. Gonzales, C., Gutierrez, C., Grande, T., 1987. Bacterial flora in bottled uncarbonated mineral drinking water. Can. J. Microbiol. 33, 1120–1125. GUDRI 1997 (Gazzetta Ufficiale della Repubblica Italiana 23 / 7 / 1997). Decreto 8 luglio 1997. Integrazioni dei criteri di valutazione della caratteristiche microbiologiche delle acque minerali naturali. Handfield, M., Simard, P., Couillard, M., Letarte, R., 1996. Aeromonas hydrophila isolated from food and drinking water: haemoagglutination, haemolysis and cytotoxicity for a human intestinal cell-line (HT-29). Appl. Environ. Microbiol. 62, 3459–3461. Havelaar, A., Toorop-Bouma, A., Medema, G., 1990. The occurrence and significance of Aeromonas in water with special reference to natural mineral water. Rivista Italiana di Igiene 50, 349–356. Hazen, T.C., Fliermans, C.B., Hirsch, R.P., Esch, P.T., 1978. Prevalence and distribution of Aeromonas hydrophila in the United States. Appl. Environ. Microbiol. 36, 731–738. Hunter, P.R., Burge, S.H., 1987. The bacteriological quality of bottled mineral waters. Epidemiol. Infect. 99, 439–443. Janda, J.M., Bottone, E.J., Skinner, C.V., Calcaterra, D., 1983. Phenotypic markers associated with gastrointestinal Aeromonas hydrophila isolates from symptomatic children. J. Clin. Microbiol. 17, 588–591. Janda, J.M., 1991. Recent advances in the study of the taxonomy, pathogenicity and infections syndromes associated with the genus Aeromonas. Clin. Microbiol. Rev. 4, 397–410. Kaper, J.B., Lockman, H., Colwell, R.R., 1981. Aeromonas hydrophila: ecology and toxigenic of isolates from an estuary. J. Appl. Bacteriol. 50, 359–377.
S. Massa et al. / International Journal of Food Microbiology 63 (2001) 169 – 173 Kirow, S.M., 1993. The public health significance of Aeromonas spp. in foods. Int. J. Food Microbiol. 20, 179–198. Kirow, S.M., 1997. Aeromonas and Plesiomonas species. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology. Fundamentals and Frontiers. ASM Press, Washington DC, USA, pp. 265–287. Krovacek, K., Peterz, M., Faris, A., Mansson, I., 1989. Enterotoxigenicity and drug sensitivity of Aeromonas hydrophila isolated from well water in Sweden: a case study. Int. J. Food Microbiol. 8, 149–154. Le Chevallier, M.W., Evans, T.M., Seidler, R.J., Daily, O.P., Merrel, B.R., Rollins, D.M., Joseph, S.W., 1982. Aeromonas sobria in chlorinated drinking water supplies. Microb. Ecol. 8, 325–333. Legnani, P., Leoni, E., Soppelsa, F., Burigo, R., 1998. The occurrence of Aeromonas spp. in drinking water supplies of an area of Dolomite mountains, Italy. J. Appl. Microbiol. 85, 271–276. Manaia, C.M., Nunes, O.C., Morais, P.V., Da Costa, M.S., 1990. Heterotrophic plate counts and isolation of bacteria from mineral waters on selective and enrichment media. J. Appl. Bacteriol. 69, 871–876. Massa, S., Fanelli, M., Brienza, M.T., Sinigaglia, M., 1998. The bacterial flora in bottled natural mineral water sold in Italy. J. Food Qual. 21, 175–185.
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Massa, S., Armuzzi, R., Canganella, F., Tosques, M., Trovatelli, L.D., 1999. Susceptibility to chlorine of Aeromonas hydrophila in water. J. Appl. Microbiol. 86, 169–173. ` J.M., 1995. EmergMerino, S., Rubires, X., Knøchel, S., Tomas, ing pathogens: Aeromonas spp. Int. J. Food Microbiol. 28, 157–168. Popoff, M., 1984. Genus III. Aeromonas Kluyver and van Niel 1936, 1938. In: Krieg, N.R., Holt, J.G. (Eds.). Bergey’s Manual of Systematic Bacteriology, Vol. 1. Williams and Wilkins, Baltimore, pp. 545–548. Quevedo-Sarmiento, J., Ramos-Cormenzana, A., Gonzales-Lopez, J., 1986. Isolation and characterization of aerobic and heterotrophic bacteria from natural springs in the Lanjaron aree (Spain). J. Appl. Bacteriol. 61, 365–372. Schwaller, P., Schmidt-Lorenz, W., 1981. La flore microbienne de ´ ´ quatre eaux mineralel non Gazeifies et mises en bouteilles. ` ´ 2eme communication. Les Pseudomonas et autres bacteries gram-negatif. Composition fine de la flore. Zentralblatt fur Bakteriologie und Hygiene I abt. Orig. C 2, 179–196. Warburton, D., Harrison, B., Crawford, C., Foster, R., Fox, C., Gour, L., Krol, P., 1998. A further review of the microbiological quality of bottled water sold in Canada: 1992–1997 survey results. Int. J. Food Microbiol. 39, 221–226.