Genetic variability and pathogenicity potential of Escherichia coli isolated from recreational water reservoirs

Research in Microbiology 158 (2007) 420e427 www.elsevier.com/locate/resmic

Genetic variability and pathogenicity potential of Escherichia coli isolated from recreational water reservoirs Renato H. Orsi a, Nancy C. Stoppe c, Maria Ineˆs Z. Sato c, Taˆnia A.T. Gomes d, Paulo I. Prado b, Gilson P. Manfio e, Laura M.M. Ottoboni a,* a

Centro de Biologia Molecular e Engenharia Gene´tica, CP 6010, Universidade Estadual de Campinas, 13083-875 Campinas, SP, Brazil b Nu´cleo de Estudos e Pesquisas Ambientais, CP 6166, Universidade Estadual de Campinas, 13084-867 Campinas, SP, Brazil c Companhia de Tecnologia de Saneamento Ambiental, Av. Prof. Frederico Hermann Jr. 345, 05459-900 S~ao Paulo, SP, Brazil d Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, S~ao Paulo, SP, Brazil e Natura Inovac¸~ao e Tecnologia de Produtos Ltda, Rod. Anhagu¨era s/n Km 30.5, 07750-000 Cajamar, SP, Brazil Received 20 November 2006; accepted 28 February 2007 Available online 15 March 2007

Abstract Contamination of recreational waters and public water supplies by Escherichia coli represents a risk for public health, since some strains can be pathogenic or propagated with other pathogenic microorganisms. In this study, two reservoirs, Billings and Guarapiranga (S~ao Paulo metropolitan area, Brazil), were investigated in order to assess E. coli diversity. Genetic typing using rep-PCR completely differentiated all strains and enabled the determination of their genetic variability. Although the same level of genetic variability was observed for strains originating from both reservoirs, randomization procedures showed that isolates from the same reservoir were more closely related to each other. Phylogenetic group frequencies in each reservoir suggested that contamination in the Billings reservoir was mostly from humans, whereas contamination in the Guarapiranga reservoir was mostly from animals. Colony blot experiments using probes from several virulence factor genes showed that both reservoirs contained potential pathogenic strains and may represent a risk to recreational or household usage of these water resources. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Escherichia coli; Water contamination; Pathogenicity

1. Introduction Urbanization is rapidly compromising the quality of fresh water resources worldwide, and since its availability is limited, efforts must be undertaken to preserve it. Hence, in December of 1991, twenty-two water resources management units (UGRHI) were created in the State of S~ao Paulo, Brazil.

* Corresponding author. E-mail addresses: [email protected] (R.H. Orsi), [email protected]. gov.br (N.C. Stoppe), [email protected] (M.I.Z. Sato), tatgomes@ ecb.epm.br (T.A.T. Gomes), [email protected] (P.I. Prado), gilsonmanfio@ natura.net (G.P. Manfio), [email protected] (L.M.M. Ottoboni). 0923-2508/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2007.02.009

Two reservoirs, namely Billings and Guarapiranga, responsible for supplying drinking water to the metropolitan area of the city of S~ao Paulo and industries in the region, were designated UGRHI number 6. The Guarapiranga and the Billings reservoirs drain an area of 631 and 560 km2, respectively, and both receive domestic and industrial effluents. The water from these reservoirs is constantly monitored by CETESB (the organization responsible for the control of environmental pollution, sewage and water quality in the State of S~ao Paulo, Brazil), since it is also used for recreation purposes. Therefore, contamination of these waters by potentially hazardous microorganisms represents a serious public health threat. The presence of Escherichia coli in recreational water is a matter of concern because some strains may be pathogenic

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or can be propagated with other pathogenic organisms. Several cases of recreational water contamination by E. coli have been described. For example, 65 waterborne-disease outbreaks associated with recreational water were reported in the United States during 2001e2002 [36]. During that period, 25% of the 30 outbreaks involving gastroenteritis were associated with toxigenic E. coli [36]. The E. coli pathotypes responsible for extraintestinal infections are UPEC (uropathogenic E. coli) and MNEC (meningitis-associated E. coli) [19]. E. coli from these pathotypes can cause hemolytic uremic syndrome, urinary tract infection, newborn meningitis and sepsis [9,19]. The intestinal pathogenic E. coli strains belong to the pathotypes ETEC (enterotoxigenic E. coli), EPEC (enteropathogenic E. coli), EIEC (enteroinvasive E. coli), EHEC (enterohemorrhagic E. coli), EAEC (enteroaggregative E. coli) and DAEC (diffusely adherent E. coli). These pathotypes have been associated with cases of mild and severe diarrhea in adults and children, mostly in developing countries [19]. In the present work, rep-PCR was used to determine genetic variability among E. coli strains isolated from the Billings and Guarapiranga reservoirs. This approach, in association with phylogenetic group determination of the strains, was used to analyze the population structure of the reservoirs. Colony blot experiments were also conducted in order to determine the pathogenic potential of the tested strains. 2. Materials and methods 2.1. E. coli strains One hundred and thirty-three strains of E. coli were isolated by CETESB from water samples from the Billings (65 isolates) and Guarapiranga (68 isolates) reservoirs, during the dry season (April to June) and rainy season (September to February) in the years 1999, 2000 and 2001. Sterile disposable bottles were used to collect water samples according to standard methods [2]. Samples were analyzed using the membrane filter technique with mTEC agar (Difco) with b-D-indoxyl glucoside (final concentration 100 mg/L) and incubated for 2 h at 35  0.5  C and 22e24 h at 44.5  0.2  C. Typical colonies (yellow) were transferred to EC broth and incubated at 44.5  C for 24 h. Positive tubes were streaked on EMB agar (Merck). Isolated colonies were tested for citrate utilization, lactose fermentation, oxidase, L-lysine decarboxylase, motility, glucose and sucrose fermentation, tryptophan deamination, indole production, urea hydrolysis and sulfide production. Isolates with an E. coli profile were inoculated into LB broth at 37  C overnight. One isolated colony from each EC positive sample was selected for further analyses. 2.2. DNA isolation Genomic DNA was isolated from the different strains using the GenomicPrep cells and tissue DNA isolation kit (Amersham Biosciences), following the specifications of the manufacturer. The DNA was stored at 4  C.

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2.3. rep-PCR amplification BOX-PCR reactions were carried out as previously described [35], with modifications. The reaction mixtures (25 ml) contained 10 ng of DNA, 50 pmol of primer BOXA1R (50 -CTACGGCAAGGCGACGCTGAGG-30 ), 1 PCR buffer, 0.1 mM of each dNTP, 2 mM MgCl2 and 0.5 U of Taq DNA polymerase (Amersham Biosciences). ERIC-PCR reactions were carried out as described by Versalovic and co-workers [34], with modifications, using the primers ERIC1R (50 -AT GTAAGCTCCTGGGGATTCAC-30 ) and ERIC2 (50 -AAGTA AGTGACTGGGGTGAGCG-30 ). The reaction mixtures (25 ml) contained 50 ng of DNA, 75 pmol of each primer, 1 PCR buffer, 0.1 mM of each dNTP, 1.5 mM MgCl2 and 0.5 U of Taq DNA polymerase (Amersham Biosciences). REP-PCR was carried out as previously described [34], with modifications. The primers used were REP1R-1 (50 -IIIICGICGICATC IGGC-30 ) and REP2-I (50 -ICGICTTATCIGGCCTAC-30 ). The amplification reactions contained 50 ng of DNA, 75 pmol of each primer, 1 PCR buffer, 0.1 mM of each dNTP, 1.5 mM MgCl2 and 0.5 U of Taq DNA polymerase (Amersham Biosciences). PCR reactions were performed in duplicate in a PT 100 thermocycler (MJ Research Inc.). The amplification conditions included an initial denaturation at 95  C for 7 min, followed by 30 cycles at 94  C for 1 min, annealing at either 53  C (BOX-PCR) or 52  C (ERIC-PCR) or 40  C (REP-PCR) for 1 min and amplification at 56  C for 4 min (BOX-PCR) or at 65  C for 8 min (ERIC-PCR) or at 65  C for 4 min (REPPCR). The final extension was at 65  C for 16 min. The amplification products were separated by electrophoresis on 1.5% agarose 1 TAE [28] gels. The gels were stained with ethidium bromide and photographed under UV light [28]. The combined BOX-, ERIC- and REP-PCR data were used in the construction of the dendrograms and in the statistical analyses. 2.4. rep-PCR data analysis The band patterns obtained for the strains by rep-PCR were digitalized and gel images were analyzed using the software GelCompar v. 4.1 (Applied Maths, Kortrijk, Belgium). The similarity between the strains was determined using the Pearson coefficient (1926) and dendrograms were constructed using the UPGMA grouping method [31]. 2.5. Phylogenetic grouping The phylogenetic group of each strain was determined as described by Clermont and co-workers [6]. PCR amplifications were carried out to identify chuA and yjaA genes and DNA fragment TSPE4.C2. The gene chuA encodes the heme transport protein ChuA [32], while yjaA encodes a hypothetical protein [1] and TSPE4.C2 is an anonymous DNA fragment of the E. coli genome [5]. The amplification products were separated in 2% agarose gel containing ethidium bromide. After electrophoresis, the gel was photographed under UV light

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and strains were assigned to phylogenetic group B2 (chuAþ, yjaAþ), D (chuAþ, yjaA), B1 (chuA, TSPE4.C2þ), or A (chuA, TSPE4.C2). Chi-square and Fisher’s exact tests were performed using Biostat v. 2.0 software [3]. In some cases, strains belonging to phylogenetic group B2 were grouped with strains belonging to phylogenetic group D, which is the closest evolutionary group to B2. The frequency of each phylogenetic group in the reservoirs and the season of the year when the strains were isolated were organized in contingency tables and analyzed by log-linear models [11,12] using Estatistica software. Confidence intervals of the means of similarities of both reservoirs were gathered after 4000 bootstraps, using Resampling Stats v. 2.0 (Resampling Stats Inc., http://www. resample.com/) software for Excel (Microsoft Corp.). To verify if there was greater similarity among strains isolated from the same reservoir than among strains isolated from different reservoirs, a randomization procedure was performed [24,30] using Resample Stats v. 2.0 software. 2.6. Virulence analysis Colony blot hybridization was used to analyze the pathogenic potential of strains [23]. Probes used in hybridizations were: bfpA (bundle-forming pilus of EPEC), an 852 bp EcoRI fragment from plasmid pMSD207; EPEC adherence factor (EAF), a 1 kb BamHIeSalI fragment from plasmid pMAR2; eae (intimin gene), a 1 kb SalIeKpnI fragment from plasmid pCVD 434; stx1 (shiga toxin 1), a 1142 bp BamHI fragment from pJN 37e19; stx2 (shiga toxin 2), an 842 bp PstI fragment from plasmid pNN111e19; INV (EIEC invasion plasmid), a 2500 bp HindIII fragment from pSF55; LT-I a 1200 bp HincI fragment from pEWD299; ST-h a 215 bp HpaII fragment from pSLM004; ST-p, a 157 bp HinfI fragment from pRIT10036; and AA (aggregative adherence plasmid), a 1000 bp XbaIe SmaI fragment from pCVD 432. 3. Results and discussion 3.1. Quantitative analyses In this work, the water from two reservoirs, Billings and Guarapiranga, located in the city of S~ao Paulo, was analyzed. Water samples were collected from several sampling sites in each reservoir (supplementary figure). The results of quantitative analysis showed that, in the Billings reservoir, sampling site B4 presented high levels of E. coli contamination (CFU/ 100 ml water  1  103) in four out of 11 samples. This site is close to an untreated sewage emission and therefore is prone to higher levels of contamination. Other sites (B1, B2 and B6) presented high levels of contamination in one sampling date. In the Guarapiranga reservoir, fourteen samples presented high levels of contamination. Sampling site G11 presented high levels of contamination in four out of seven samples. Sampling site G4 had three out of four samples with high

contamination levels. These two sites receive illegal sewage via small streams in the area. 3.2. rep-PCR Genomic fingerprints were generated by BOX-, ERIC-, and REP-PCR for 65 E. coli strains isolated from the Billings reservoir and 68 strains isolated from the Guarapiranga reservoir. The amplification profiles obtained using BOXA1R, ERIC1R/ ERIC2 and REP1R-1/REP2-I primers were complex. BOXPCR yielded fingerprints with bands ranging from 298 to 4000 bp, whereas ERIC- and REP-PCR yielded bands ranging from 220 to 5000 bp. The combined BOX-, ERIC- and REP-PCR data were subjected to cluster analyses using the Pearson coefficient and the resulting similarity matrix was used to generate UPGMA dendrograms, as shown in Fig. 1 (Billings reservoir) and Fig. 2 (Guarapiranga reservoir). Strong genetic variability was observed among strains isolated from the Billings reservoir (Fig. 1). Two strains, namely 3129 and 5675 (64% similarity), were the most divergent, exhibiting the lowest similarity compared to the other tested strains (30%). Some strains were highly related (similarity values above 80%), namely 18 and 94, 3130 and 5060, 2285 and 5676, and 2148 and 3126. Strains isolated from the Guarapiranga reservoir also presented a high level of genetic variability (Fig. 2). Strains 2156 and 108 were the most divergent, exhibiting a similarity value of 19.6% with the remaining strains. The strains that showed the highest similarities were 5642 and 5646 (90.5% similarity), 2166 and 2265 (90.1%), 5647 and 1539 (87.2%), 1540 and 2272 (86.7%), 2154 and 2158 (85.3%), 2263 and 2273 (85.3%) and 1548 and 1549 (83.8%). To test the hypothesis that strains isolated from the same reservoir were more closely related to each other than strains isolated from different reservoirs, a permutation procedure was carried out [24,30]. The 133 strains were randomly assigned to two groups of the same size as the original groups (each reservoir) and, for each random group, internal similarity (Si) was calculated as the mean genetic similarity among all strain pairs. The mean of the two Si was then used as an index of overall intragroup similarity. Permutations were carried out 500 times using Resampling Stats 2.0 software. The proportion of randomizations that showed Si values greater than or equal to the observed value were used to estimate the probability of no differentiation due to the source of isolation (reservoir). Only thirteen randomizations had overall intragroup similarity greater than that observed, indicating that strains isolated from the same reservoir were significantly more similar to each other than strains isolated from different reservoirs ( p ¼ 13/500 ¼ 0.026). We speculate that no migration of strains from one reservoir to the other or different contamination events in each reservoir could be the reason for this clustering. Differences in Si between reservoirs were tested by checking overlap in their 95% confidence intervals. These intervals were estimated by performing 4000 bootstrap resamplings [8,10]. The difference between Si was less than one, and their

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Fig. 1. Dendrogram based on UPGMA cluster analysis of combined BOX-, ERIC- and REP-PCR data from strains isolated from water samples from the Billings reservoir; percent similarity is given at the top. The names of the analyzed strains (given by numbers) and their phylogenetic groups (A, B1, B2 and D) are given at the right side.

confidence intervals overlapped extensively (Billings Si ¼ 39.3  0.7; Guarapiranga Si ¼ 40.6  0.8; Total Si ¼ 40.0), indicating that genetic similarities among strains did not vary between reservoirs. This result suggests that both

reservoirs were contaminated with strains sharing similar levels of variability. To evaluate genetic diversity in each reservoir, relatedness data (Figs. 1 and 2) were used to plot the logarithm of

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Fig. 2. Dendrogram based on UPGMA cluster analysis of combined BOX-, ERIC- and REP-PCR data from strains isolated from water samples from the Guarapiranga reservoir; percent similarity is given at the top. The names of the analyzed strains (given by numbers) and their phylogenetic groups (A, B1, B2 and D) are given at the right side.

cumulative numbers of strains in relation to similarity (Fig. 3). Constant rates of strain origin and extinction would result in an exponential distribution of the divergence over time [25]. In our case, the time between successive divergent events diminishes progressively as the distance from the root of the tree increases. Hence, under the assumption that dissimilarity

increases proportionally to time, the plot will show a straight line [25]. As shown in Fig. 3, none of the samples conformed to this null hypothesis. There was an excess of divergent strains, indicated by the curve above the straight line in both reservoirs, suggesting that many contamination events occurred in these waters.

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Table 1 Chi-square deviancesa of the log-linear model that test the correlation for the interaction between phylogenetic group and season, estimated by log-linear models Season

Reservoir

Dry

Billings Guarapiranga Billings Guarapiranga

Rainy a

Fig. 3. Number of strains at each divergence level for Billings and Guarapiranga reservoirs. The line represents the null hypothesis of constant rates of origin and extinction of strains [25].

3.3. Phylogenetic group determination The 133 E. coli strains isolated from the Billings and Guarapiranga reservoirs were assigned to four phylogenetic groups, A, B1, B2 and D, according to the methodology described by Clermont and co-workers [6]. Strains from the same phylogenetic group were present in clusters obtained by BOX-, ERIC- and REP-PCR combined analysis (Figs. 1 and 2). Among strains isolated from the Billings reservoir, 28 (43.1%) were assigned to phylogenetic group A, 17 (26.2%) to B1, 5 (7.7%) to B2 and 15 (23%) to D. Strains isolated from the Guarapiranga reservoir were assigned to group A (44 strains, 64.7%), group B1 (15 strains, 22.1%), group B2 (3 strains, 4.4%) and D (6 strains, 8.8%). Differences in the numbers of strains belonging to each phylogenetic group in the reservoirs and sampling seasons (dry and rainy) were tested by log-linear models [7,11,12]. The log-linear models showed a significant association between phylogenetic group and season (c2 ¼ 6.59; d.f. ¼ 2; p ¼ 0.037). In the dry season, there was a greater frequency of group A strains and a lower frequency of groups B2 and D strains (Table 1). The models also showed a significant association between phylogenetic groups and the reservoirs (c2 ¼ 7.82; d.f. ¼ 2; p ¼ 0.020). Fewer strains from group A and more from groups B2 and D were observed in the Billings reservoir, whereas the opposite was observed in the Guarapiranga reservoir (Table 2). A significant association between phylogenetic group and the presence of rain 24 h prior to sampling was observed in the Billings reservoir ( p ¼ 0.002, Fisher’s exact test) but not in the Guarapiranga reservoir ( p ¼ 0.961, Fisher’s exact test). This suggests that the rain might have changed the source of E. coli contamination in the Billings reservoir. Goullet and Picard [16] showed that the number of strains from group B2 is different among commensal strains isolated

Phylogenetic group A

B1

B2 þ D

0.479 1.031 0.385 0.829

0.261 0.046 0.210 0.037

1.018 1.348 0.818 1.084

Deviance ¼ (observed  expected)/(expected)1/2.

from human (9%) and animal (1.6%) feces. The percentage of strains from group B2 isolated from both reservoirs was intermediary (Billings ¼ 7.69%; Guarapiranga ¼ 4.41%) in relation to the percentages described by Goullet and Picard [16]. The frequencies of B2 strains in both reservoirs did not differ from the frequency of B2 in humans (9%) (Billings c2 ¼ 0.14, p ¼ 0.830; Guarapiranga c2 ¼ 1.75, p ¼ 0.216), but differed significantly from 1.6% in the Billings reservoir (c2 ¼ 15.3, p ¼ 0.007), and was marginally significantly in the Guarapiranga reservoir (c2 ¼ 3.4, p ¼ 0.086). Therefore, we speculate that the main source of contamination in the Billings reservoir was human, while animal contamination might have been more important in the Guarapiranga reservoir. Moreover, the distribution of chuA among 304 E. coli strains from different origins has been analyzed [18]. A 30% frequency of chuA was observed among isolates from the environment (river and surface water) in Munich, Germany. It was suggested that ChuA might be involved in human host colonization. In our work, 20 out of 65 (30.7%) strains isolated from the Billings reservoir harbored chuA, a frequency that is no different from 30% (c2 ¼ 0.02, p ¼ 0.999). In the Guarapiranga reservoir, however, chuAþ strains represented 9 out of 68 (13.23%), which was significantly lower than 30% (c2 ¼ 9.1, p ¼ 0.002). Hence, this data also suggested that the prevalence of strains from human hosts was higher in the Billings reservoir. 3.4. Virulence factor mapping The pathogenic potential of strains isolated from the Billings and Guarapiranga reservoirs was determined by colony blot hybridizations. Three strains (94, 878 and 881) isolated from the Billings reservoir and three (868, 109 and 2265) from the Table 2 Chi-square deviancesa for the interaction between phylogenetic group and reservoirs, estimated by log-linear models Season

Reservoir

Dry

Billings Guarapiranga Billings Guarapiranga

Rainy a

Phylogenetic group A

B1

B2 þ D

0.994 0.972 0.702 0.687

0.055 0.054 0.391 0.382

1.207 1.180 1.120 1.095

Deviance ¼ (observed  expected)/(expected)1/2.

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Guarapiranga reservoir hybridized with the EAEC probe AA, which targets the aggregative adherence plasmid, specific to this pathotype. EAEC is considered an emerging pathogen in both developing and industrialized countries [19], and was found to be the most prevalent E. coli pathotype among children with diarrhea in Brazil, suggesting that this pathotype is an important enteric pathogen in the community investigated [27,29]. This pathotype can cause infant diarrhea, traveler’s diarrhea and persistent diarrhea in immunocompromised adults [17]. Two other strains (2264 and 1538) isolated from the Guarapiranga reservoir hybridized with the LT-I probe (heatlabile enterotoxin I gene), specific to ETEC, which can cause liquid diarrhea without blood, and vomiting [20]. ETEC is the most common cause of waterborne diarrhea among the E. coli pathotypes [14]. Strains 4102 and 858 hybridized with the eae probe (intimin gene), but were devoid of the stx1, stx2, EAF and bfpA probe sequences. These properties are characteristic of the subgroup of EPEC designated atypical EPEC, which seems to be less virulent than typical EPEC [33]. Nevertheless, this pathotype is frequently found in many states in Brazil [15] and was the most prevalent pathotype among children with acute endemic diarrhea in Salvador, Bahia, Brazil [13]. In contrast to typical EPEC, both animals and humans can be reservoirs for atypical EPEC [33]. This is consistent with phylogenetic group results, suggesting that animals were the main source of contamination in the Guarapiranga reservoir. One strain (2156) hybridized with the stx2 probe (shiga toxin 2 gene) of VTEC. Contamination of a water supply in Scotland by E. coli O157 has been reported where four out of the six infected people harbored strains that carried only shiga toxin 2 [22]. VTEC can cause watery diarrhea, sometimes with the presence of blood. Approximately 10% of infant intestinal infections develop in an extraintestinal infection [20]. Two strains (878 and 881) harboring the AA plasmid were isolated the same day (21 February 2000) in the Billings reservoir. Among strains from the Guarapiranga reservoir harboring a virulence factor determinant, only two (858 and 2265) were sampled at the same location. However, these two strains harbored different virulence factor determinants: eae and AA, respectively. None of the strains sampled the same day in different locations presented the same virulence factor, suggesting that potentially pathogenic strains contaminated the reservoirs independently. In conclusion, strains isolated from Billings and Guarapiranga reservoirs were derived from distinct populations, probably restricted to each reservoir. Moreover, both reservoirs accommodate strains with a high degree of genetic variability, probably due to different contamination events. The presence of virulence markers such as stx2 and eae suggests that the Billings and Guarapiranga reservoirs have been contaminated with pathogenic isolates that pose a significant risk to the populations that use these waters for recreation and consumption. Four out of six isolates associated with a waterborne outbreak in Scotland harbored shiga toxin 2 [22], the same found in strain 2156 in our study. In Japan, eae-positive enteropathogenic E. coli isolates were associated with a waterborne outbreak. However, 10 out of 11 isolates were indistinguishable by RAPD subtyping [4].

The higher diversity and number of virulence factors in the Guarapiranga reservoir might be related to the source of contamination in this reservoir, which seems to be mainly animal. It has previously been shown that animals such as ruminants and rabbits are reservoirs of human-pathogenic E. coli [26,21]. Taken together, results obtained in this work suggest that appropriate treatment of sewage that flows to the Billings and Guarapiranga reservoirs is necessary to guarantee the quality of these waters. Acknowledgments This work was supported by grant 2000/05721-8 from the Fundac¸~ao de Amparo a` Pesquisa do Estado de S~ao Paulo (FAPESP). R.H.O. had a fellowship from CAPES. L.M.M.O. had a research fellowship from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). We thank Dr. Moˆnica A. M. Vieira (Escola Paulista de Medicina) for carrying out colony blot hybridizations.

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