Abstract
Bacteriophages are the most abundant and genetically diverse viruses on Earth, with complex ecology in both quantitative and qualitative terms. Somatic coliphages (SC) have been reported to be good indicators of fecal pollution in seawater. This study focused on determining the concentration of SC and their diversity by electron microscopy of seawater, plankton, and bivalve samples collected at three coastal regions in São Paulo, Brazil. The SC counts varied from <1 to 3.4 × 103 PFU/100 ml in seawater (73 samples tested), from <1 to 4.7 × 102 PFU/g in plankton (46 samples tested), and from <1 to 2.2 × 101 PFU/g in bivalves (11 samples tested). In seawater samples, a relationship between the thermotolerant coliforms and Escherichia coli and SC was observed at the three regions (P = 0.0001) according to the anthropogenic activities present at each region. However, SC were found in plankton samples from three regions: Baixada Santista (17/20), Canal de São Sebastião (6/14), and Ubatuba (3/12). In seawater samples collected from Baixada Santista, four morphotypes were observed: A1 (4.5%), B1 (50%), C1 (36.4%), and D1 (9.1%). One coliphage, Siphoviridae type T1, had the longest tail: between 939 and 995 nm. In plankton samples, Siphoviridae (65.8%), Podoviridae (15.8%), Microviridae (15.8%), and Myoviridae (2.6%) were found. In bivalves, only the morphotype B1 was observed. These SC were associated with enteric hosts: enterobacteria, E. coli, Proteus, Salmonella, and Yersinia. Baixada Santista is an area containing a high level of fecal pollution compared to those in the Canal de São Sebastião and Ubatuba. This is the first report of coliphage diversity in seawater, plankton, and bivalve samples collected from São Paulo coastal regions. A better characterization of SC diversity in coastal environments will help with the management and evaluation of the microbiological risks for recreation, seafood cultivation, and consumption.
INTRODUCTION
Bacteriophages have been found to infect bacterial hosts in almost any aquatic or terrestrial habitat where such bacteria can exist. Marine viruses were first described by Spencer (43), though they were largely ignored for 3 decades because of the relatively low abundances inferred on the basis of culture-based assays (12). However, more recently, it has been recognized that viruses are extremely abundant in aquatic environments (11, 17, 40, 46, 47). The viruses may exceed the concentration of bacteria by up to 100 times, with their total numbers estimated to be 1031 in the biosphere (12).
Undoubtedly, the largest reservoir of viruses is in the oceans, where they range from 104 to over 107/ml in the water column and up to 108/ml in the sediment (37, 48). This fact has resulted in a reevaluation of the role of viruses, and particularly bacteriophages, in the microbial ecology of marine environments, for which extensive efforts have focused on understanding the role of viruses in horizontal gene transfer and microbial mortality and on the consequent impacts on microbial abundance, diversity, and community structure (39). Since 1959, more than 5,000 phages have been classified by electron microscopy (5).
Three bacterial viruses were proposed as potential indicators for water quality and to model human enteric viruses: somatic coliphages, male-specific RNA coliphages, and phages infecting Bacteroides fragilis (20, 23a). However, somatic coliphage became the most widely used due to its simple, inexpensive, and rapid methodology. The presence of somatic coliphages was associated with the intensity of fecal contamination in freshwater, lake, seawater, bathing water, sewage-polluted water, raw sewage, sewage sludge, reclaimed water, and drinking water (8, 18, 19, 20, 23, 24, 29, 30, 31).
Somatic coliphages infecting Escherichia coli, via the cell wall, belong to four families: three-tailed phages (order Caudovirales) having double-stranded DNA (dsDNA) genomes, including Myoviridae (long contractile tails), Siphoviridae (long noncontractile tails), and Podoviridae (short contractile tails), and one nontailed member of the Microviridae, as single-stranded DNA (ssDNA) phages (5, 6, 20, 45). The detailed morphological studies of phages isolated from different habitats and infecting various host bacteria were described, and direct electron microscopy was used to make a general classification based on size and morphology (9).
Human activities are significantly altering coastal regions, which results in modification of the marine ecosystem and its biodiversity. Although coliphages were proposed as a parameter to measure water quality, the ecology and diversity of somatic coliphages in natural ecosystems remain little understood (20, 40).
The main objective of this study was to determine the concentration, prevalence, and diversity of somatic coliphages (SC) in seawater, plankton, and bivalve samples collected from three coastal regions of São Paulo state, Brazil (Canal de São Sebastião, Baixada Santista, and Ubatuba), with different levels of anthropogenic activities.
MATERIALS AND METHODS
Sites and sample collection.
Seawater and plankton samples were collected from three areas located in the coastal region of São Paulo state, in the southeast of Brazil. In each area, two or three collection sites were selected (Fig. 1).
Fig. 1.
Map for sampling collection.
Area 1, Canal de São Sebastião (SS), includes 25 km of land, three outfalls, a petroleum terminal, a small port, and an urban community of 67,000 inhabitants (25). The seawater samples were collected monthly from August 2005 to March 2007 (20 months), at two sites: SS1 (23°49′50"S, 45°25′20"W) and SS2 (23°49′56"S, 45°25′51"W). Plankton samples were collected only during the summer: from February to May (2006, 4 months) and from January to March (2007, 3 months) (Fig. 1).
Area 2, Baixada Santista (BS), is comprised of two channels, Santos and São Vicente, with 40 km of land. Santos is one of the 10 most populated cities in the São Paulo state, with 417,000 inhabitants. It has only one submarine outfall to dispose of all domestic effluent and seven primary treated sewage channels that are discharged into Santos Bay. This area supports the main and major port of Brazil and the industrial pole of Cubatão. The area is affected by nutrients and heavy metals because of the drag effects of the high frequency of ship transportation in the channel (15). Three sites were determined for sampling: BS1 (23°59′13"S, 46°22′26"W) in front of São Vicente; BS2, a preserved area (24°02′25"S, 46°19′20"W); and BS3, the exit of the Santos channel (23°59′57"S, 46°18′39"W). The samples were collected during the summer: from February to May (2006, 4 months) and from January to March (2007, 3 months) (Fig. 1).
Area 3, Ubatuba (U), a small bay for tourism and sailing ships, is characterized by scarce inorganic nutrients: it has an oligotrophic to mesotrophic environment. The city has 75,000 inhabitants, and the population increases during the summer (25). Two sites were selected for sample collection from February to April (2006, 3 months) and from January to March (2007, 3 months): U1 (23°30′02"S, 45°07′07") and U2 (23°30′41"S, 45°06′04") (Fig. 1).
Sampling.
Approximately 1.55 to 7.16 km away from the coast (GPS), 5 liters of seawater was collected 10 cm under the surface, in sterile polypropylene bottles. Physicochemical (temperature, pH, salinity, and conductivity) parameters were determined using a multiparameter apparatus, the Hach CO150 (Hach Company). Plankton samples were collected using a 64-μm-pore-size mesh net and were drawn for 5 min. All samples were transported under refrigeration to the laboratory and processed. Additionally, 11 samples of bivalves were collected from the surrounding areas of sampling collection, during summer 2006 only.
Microbial quality of seawater samples.
To evaluate the microbial quality of the seawater samples, thermotolerant coliforms (TC) and Escherichia coli counts were determined using the membrane filter technique and MFC agar (Difco). Blue colonies from MFC agar were submitted to biochemical identification of E. coli (21).
Somatic coliphage counting in seawater, plankton, and bivalve samples.
Seawater samples were centrifuged at 3,000 × g for 30 min. Plankton samples were concentrated by centrifugation; 1 g of each sample was macerated with a tissue homogenizer, and 1:10 and 1:100 dilutions were made in 0.85% saline solution. Bivalve samples (25 g) were mixed with 225 ml of saline solution and homogenized in a stomacher (Lab Blender). In all cases, the supernatant was used for the quantification of somatic coliphages. The counting was performed by following the methodology recommended by the APHA (21) using the host strain E. coli C (ATCC 13706). The plaques were counted, and the numbers were expressed as PFU/100 ml or PFU/g of sample (21).
Coliphage isolation and purification.
Individual plaques of each sample were observed, their diameters were measured with a caliper, and their macroscopic characteristics were recorded. Each selected plaque was transferred using the point of a sterile toothpick or inoculation needle to a tube containing 5.0 ml of Trypticase soy broth (TSB) and 1.0 ml of the host bacterial culture, grown previously for 3 h at 35 ± 0.5°C (at an optical density [OD] of 0.5 at 520 nm). The tube was incubated at 37°C for 3 h with shaking and then centrifuged at 3,000 × g for 30 min (Eppendorf 5403). Then, the supernatant was filtered by a 0.22-μm-pore-size filter (Millipore). This plaque purification was performed at least twice. Finally, the concentration of phage particles was determined, and all phage stock had a concentration from 108 to 1011 PFU/ml. The samples were stored at 4°C, protected from light.
Morphological coliphage characterization.
The purified somatic coliphages were examined by electron microscopy and characterized according to the ultrastructure of the virus particle, as outlined by the International Committee for Taxonomy of Viruses (34). The phage suspensions were tested by lysis and submitted to a negative staining technique with 2% potassium phosphotungstate (PTK), pH 6.4 (12). The grids were examined by JEOL JEM-1011 electron microscopy, operating at 60 kV, and the electron micrographics were recorded with a charge-coupled-device (CCD) camera with a magnification of ×150,000. On average, five measurements were made for each phage particle examined.
Statistical analysis.
The Kruskal-Wallis test was applied for nonparametric variables, using an alpha risk of 5% and the Spearman correlation to establish the relationship between the microbiological parameters, with P values of ≤0.05 and r values between 0 and 1. The number of somatic coliphages was used as a binary variable, and when they were not detected, a value of zero was assumed. The box plot was obtained using the Tukey's hinges method.
RESULTS
A total of 130 samples collected from three areas of the coastal region of São Paulo state, Brazil, were analyzed: 73 seawater, 46 plankton, and 11 bivalve samples.
In the seawater samples, the temperature ranged from 15.2 to 31.9°C, the salinity from 15.5 to 35‰, the pH from 6.5 to 8.6, and the conductivity from 14.0 to 49.9 ms (Table 1). At Baixada Santista, high concentrations of thermotolerant coliforms and E. coli (8.4 × 103 CFU/100 ml) were found at frequencies of 95.2% and 61.9%, respectively (Table 1). The somatic coliphages varied from <1 to 3.4 × 103 PFU/100 ml (76.2%). At the Canal de São Sebastião, E. coli was present in 27.5% of the samples, with a maximal concentration of 6.2 × 101 CFU/100 ml. The concentration of somatic coliphages ranged from <1 to 1.5 × 101 PFU/100 ml (10%). At the Ubatuba area TC, E. coli and somatic coliphages were absent (<1 PFU/100 ml) (Table 1; Fig. 2).
Table 1.
Description, by area studied, of coliphages isolated from coastal regions of São Paulo state, Brazil (2006 and 2007)a
| Sample group and parameter | Results |
||
|---|---|---|---|
| Canal de São Sebastião (SS) | Baixada Santista (BS) | Ubatuba (U) | |
| Seawater samples | |||
| No. of samples | 40 | 21 | 12 |
| Temp (°C) | 21.5 to 26.3 | 15.2 to 28.5 | 25.3 to 31.9 |
| pH | 8.1 to 8.6 | 6.5 to 8.0 | 7.8 to 8.0 |
| Salinity (‰) | 34 to 35 | 15.5 to 31.8 | 26.4 to 31.7 |
| Conductivity (ms) | 37.6 to 49.4 | 14.0 to 48.8 | 41.0 to 49.9 |
| Concn (%) of thermotolerant coliforms (CFU/100 ml) | <1 to 2.9 × 102 (52.5) | <1 to 8.4 × 103 (95.2) | <1 to 1 (8.3) |
| Concn (%) of E. coli (CFU/100 ml) | <1 to 6.2 × 101 (27.5) | <1 to 8.4 × 103 (61.9) | <1 |
| Concn (%) of somatic coliphages (PFU/100 ml) | <1 to 1.5 × 101 (10) | <1 to 3.4 × 103 (76.2) | <1 |
| No. of coliphages isolated | 0 | 22 | 0 |
| Plankton samples | |||
| No. of samples | 14 | 20 | 12 |
| Concn (%) of somatic coliphages (PFU/g) | <1 to 4.2 × 102 (42.8) | <1 to 4.7 ×102 (85) | <1 to 1.0 × 101 (25) |
| No. of coliphages isolated | 12 | 23 | 3 |
| Bivalve samples | |||
| No. of samples | 4 | 4 | 3 |
| Concn (no./total no.) of somatic coliphages (PFU/g) | <1 to 2.0 (2/4) | <1 to 2.2 × 101 (2/4) | <1 to 1.4 × 101 (1/3) |
| No. of coliphages isolated | 1 | 2b | 1 |
Values given include numbers of samples, minimal and maximal values for physical-chemical and microbiological parameters, and numbers of coliphages isolated.
The two isolates from Baixada Santista died and were therefore not studied further.
Fig. 2.
Box plot of thermotolerant coliforms and Escherichia coli.
Somatic coliphages correlated positively with TC and E. coli (P = 0.0001) and negatively with temperature (−0.336, P = 0.004).
Based on the microbiological parameters of concentration and frequency obtained in this study in seawater samples and the characteristics for anthropogenic activities (AA) in each area, the three areas studied were classified as the following: Baixada Santista as an area with high AA (HAA); Canal de São Sebastião as medium AA (MAA), and Ubatuba as low AA (LAA). These categories are in line with the brief description reported previously. The results for somatic coliphages will be presented and discussed by area and type of sample.
Somatic coliphages were most frequent in plankton samples in the three regions studied: Baixada Santista (85%), Canal de São Sebastião (42.8%), and Ubatuba (25%); however, the concentration varied only from <1 to 4.7 × 102 PFU/g (Table 1). The highest counts of somatic coliphages were observed at Baixada Santista (4.7 × 102 PFU/g) and Canal de São Sebastião (4.2 × 102 PFU/g). In the Ubatuba area, the region with the lowest pollution levels and an absence of TC and somatic coliphages in all seawater samples, 25% of plankton samples showed a presence of coliphages with a maximal count of 10 PFU/g.
Only five bivalves, from a total of 11 samples, had somatic coliphages, varying from <1 to 2.2 × 10 PFU/g (Table 1).
A total of 62 somatic coliphages were submitted to reisolation and purification using the host bacterium E. coli C (ATCC 13706); 22 isolates were from seawater collected at Baixada Santista, and 38 were from plankton (23 from BS, 12 from SS, and 3 from U). Only two coliphages isolated from bivalves were included (1 from SS and 1 from U).
The highest number of somatic coliphages was observed at Baixada Santista: 45 (23 from plankton and 22 from seawater samples). The somatic coliphage frequencies varied according to the degree of pollution present in the area where they were isolated (Table 1).
Somatic coliphages isolated from seawater samples.
It was not possible to isolate coliphages from seawater samples collected at Canal de São Sebastião (20 months) and Ubatuba (summer 2006 and summer 2007). However, from 21 seawater samples collected at Baixada Santista, during the 2006 and 2007 summers, 22 coliphages were obtained; 12 were from site BS3, the exit of Santos channel, and 8 were from site BS1, near São Vicente. Only two somatic coliphage isolates were obtained from control site BS2, the area preserved from anthropogenic activities.
According to the morphological characterization by electron microscopy, 50% of somatic coliphages belonged to Siphoviridae, 36.4% to Podoviridae, 9.1% to Microviridae, and 4.5% to Myoviridae (Table 2). Various morphologies of plaque lysis with different sizes were observed, ranging from 0.3 to 4 mm in diameter for Siphoviridae, from 0.48 to 8 mm for Podoviridae, and from 2.0 to 8 mm for Microviridae. One plaque, belonging to the family Myoviridae, had a diameter of 0.6 mm. The somatic coliphages identified as Podoviridae and Siphoviridae by electron microscopy showed plaques with and without a halo and with smooth or irregular edges. In other plaques, no halo was observed, and the edges were always regular.
Table 2.
Mean sizes of somatic coliphages observed by electron microscopy isolated from seawater samples at Baixada Santista, São Paulo, Brazil, during summer 2006 and 2007
| Site | Familya | No. of isolates | Morphotype | Type | Head length × diam (nm) | Tail length × width (nm) |
|---|---|---|---|---|---|---|
| BS1 | Siphoviridae (12.5%) | 1 | B1 | T1 | 58 × 54 | 131 × 10 |
| Podoviridae (62.5%) | 1 | C1 | sd | 56 × 56 | 0 × 0 | |
| 2 | C1 | N4 | 48 × 48 | 24 × 26 | ||
| 1 | C1 | 1412 | 45 × 49 | 59 × 17 | ||
| 1 | C1 | 7480b | 61 × 56 | 33 × 15 | ||
| Microviridae (25%) | 2 | D1 | φX174 | 53 × 53 | 12 × 11 | |
| BS2 | Siphoviridae (50%) | 1 | B1 | T1 | 54 × 51 | 121 × 6 |
| Podoviridae (50%) | 1 | C1 | N4 | 54 × 56 | 18 filaments | |
| BS3 | Siphoviridae (75%) | 8 | B1 | T1 | 58 × 58 | 116 × 10 |
| 1 | B1 | Vi II | 76 × 76 | 175 × 14 | ||
| Podoviridae (17%) | 1 | C1 | N4 | 58 × 58 | 0 × 0 | |
| 1 | C1 | sd | 54 × 50 | 13 × 13 | ||
| Myoviridae (8%) | 1 | A1 | YerA41 | 71 × 70 | 112 × 23 |
Numbers in parentheses represent the percentages of total isolates from each site.
The highest numbers of somatic coliphages (13) were obtained from site BS3, the region with domestic/industrial pollution. Here, 100% of samples contained TC and SC: Siphoviridae (75%), Podoviridae (17%), and Myoviridae (8%). At site BS1, the region with domestic pollution and with 100% of samples containing TC and SC, a higher frequency of Podoviridae (62.5%) was observed, followed by Microviridae (25%) and Siphoviridae (12.5%). At site BS2, considered to be a preserved area with low counts of somatic coliphages, only two coliphages belonging to the families Siphoviridae (1) and Podoviridae (1) (Table 2 and Fig. 3 A and D) were obtained.
Fig. 3.
Transmission electron micrographs of negatively stained coliphages. (A to C) Siphoviridae family (type T1). (A) Coliphage isolated from seawater (Baixada Santista). (B) Coliphage isolated from plankton (São Sebastião). (C) Coliphage isolated from bivalves (Ubatuba). (D) Podoviridae family type N4, isolated from seawater (Baixada Santista). (E) Microviridae family type φX174, isolated from plankton (Baixada Santista). (F) Myoviridae family type YerA41, isolated from plankton (Baixada Santista).
The observed head and tail sizes of somatic coliphages in seawater samples are shown in Table 2. Siphoviridae type T1 was the most frequent (80%), and on average, the phages observed at site BS3 had a head size (length by diameter) of 58 by 58 nm and a tail size (length by width) of 116 by 10 nm. One somatic coliphage had an especially long tail, between 939 and 995 nm (Fig. 3A). This size was not considered for the average in Table 2. Some very large phages with isometric heads and long, noncontractile, flexible tails were observed; the tails were measured to be 121 by 6 nm (BS2) and 131 by 10 nm (BS1) (Table 2). The somatic coliphage types φX174 and YerA41 were observed at sites BS1 and BS3, areas with fecal contamination, from seawater samples collected during summer 2007 at Baixada Santista.
Coliphages isolated from plankton samples.
A total of 38 somatic coliphages were obtained from plankton samples: 23 from Baixada Santista (BS), 12 from Canal de São Sebastião, and 3 from the Ubatuba region (Table 3). At Baixada Santista, site BS1, high coliphage diversity was observed. The families found were Siphoviridae (68.8%), represented by type T1 (Fig. 3B), Podoviridae (18.8%), with the morphological types sd and N4, Microviridae (6.2%), with type φX174, and Myoviridae (6.2%), with the type YerA41 (Table 3 and Fig. 3F). At site BS2, only Microviridae (type φX174) was found (Fig. 3E), and at site BS3, only Siphoviridae (types Vi II and T1) was observed. In the plankton samples, various morphologies of lysis plaques with different sizes were observed, ranging from 0.8 mm to 7 mm in diameter for Siphoviridae, 1.4 to 5.2 mm for Podoviridae, and 0.6 to 2.5 mm for Microviridae. One plaque belonging to the family Myoviridae had a diameter of 2 mm. The presence or absence of halos and smooth or irregular edges were observed in Podoviridae and Siphoviridae. In Myoviridae the plaques were clear and regular.
Table 3.
Mean sizes of somatic coliphages observed by electron microscopy from plankton samples by area, site studied, and family in coastal regions of São Paulo state, Brazil (2006 and 2007)
| Area and site (no. of isolates) | Family | No. of isolates | Morphotype | Type | Head length × diam (nm) | Tail length × width (nm) |
|---|---|---|---|---|---|---|
| Canal de São Sebastião | ||||||
| SS1 (8) | Siphoviridae | 3 | B1 | T1 | 58 × 62 | 211 × 9 |
| 1 | B1 | Vi II | 70 × 78 | 175 × 11 | ||
| Microviridae | 3 | D1 | φX174 | 26 × 25 | 0 × 0 | |
| Podoviridae | 1 | C1 | N4 | 56 × 57 | 13 × 9 | |
| SS2 (4) | Siphoviridae | 2 | B1 | T1 | 58 × 59 | 148 × 8 |
| Microviridae | 1 | D1 | φX174 | 24 × 23 | 0 × 0 | |
| Podoviridae | 1 | C1 | N4 | 55 × 54 | 0 × 0 | |
| Baixada Santista | ||||||
| BS1 (16) | Siphoviridae | 11 | B1 | T1 | 58 × 57 | 154 × 10 |
| Podoviridae | 2 | C1 | sd | 53 × 49 | 16 × 28 | |
| 1 | C1 | N4 | 47 × 48 | 16 × 22 | ||
| Microviridae | 1 | D1 | φX174 | 24 × 24 | 0 × 0 | |
| Myoviridae | 1 | A1 | YerA41 | 66 × 62 | 104 × 19 | |
| BS2 | Microviridae | 1 | D1 | φX174 | 28 × 26 | 0 × 0 |
| BS3 (6) | Siphoviridae | 4 | B1 | T1 | 56 × 56 | 108 × 9 |
| 2 | B1 | Vi II | 55 × 46 | 121 × 8 | ||
| Ubatuba | ||||||
| U1 | Podoviridae | 1 | C1 | sd | 61 × 54 | 93 × 7 |
| U2 | Siphoviridae | 2 | B1 | T1 | 62 × 65 | 127 × 7 |
In the Canal de São Sebastião, it was possible to isolate eight coliphages from site SS1 and four from site SS2. At both sites, the predominant family was Siphoviridae, represented by types T1 and Vi II. However, Microviridae of type φX174 alone was also observed, as well as Podoviridae, of the morphological type N4. Myoviridae was not observed (Table 3). The morphology of plaques was diverse, with diameters varying from 0.9 mm to 1.5 mm in Siphoviridae, 1.0 to 1.2 mm in Podoviridae, and 1.1 to 1.3 mm in the Microviridae family. All plaques were clear and regular.
At the Ubatuba area, site U1, Podoviridae (type sd) was observed, and at site U2, Siphoviridae (type T1) was observed (Table 3). The plaque diameters varied from 0.9 to 1 mm for Siphoviridae, and a unique isolated plaque belonging to Podoviridae had a diameter of 1 mm. The three isolated coliphages exhibited clear and regular plaques. In this area, where seawater samples were free of fecal pollution during the sampling, one coliphage belonging to the Podoviridae family, presenting a substantial tail length (93 nm) and small tail width (7 nm) (Table 3), was observed.
Coliphages isolated from bivalve samples.
Only two coliphages were isolated and analyzed from the 11 summer 2006 bivalve samples: one from the Canal de São Sebastião area (Siphoviridae, type Vi II) and one from the Ubatuba area (Siphoviridae, type T1) (Fig. 3C). The diameters of the plaques were 3.5 mm for type Vi II and 1.5 mm for type T1. The phage isolated from Ubatuba had the longest tail, 135 by 8 nm (Table 4).
Table 4.
Morphological characterization of somatic coliphages of the family Siphoviridae isolated from bivalve samples by area studied in coastal regions of São Paulo, Brazil, summer 2006
| Area | No. of coliphages | Morphotype | Type | Head length × diam (nm) | Tail length × width (nm) |
|---|---|---|---|---|---|
| Baixada Santista | 2a | ||||
| Canal de São Sebastião | 1 | B1 | Vi II | 51 × 52 | 70 × 10 |
| Ubatuba | 1 | B1 | T1 | 60 × 59 | 135 × 8 |
The two isolates from Baixada Santista died and were therefore not studied further.
The electron micrographics obtained in this study were effective in revealing the details of the ultrastructure of viral particles, such as the spikes and tail fibers of the somatic coliphages belonging to the family Myoviridae from the seawater and plankton samples (Fig. 3F).
DISCUSSION
The total abundance of viruses in natural aquatic environments is typically reported to be high (12, 36, 37, 46, 47, 48). Variations in virus abundance in time and space, from high values during productive seasons in surface waters, near-shore waters, and eutrophic waters to low values during unproductive seasons and in deep, offshore, and oligotrophic waters, have been observed (44).
Studies of phage distribution in the environment are mostly performed either through direct observation by electron microscopy or via molecular techniques (12, 14, 17, 24, 26, 28, 39). However, these approaches do not provide a complete picture about the infectivity of the bacterial host or whether they infect a given host bacterium. These are key issues when somatic coliphages are used as indicators. Somatic coliphages are useful indicators of both fecal pollution and the presence of enteric viruses in water environments (20, 23a). However, little is known about the diversity of somatic coliphages in areas with different levels of fecal contamination.
We selected three areas in the coastal region of the São Paulo state classified according to the criteria established by the Brazilian resolution CONAMA no. 274/2000 for bathing water quality using indicative sampling for fecal contamination (16). Our results showed a proportional relationship between the presence of TC, E. coli, and SC and the degree of anthropogenic pollution in the three areas studied (P = 0.0001). Somatic coliphages have been detected in high numbers as a result of fecal contamination in rivers, lakes, seawater, natural bodies of water, and sewage sludge samples in tropical and temperate regions (18, 19, 20, 23a, 29, 30, 31). However, coliphages found in bathing water have shown a slightly weaker correlation to E. coli and bacteriophages (24).
The somatic coliphage counts varied from <1 to 3.4 × 103 PFU/100 ml in seawater, from <1 to 4.7 × 102 PFU/g in plankton, and from <1 to 2.2 × 101 PFU/g in bivalve samples.
The low counts of somatic coliphages obtained in this study using the traditional plate method with seawater and plankton samples could be due to significant biases in our sample types and the concentration of environmental phage populations. Several authors have unsuccessfully tried to isolate phages through direct methods, and others, although occasionally successful, did not publish their findings (4). Tailed phages, for example, may be overrepresented because they often make more dramatic plaques than the other phages and, therefore, beckon more alluringly to the scientist looking to isolate a new phage. A related issue is seen in the Bacillus phage G, the largest known phage at a genome size of 500 kb: the phage G virions are so big that they do not diffuse well through agar, so plaques are too small to be seen unless unusually low concentrations of top agar are used in plaque assays (42).
Bacteriophages, as models for viruses in water, are being applied worldwide; however, there are disagreements about the methodology, the host range breadth and replication of coliphages in the environment, the presence of prophages, and the physical-chemical characteristics of the environment matrix (20, 24, 32, 33, 40). Phages in aquatic environments are unstable and therefore have short infective half-lives. Decay rates of 5 to 30% per hour are not uncommon (41, 48). The factors controlling viral activity and abundance in natural waters are elusive, but it seems reasonable to presume that the physiological conditions and activity of host populations play important roles. Low-growth conditions such as nutrient or carbon limitations will, in general, impede lytic processes so that infected cells do not produce new viruses or lyse, or cell lysis will be delayed and burst size reduced (32, 33, 36, 46).
Coastal regions are being exposed to continuous changes due to increasing anthropogenic activities. Out of the three regions studied, one area was the most critical: Baixada Santista, with a maximal concentration of somatic coliphages (1,000 PFU/100 ml). This could be related to the high input of nutrients and heavy metals through the channels of São Vicente and Santos. Currently, the main sources of contamination of the estuary of Santos and São Vicente are the Port of Santos, the industrial pole of Cubatão, the illegal dumping of sewage, the primary treated sewage, and the emissary of Santos, which is 4 km long and dumps, within the bay, tons of domestic sewage containing a high load of nutrients, heavy metals, and surfactants from the city of Santos (2, 13). At Baixada Santista there is evidence that a great deal of organic matter exists in seawater: it is a complex mixture of organic compounds with diverse chemical compositions and physical structures. For heterotrophic bacteria in the oceans, organic matter not only serves as a source of carbon and nutrients but also provides attachment surfaces, depending on its physical dimensions. The complexity in the composition and structure of organic matter, along with variable supply regimes, is probably one of the major factors helping to maintain a high diversity of prokaryote communities in the oceans. Interactions of organic matter and bacteria also exert a large influence on the major properties and patterns of ecosystems, including primary production, food web organization, and biogeochemical fluxes (35). The highest concentrations of somatic coliphages were observed mainly in February and March, during the summer period, and related to the vacation period. However, site BS2 showed lower levels of fecal contamination, in line with the original hypothesis that the area is clean and can be used as a control. Data on bathing water quality at Baixada Santista's beaches obtained from CETESB, the Brazilian environmental agency, from 2006 and 2007 showed that the quality was bad in 54% and 60% of the samples, respectively (15).
Somatic coliphage diversity. (i) Seawater samples.
Our results on somatic coliphage diversity from seawater samples collected at Baixada Santista can be compared with those obtained by Ackermann and Nguyen (8) by using electron microscopy to study the distribution of coliphages in sewage water samples. The phages found belonged to the families Myoviridae, Siphoviridae, Podoviridae, and Microviridae. RNA cubic phages and filamentous phages were not detected. In our study, regarding the three sites at Baixada Santista, four morphotypes were observed: A1 (4.5%), B1 (50%), C1 (36.4%), and D1 (9.1%), as described by Ackermann and Eisenstark (7). It is difficult to do the comparison since many studies used other bacterial hosts, different methods of isolation, or different samples in their comparisons. Our results agree with findings concerning the diversity in lakes, where the bacteriophages belonged to Siphoviridae (50.4%), Myoviridae (17.6%), and Podoviridae (19.2%), and 12.8% were of uncertain classification (17). However, our results disagree with those of studies in freshwater samples in São Paulo, Brazil, with minimal fecal contamination present and with somatic coliphages belonging to the families Myoviridae (47.7%), Podoviridae (36.4%), and Siphoviridae (15.4%) (38).
A variety of morphology was observed in the population of phages isolated from seawater. The Siphoviridae showed types T1 (enteric host, E. coli) and Vi II (enteric host, Salmonella). The Podoviridae showed types N4, sd (enteric host, E. coli), 1412 (enteric host, Salmonella), and 7480b (enteric host, Proteus). The morphotype D1 (Microviridae), type φX174 (enteric host, enterobacteria), and YerA41 (enteric host, Yersinia), both isolated in March 2007, were observed at sites BS1 and BS3, respectively—both sites with the highest anthropogenic activities. Only two somatic coliphages were isolated from site BS2, the control clean site for the region: B1, with a tail length and width of 121 by 6 nm, and C1, containing 18 filaments. We are describing here for the first time a somatic coliphage, Siphoviridae type T1, with a long tail from 939 to 995 nm. More studies are being done to explain the coliphage's survival in an injurious environment.
(ii) Plankton samples.
It was possible to obtain somatic coliphages isolated from the three areas in this type of sample. However, a high number of SC isolates was obtained from Baixada Santista compared with numbers from other two regions. Four families were present in the plankton samples: Siphoviridae (65.8%), Microviridae (15.8%), Podoviridae (15.8%), and Myoviridae (2.6%). Somatic coliphages belonging to the Myoviridae type YerA41 (enteric host, Yersinia) were found only at Baixada Santista, the area associated with high fecal contamination in seawater samples at site BS1.
The morphotype D1, type φX174, was the first virus found to have single-stranded DNA. In our study, it was found in seawater (9.1%) and plankton (15.8%) samples collected from the coastal region of São Paulo state, Brazil.
(iii) Bivalve samples.
Only two somatic coliphages were isolated: morphotype B1, types Vi II and T1, from the Canal de São Sebastião. There was no SC studied from Baixada Santista because the two isolates died and were not recoverable.
Out of the 5,100 bacterial viruses, 96% are tailed and only 3.6% are cubic, filamentous, or pleomorphic. The phages with tails are unevenly distributed among the families Siphoviridae (61%), Myoviridae (25%), and Podoviridae (14%) (3, 6). In our study, 90.3% of coliphages were tailed. A high frequency of type B1 somatic coliphages isolated from seawater (50%) and plankton (65.8%) samples was observed. The frequencies of type A1 in samples isolated from seawater (4.5%) and plankton (2.6%) and of type C1 in seawater (36.4%) and plankton (15.8%) samples are different and could be related to the host. All coliphages isolated are being studied for the presence of genes that codify for virulence-associated factors in E. coli and toxigenic Vibrio cholerae. This could help to better characterize the sources from which they were isolated.
The distribution pattern of phage morphologies may be influenced by the selection of phage isolates. Frequently, the isolation for phage diversity studies is based on the selection of plaque morphology, although it is known that there is no relationship between coliphage family and plaque morphology, as we also observed (8, 38). Detailed morphological studies of phages, from different natural habitats, infecting specific strains of host bacteria have been described, and direct electron microscopy, without enrichment, was used only for general classifications of native marine viruses based on size and morphology (8).
As discussed previously, the number of bacteriophages and the types of phages detected may be influenced by the assay medium, the composition of the medium, the host bacteria (23), the availability of phage receptors, and the composition of water ecosystems (24, 32). In the present study, we used a standardized technique and host strain. However, our results indicate that data on the morphological diversity of infectious coliphages from various studies can be compared only if the same assay conditions were used. Regarding the characterization of the areas studied, we found that the frequency of somatic coliphages had a negative correlation with temperature (−0.336, P = 0.004). Other factors that could contribute to the SC concentration are rainfall, the level of nutrients, and salinity, as was shown when the coliphage occurrence decreased from inland to the coastal ocean and in the significant difference between saltwater and freshwater samples (40).
Viruses are potentially dangerous contaminants of seawater, mainly when products of human activities are polluting the ecosystems. The coastal areas are being exposed to continuous changes due to increasing of anthropogenic activities, and many bodies of water constantly receive a significant amount of treated, partially treated, or untreated sewage, as well as discharge of ballast water, which severely depletes the water quality of those water bodies used for drinking, irrigation, seafood cultivation, and recreational purposes. Maintaining and improving the health of marine life will translate into a better quality of life for humans, animals, and ecosystems.
Currently, somatic coliphages are considered to be one of the best indicators of the presence of enteric viruses, and the diversity and concentrations obtained in this study continue to support their use. Some authors have suggested the use of an enrichment method because of a significant correlation between somatic coliphages, total enteric viruses, and adenoviruses (11). Others proposed the use of one bacterial indicator and somatic coliphages, which could be more informative than two bacterial indicators (22, 29), and the observation of somatic coliphage replication must not be argued as a limitation (27).
Bacteriophages are now being recognized as important vectors for genomic reshuffling and are having a significant impact on the understanding of genetic mechanisms for phage evolution, genetic diversity, and the genetic structure of phage populations (1).
A better characterization of the diversity of somatic coliphages isolated from coastal aquatic environments will help to contribute to the management and evaluation of microbiological risk for bathing and/or seafood cultivation and consumption.
This is the first report in Brazil showing the abundance and diversity of somatic coliphages in seawater, plankton, and bivalve samples collected from three areas of the São Paulo state coast, each with different levels of human activities. Baixada Santista is an area containing high fecal pollution and virus hazards compared to those of the Canal de São Sebastião and Ubatuba regions. For the first time, an SC Siphoviridae with a long tail has been described.
ACKNOWLEDGMENTS
This work was supported by a grant from CNPq and FAPESP. We thank the following for support of the sample collection: CEBIMar, USP; Base IO/USP de Ubatuba; and Corpo de Bombeiros de Santos. E.M.B.-R. acknowledges a Ph.D. fellowship from CNPq.
We thank Zelma F. Marinho for technical assistance.
Footnotes
Published ahead of print on 29 April 2011.
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