Environmental Research 127 (2013) 56–62
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Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli strains isolated from environmental waters in the Mediterranean area S. Rodríguez-Martínez, S. Cervero-Aragó, I. Gil-Martin, R. Araujo n Departament de Microbiologia, Universitat de Barcelona, Av. Diagonal 643, Barcelona 08028, Spain
art ic l e i nf o
a b s t r a c t
Article history: Received 18 April 2013 Received in revised form 22 August 2013 Accepted 2 October 2013 Available online 8 November 2013
Campylobacter jejuni and Campylobacter coli are important animal-related waterborne pathogens that are distributed worldwide. To further understand Campylobacter populations in water from the Mediterranean area, the genetic diversity of environmental strains was analyzed using multilocus sequence typing (MLST). MLST was also used to determine the potential geographical differences between these bacterial strains and other campylobacters isolated worldwide. The typing study was conducted using 58 strains isolated from the Llobregat river and other water sources, such as urban sewage, animal wastewater and clinical samples. Thirty-nine different sequence types were obtained; eight of these sequences were described for the first time in this study, suggesting the presence of local strains. The identified C. jejuni strains were the most diverse population, whereas the identified C. coli strains showed a high clonal structure, which clustered most of the sequence types into a few clonal complexes. The strains were not exclusively related to specific water sources. However, comparing the identified strains with an international database showed that most of the Mediterranean strains that were exclusively isolated from environmental waters have previously been isolated from similar sources, particularly those obtained from river water. Additional studies, including those in different geographical areas using a wide range of Campylobacter sources, are required to improve the global knowledge concerning Campylobacter dissemination in the environment. & 2013 Published by Elsevier Inc.
Keywords: Campylobacter jejuni Campylobacter coli MLST River Urban sewage and animal sewage
1. Introduction Thermotolerant Campylobacter species are the major cause of bacterial gastroenteritis in Europe and most developed countries worldwide (Ailes et al., 2008; European Food Safety Authoriry, 2010; Mickan et al., 2007). It is estimated that over 95% of Campylobacter spp. infections are caused by Campylobacter jejuni and Campylobacter coli (Butzler, 2004; Nachamkin et al., 2000). These pathogens are typically located in the intestinal biota of many warm-blooded animal species, particularly birds (Newell et al., 2001; Skirrow, 1994), which excrete these bacteria in feces and pollute nearby environments. Food produced from these animals and polluted waters are considered major sources of Campylobacter transmission to humans (Koenraad et al., 1997; Savill et al., 2003). Although Campylobacter are not considered aquatic microorganisms, many species have been isolated from river water worldwide, and the presence of these bacteria is considered a sign of recent fecal contamination (Jones, 2001). Common sources of pollution include
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Corresponding author. Fax: þ34 934039047. E-mail address: [email protected] (R. Araujo).
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the discharge of effluents from untreated urban sewage, local food industries, agricultural run-off and livestock waste, and other environmental inputs, such as those from wild animals or birds living close to the water source (Hokajarvi et al., 2013). Most studies on the presence and distribution of Campylobacter species have been conducted in Northern European countries, including the UK, USA and New Zealand (Devane et al., 2005; Eyles et al., 2003; Obiri-Danso and Jones, 1999; Sails et al., 2002; Savill et al., 2001; Vereen et al., 2007). Information regarding the occurrence of Campylobacter in the Mediterranean area is scarce. Indeed, our group has previously published one of the few studies focused on this area (Rodríguez and Araujo, 2010). In a 2-year study, we monitored the occurrence of Campylobacter species in the Llobregat River and at the most probable sources of pollution in the surrounding area, including poultry wastewater, pig slurries and urban sewage (Hundesa et al., 2009). In this work, strains from those origins, together with some clinical strains, were analyzed in order to see whether a genetic relationship existed between these microorganisms. Multiple methods can be used to determine epidemiological relationships between microbial isolates (Foley et al., 2009). Different typing methods have been described for Campylobacter (Wassenaar and Newell, 2000). The most frequently used are
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pulse-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). MLST is a technique based on sequencing gene fragments of housekeeping loci. This discriminatory procedure has some advantages over other molecular methods, such as the unambiguous nature of the nucleotide sequence data, the employment of reproducible technology (Maiden et al., 1998) and the fact that data can easily be shared electronically between laboratories, facilitating comparisons of Campylobacter sequences from strains isolated worldwide (http://pubmlst.org/campylobacter/) (Dingle et al., 2005; Foley et al., 2009). Thus, using MLST, we addressed the following aims of the investigation: (i) to type and analyze the genetic diversity of the Campylobacter strains collected from environmental waters and some clinical samples; (ii) to compare the Mediterranean strains with those from the international database; and (iii) to add our collection of strains isolated from the Mediterranean area to the existing database.
2. Material and methods 2.1. Campylobacter isolates Campylobacter strains were isolated during a 2-year study of environmental water in a Mediterranean area (Rodríguez and Araujo, 2010). Briefly, appropriate volumes of the water samples were analyzed using the most probable number (MPN). The samples were concentrated and inoculated in tubes containing the Preston enrichment broth (Oxoid, Basingstoke, UK) and incubated at 42 1C for 48 h under microaerobic conditions. The samples were subcultured from the Preston broth tubes to the Karmali agar plates (Scharlab, Barcelona, Spain) and incubated for 48 h at 42 1C under microaerobic conditions using the Oxoid CampyGen™ system (Oxoid, Basingstoke, UK). The plates were examined for the presence of Campylobacter colonies, and the isolates were identified as C. jejuni or C. coli using multiplex PCR according to the methods of Wong et al. (2004). The strains were stored at 80 1C in Brain Heart Infusion broth (Difco, New Jersey, USA) containing 20% glycerol. A total of 26 C. jejuni and 32 C. coli isolates were typed. The samples comprised 21 isolates from Llobregat river water (6 C. jejuni and 15 C. coli), 18 isolates from urban sewage water (7 C. jejuni and 11 C. coli), 8 C. jejuni strains from poultry wastewater and 5 C. coli strains from pig slurry. Six strains (5 C. jejuni and 1 C. coli) from clinical cases of gastroenteritis were also included in the analysis as an outside group not isolated from water. The samples were in-kind donations from Dr. Bartolomé from the Vall d´Hebron Hospital of Barcelona. All strains were cultured on Karmali agar plates (Scharlab, Barcelona, Spain) at 42 1C for 2 days under microaerobic conditions in a jar using the Oxoid CampyGen™ system (Oxoid, Basingstoke, UK). Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega, Madison, Wis.). 2.2. Multilocus sequence typing Multilocus sequence typing (MLST) of seven housekeeping genes (aspA, glnA, gltA, glyA, pgm, tkt and uncA) was performed according to previously described protocols (Devane et al., 2005; Dingle et al., 2001). The DNA was amplified using the Illustra™ PureTaq Ready-to-Go PCR Bead Kit (GE Healthcare, Buckinghamshire, UK). The PCR amplicons were purified using the Invisorb Fragment Clean Up kit (Invitek GmbH, Germany). The nucleotide sequences were determined using the BigDye Terminator v2 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, USA). The reaction products were detected using an ABI Prism 3700 Capillary Sequencer (Applied Biosystems, USA). The sequence files were edited using BioEdit v. 7.1.3 software (Hall, 1999). The allele numbers, sequence types (STs) and clonal complexes were assigned through comparison of the obtained sequences with the Campylobacter MLST database (http://pubmlst.org/campylobacter/) (Jolley and Maiden, 2010). Novel STs were submitted to this MLST database for number designation. Information concerning Campylobacter strains isolated worldwide was also obtained from the same database. Neighbor-joining trees of the concatenated gene sequences of each isolate were constructed using MEGA v. 5.0 (Tamura et al., 2011) with Kimura 2 parameters and 1000 bootstrap simulations.
3. Results 3.1. Sequence typing of Campylobacter strains Strains from five different origins (Llobregat river water, urban sewage, poultry wastewater, pig slurries and clinical samples)
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were analyzed. The results of the MLST analysis are summarized in Table 1. A total of 39 different STs were identified among the 58 strains analyzed. Among these, 20.5% of the STs were identified for the first time and submitted to the database (Table 2). These sequences originated from new combinations of previously described alleles. A total of 22 different STs were detected among the 26 C. jejuni strains. Most of the C. jejuni STs occurred only once in the dataset, except for ST-50, which occurred four times (three clinical strains and one urban sewage strain), and ST-441, which occurred twice (one river water strain and one urban sewage strain). C. coli presented lower ST diversity, with 17 STs among the 32 isolated strains. The predominant C. coli STs were ST-825, which was isolated from river water and pig slurry, and ST-1771, which was isolated from river water. The predominant STs differed depending on the water origin. ST-1771 and ST-1766 were the predominant isolates in river water (5/15 and 4/15 isolates, respectively), ST-825 was the predominant isolate in urban sewage (4/11), and a similar number of STs were isolated from pig slurries (1/5). A comparison of our data with the Campylobacter MLST database resulted in the assignment of STs to a ST-clonal complex (Table 1). ST-clonal complex groups STs that share four or more alleles. Among the 22 C. jejuni STs, eight of them could not be assigned to any complex. Only six STs are grouped in the same two clonal complexes: ST-complex 45 and ST-complex 21. Four STs are grouped in ST-complex 45: ST-45 and ST-5168, isolated from urban sewage, and ST-2663 and ST-3730, both isolated from poultry wastewater. And two STs are grouped in ST-complex 21: ST-50, isolated from urban sewage and clinical samples, and ST-760, isolated from poultry wastewater. Most of the C. coli STs obtained (11/17) were assigned to clonal complex ST-828. These STs were ST-1009, ST-1689 and ST-5169, which were isolated exclusively from urban sewage; ST-890, ST1595 and ST-2713, which were isolated from pig slurries; ST-825 and ST-854, which were isolated from urban sewage and pig slurries; and ST-827, which was isolated from urban sewage and clinical samples. Notably, none of the strains isolated from river water belonged to the 828-complex. The remaining nine STs obtained could not be assigned to any clonal complex.
3.2. Phylogeny and clustering of the isolates A neighbor-joining tree was constructed using the 3309-bp concatenated MLST allele sequences for the two species of Campylobacter analyzed; bootstrapping values of 100% were obtained for the branch separating the two species after 1000 repetitions (data not shown). Therefore, two new neighbor-joining trees were constructed according to species (Fig. 1). We obtained a tree that divided the C. coli isolates into two large groups. The first cluster grouped the isolates obtained from river water and urban sewage. The relationship between these STs was highly variable; remarkably, the ST-4356 obtained from river water was the most genetically divergent sequence. The second major cluster grouped the isolates collected from urban sewage, pig slurries and a clinical sample as well as the reference strain. The bootstrap values within this second cluster were higher than those in the first cluster of STs. The tree obtained for C. jejuni STs showed higher divergence between the STs than the C. coli tree. Although there was no clear division into clusters according to ST origin, various patterns were observed. The tree showed three different associations between poultry wastewater and urban sewage STs: ST-3730 with ST-45, ST760 with ST-50, and ST-305 with ST-969. We also observed a genetic relationship between ST-572 and ST-429, which were isolated from river water and poultry wastewater, respectively.
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Table 1 Distribution of STs among Campylobacter jejuni and Campylobacter coli isolates from environmental waters. N: number of isolates; NI: not isolated from this source; and ND: not defined. In bold, STs isolated in the study submitted for the first time in the dataset. Campylobacter jejuni
Source
River water (N ¼ 21)
Urban sewage wáter (N ¼18)
Campylobacter coli
ST (no. isolates)
ST-clonal complex
Database identity no.
ST (no. isolates/no. isolates with common origin)
ST-clonal complex
Database identity number
177 (1) 441 (1) 572 (1) 823 (1) 1223 (1) 4355 (1)
177 ND 206 ND 1275 ND
8229 5140 5203 12760 12761 5204
1764 (2) 1765 (1) 1766 (4)
ND ND ND
1771 (5)
ND
2272 (1) 4356 (1) 5603 (1)
ND ND ND
5206, 12767 5205 5208, 12769, 12770, 12771 5207, 5209, 5210, 5211, 12768 12772 8212 12773
22 (1) 45 (1) 50 (1) 441 (1) 969 (1) 2236 (1) 5168 (1)
22 45 21 ND 354 353 45
12753 12774 12754 12757 8228 8227 11143
825 (4)
828
827 (1) 854 (2) 1009 (1) 1689 (1) 5169 (1) 5170 (1)
828 828 ND 828 828 ND
Poultry wastewater (N ¼8)
305 (1) 429 (1) 466 (1) 760 (1) 2331 (1) 2663 (1) 3730 (1) 5602 (1)
574 48 ND 21 ND 45 45 ND
12756 5220 12758 12759 5221 12762 12763 12765
NI
NI
Pig slurry (N ¼ 5)
NI
NI
825 (1) 854 (1) 890 (1) 1595 (1) 2713 (1)
828 828 828 828 828
5226 5225 5224 5222 5223
Clinical samples (N¼ 6)
50 (3)
21
827 (1/1)
828
5227
3030 (1) 5601 (1)
ND ND
5228 5230 12755 5229 12764
5212, 5213, 5214, 5215 5218 5216, 5217 12766 5219 11144 11145
Table 2 New Campylobacter STs identified in this study. Clonal complex
C. jejuni
C. coli
Unassigned ST-45 Unassigned Unassigned Unassigned ST-828 Unassigned Unassigned
ST
4355 5168 5601 5602 4356 5169 5170 5603
Allelic profile Asp
Gln
Glt
Gly
Pgm
Tkt
Unc
1 2 2 7 136 33 72 136
172 7 257 1 104 38 39 188
95 1 16 12 88 30 69 69
62 4 243 2 113 82 277 113
43 1 23 2 266 118 143 270
32 7 3 3 238 225 35 277
147 1 12 5 95 17 98 152
3.3. Comparison of the studied strains with the MLST database Fifty-eight isolates, obtained from different origins, were uploaded to the PubMLST database to add our strains to the worldwide statistics (Tables 1 and 3). The information in the database was used to determine whether the Campylobacter
No. of isolates
Source
1 1 1 1 1 1 3 1
River water Urban sewage Clinical Poultry wastewater River Urban sewage Urban sewage River
strains analyzed in the present study are exclusive to the Mediterranean area or share genetic similarities with other isolates. A total of four C. jejuni STs were exclusively isolated in this study: ST4355 (river water), ST-5168 (urban sewage), ST-5601 (clinical samples) and ST-5602 (poultry wastewater). Two C. coli strains isolated from river water, ST-4356 and ST-5603, and two C. coli
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Fig. 1. Radial neighbor-joining trees constructed from 3309-bp concatenated MLST allele sequences of C. jejuni (A) and C. coli (B). Bootstrap of 1000 repetitions. Only the bootstrap values higher than 60% are shown in the trees. RW: river water; UW: urban sewage; PW: poultry wastewater; PS: pig slurries; and CS: clinical samples.
Table 3 International identified strains in PubMLST Campylobacter database. N: number of strains in our study, IN: number of strains described in the international database. C. jejuni
River water
Urban sewage
C. coli
ST (N/IN)
Sources
Countries
177 441 572 823 1223 4355
B, Ch, H, Sd, W Ch, H Ch, H, L No specified B, W
CAN, GBR CAN, ESP, LUX, GBR BEL, DEU, GRC, LUX, NLD, GBR DEU CAN, USA
(1/56) (1/34) (1/127) (1/2) (1/4) (1/1)
22 (1/166) Ch, F, H, Hb, L, M
45 (1/593) B, Ch, H, Hb, L, M, Tk, Sl, W 50 (1/593) Ch, H, Hb, L, Tk, M, dW 441 (1/34) 969 (1/5) H 2236 (1/3) H 5168 (1/1) Clinical
Poultry wastewater
50 (3/593) 3030 (1/3) Ch 5601 (1/1) 305 (1/13) 429 (1/16) 466 760 2331 2663 3730 5602
(1/6) (1/14) (1/3) (1/3) (1/2) (1/1)
River water
Urban AUS, BEL, CAN, CUW, DEU, DNK, GRC, ITA, JPN, NLD, GBR, USA, THA, sewage ZAF AUS, BEL, CAN, CUW, DEU, DNK, FIN, FRA, GRC, JPN, NLD, SWE, GBR, USA AUS, BEL, CAN, CUW, DEU, DNK, FRA, GRC, NLD; CHE, THA, GBR; USA BEL, GBR GBR Clinical
ST (N/IN)
Sources
Countries
1764 1765 1771 1766 2272 4356 5603
W W W W W
GBR GBR GBR LUX, GBR GBR
Ch, H, Pg
NLD, CHE, GBR, USA
(2/4) (1/2) (5/6) (4/6) (1/2) (1/1) (1/1)
825 (4/83)
827 (1/156) Ch, F, H, Pg ESP, LUX, NLD, GBR, USA 854 (2/157) Ch, H, Pg, W 1009 (1/15) Ch, H 1689 (1/7) Ch, H 5169 (1/1) 5170 (1/1)
FRA, LUX, NLD, CHE, GBR, USA NLD, GBR ESP, GBR
827 (1/156) Ch, F, H, Pg ESP, LUX, NLD, GBR, USA
BEL, ITA
Ch, H B, Ch, H
GRC, JPN, NDL, THA, GBR NLD; USA
H H H Ch, H Ch
NLD; GBR DEU, NLD, GBR ESP, GBR LUX, GBR USA
Pig slurries
825 (1/83) Ch, H, Pg 854 (1/157) Ch, H, Pg, W 890 (1/11) Ch, L, Pg 1595 (1/6) H 2713 (1/2) Pg
NLD, CHE, GBR, USA FRA, LUX, NLD, CHE, GBR, USA CAN, USA DNK, GBR, LUX GBR
Sources: B: wild birds; Ch: Chicken; F: farm; H: Human stool; Hb: Human Blood; L: Livestock (beef, cattle, dog, goat, lamb or sheep); M: cow's milk; Sd: Sand (beach); Sl: Soil; Pg: Pig; Tk: Turkey: W: environmental waters; and dW: drinking water. Countries: official codes according to ISO 3166-1 alpha-3 (Anonymous, 1999).
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strains isolated from urban sewage, ST-5169 and ST-5170, were also unique to this study. As the PubMLST Campylobacter database also includes the origin of all uploaded strains, it was possible to analyze any coincidences between the strains isolated in the present studies and other strains. Only two of the seven C. jejuni STs that were isolated from the Llobregat river, ST-177 and ST-1223, were previously isolated from environmental waters. However, all identified C. coli STs were previously identified exclusively from environmental waters, although Campylobacter species are not considered aquatic microorganisms.
4. Discussion Campylobacters are commonly found in aquatic environments including rivers, streams and lakes (Bolton et al., 1987; Carter et al., 1987; Popowski et al., 1997). Although there are several studies on the occurrence of Campylobacter species in water worldwide, few studies have focused on the Mediterranean area (Alonso and Alonso, 1993; Baffone et al., 1995; Moreno et al., 2003). In a previous study, we focused on the occurrence of Campylobacter in the Llobregat River (Rodríguez and Araujo, 2010). The river is located in the northeast of Spain in a highly populated region with problems associated with water availability. We showed that C. jejuni and C. coli are distributed in the surface water of the studied area, with C. coli being the predominant species. However, the highest counts of campylobacter were observed in pig slurry, poultry wastewater and urban sewage, which have been considered the most probable sources of pollution in the area (Hundesa et al., 2009; Rodríguez and Araujo, 2010). This 2-year study facilitated the compilation of a Campylobacter strain collection isolated from different aquatic environments in the same geographical area. To better understand the Campylobacter dissemination in the Mediterranean area, molecular typing was performed using multilocus sequence typing (MLST). MLST has been effectively applied to Campylobacter strains of human and animal origin (Colles et al., 2003; Dingle et al., 2005; Griekspoor et al., 2010; Kinana et al., 2007; Kärenlampi et al., 2007; Oporto et al., 2011). However, only a few studies have included water samples (Clark et al., 2012; Magnússon et al., 2011; McTavish et al., 2009). Indeed, the present study and the study conducted by Hokajarvi et al. (2013) are the first to completely focus on water samples. Our results were consistent with those from previous studies describing high genotypic diversity in Campylobacter populations (Dingle et al., 2002; Manning et al., 2003; Wassenaar and Newell, 2000). In the current study, 39 STs were identified out of 58 strains analyzed, indicating a high ST diversity in the Campylobacter population in the sampled Mediterranean area. Eight of these STs were added to the MLST database for the first time. Four STs were C. jejuni isolates: ST-4355, ST-5601, ST-5168 and ST-5602, which were isolated from river water, clinical samples, urban sewage, and poultry wastewater, respectively. The other four STs were C. coli isolates: ST-4356 and ST-5603, which were isolated from river water, and ST-5169 and ST-5170, which were isolated from urban sewage. These results indicate that some strains could be associated with certain geographical areas (Sheppard, 2010). C. jejuni has a diverse genetic nature and a weak clonal population structure (Dingle et al., 2001; Suerbaum et al., 2001), whereas C. coli has lower allelic diversity (Kinana et al., 2007), which is consistent with the observations reported in the present study. Within the C. jejuni isolates, only two STs were detected twice, ST-441 (isolated from river water and urban sewage) and ST-50 (from urban sewage and clinical samples); the latter ST is one of the most frequently reported STs worldwide (Gripp et al.,
2011). Although some of the C. jejuni STs could be assigned to two ST-complexes, the neighbor-joining tree that was constructed using the concatenated gene sequences showed no strong clusters, confirming the high diversity of this species. In contrast, C. coli presented a more clonal structure. Most of the strains corresponded to the ST-828 clonal complex, in which the most frequent isolate was ST-825. However, ST-1771, the most frequently isolated strain from river water, was not included in the ST-828 clonal complex. Due to the limited number of identified strains and the high genetic diversity of these species, an association between the identified STs of the current study and their isolation origins was not observed; therefore, we compared the Mediterranean dataset obtained in the present study against the international Campylobacter PubMLST database. Colles et al. (2003) and Dingle et al. (2002) previously demonstrated that some C. jejuni STs are widely distributed between different hosts, whereas others STs are specific to a certain host. C. jejuni STs identified from Mediterranean river water were also identified worldwide from wild birds, chickens, human stools, livestock and environmental water. Similarly, the strains identified in urban sewage water had different origins in the international dataset, whereas poultry wastewater strains were more specific to avian hosts, primarily chickens and wild birds and more sporadically from human samples. There is some disagreement regarding the relationship between C. coli STs and the source of pollution or host origin. While Dingle et al. (2005) observed no clear association, Miller et al. (2006), Litrup et al. (2007) and Lang et al. (2010) observed some overlap between the isolates, showing a common origin. In the present study, all C. coli STs identified from the Mediterranean river were previously and exclusively isolated from environmental water. These international isolates have not been associated with any host or source of pollution. The remaining isolates from urban sewage, clinical samples and pig slurries have been isolated from similar origins worldwide. The geographical effects, niche adaptation and host immune selection could determine the clonal structure of the strains (Kärenlampi et al., 2007). These factors could explain why the river water strains differed from the other types of isolates. MLST is a useful tool for studying strain diversity, particularly for comparing geographical areas, using the international database. However, this technique has some disadvantages because a pure culture is required for the application of this method. The fact that all strains typed required a culture step might favor the isolation of some strains over others. Moreover, potential viable but not culturable that were stressed under harsh environmental conditions could not be included. Thus, as Colles et al. (2003) suggested, isolates with STs identified exclusively in river water are most likely strains that have recently entered the river, are better adapted for long-term survival in this habitat or grow better in artificial culture media. In summary, the genetic diversity of Campylobacter environment in the Mediterranean area was consistent with that reported by other authors worldwide. The C. jejuni population showed a highly diverse genetic nature, whereas the C. coli isolates exhibited a more clonal population structure. There was no association between Campylobacter strains and the isolation source in the studied area. Thus, further analyses with more strains are needed. A comparison of the Mediterranean isolates with strains in the international database showed that most of these species have previously been isolated from similar sources. However, in the case of river water isolates, most of the STs (71%) have been exclusively isolated from environmental waters. More studies, focused in different geographical areas, which include a wide range of Campylobacter sources, are required to increase the
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number and diversity of strains in the international database. This collective effort will ultimately improve the global knowledge concerning Campylobacter dissemination in the environment and consequently expand the current understanding of the associated health risks.
Acknowledgments This study was supported through funding from the Generalitat de Catalunya (2005SGR00592), the “Xarxa de Referència en Biotecnologia de la Generalitat de Catalunya (XRB)” and L'Institut de l'aigua (Universitat de Barcelona). Sarah Rodríguez received a fellowship from the Universitat de Barcelona. The authors would like to thank Dr. Bartolomé for the clinical strains, J.A. Oliva for assistance with sampling and J. Balañà, S. Fresno and J.A. López-Portolés for analysis and technical support.
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Environmental Research journal homepage: www.elsevier.com/locate/envres
Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli strains isolated from environmental waters in the Mediterranean area S. Rodríguez-Martínez, S. Cervero-Aragó, I. Gil-Martin, R. Araujo n Departament de Microbiologia, Universitat de Barcelona, Av. Diagonal 643, Barcelona 08028, Spain
art ic l e i nf o
a b s t r a c t
Article history: Received 18 April 2013 Received in revised form 22 August 2013 Accepted 2 October 2013 Available online 8 November 2013
Campylobacter jejuni and Campylobacter coli are important animal-related waterborne pathogens that are distributed worldwide. To further understand Campylobacter populations in water from the Mediterranean area, the genetic diversity of environmental strains was analyzed using multilocus sequence typing (MLST). MLST was also used to determine the potential geographical differences between these bacterial strains and other campylobacters isolated worldwide. The typing study was conducted using 58 strains isolated from the Llobregat river and other water sources, such as urban sewage, animal wastewater and clinical samples. Thirty-nine different sequence types were obtained; eight of these sequences were described for the first time in this study, suggesting the presence of local strains. The identified C. jejuni strains were the most diverse population, whereas the identified C. coli strains showed a high clonal structure, which clustered most of the sequence types into a few clonal complexes. The strains were not exclusively related to specific water sources. However, comparing the identified strains with an international database showed that most of the Mediterranean strains that were exclusively isolated from environmental waters have previously been isolated from similar sources, particularly those obtained from river water. Additional studies, including those in different geographical areas using a wide range of Campylobacter sources, are required to improve the global knowledge concerning Campylobacter dissemination in the environment. & 2013 Published by Elsevier Inc.
Keywords: Campylobacter jejuni Campylobacter coli MLST River Urban sewage and animal sewage
1. Introduction Thermotolerant Campylobacter species are the major cause of bacterial gastroenteritis in Europe and most developed countries worldwide (Ailes et al., 2008; European Food Safety Authoriry, 2010; Mickan et al., 2007). It is estimated that over 95% of Campylobacter spp. infections are caused by Campylobacter jejuni and Campylobacter coli (Butzler, 2004; Nachamkin et al., 2000). These pathogens are typically located in the intestinal biota of many warm-blooded animal species, particularly birds (Newell et al., 2001; Skirrow, 1994), which excrete these bacteria in feces and pollute nearby environments. Food produced from these animals and polluted waters are considered major sources of Campylobacter transmission to humans (Koenraad et al., 1997; Savill et al., 2003). Although Campylobacter are not considered aquatic microorganisms, many species have been isolated from river water worldwide, and the presence of these bacteria is considered a sign of recent fecal contamination (Jones, 2001). Common sources of pollution include
n
Corresponding author. Fax: þ34 934039047. E-mail address: [email protected] (R. Araujo).
0013-9351/$ - see front matter & 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.envres.2013.10.003
the discharge of effluents from untreated urban sewage, local food industries, agricultural run-off and livestock waste, and other environmental inputs, such as those from wild animals or birds living close to the water source (Hokajarvi et al., 2013). Most studies on the presence and distribution of Campylobacter species have been conducted in Northern European countries, including the UK, USA and New Zealand (Devane et al., 2005; Eyles et al., 2003; Obiri-Danso and Jones, 1999; Sails et al., 2002; Savill et al., 2001; Vereen et al., 2007). Information regarding the occurrence of Campylobacter in the Mediterranean area is scarce. Indeed, our group has previously published one of the few studies focused on this area (Rodríguez and Araujo, 2010). In a 2-year study, we monitored the occurrence of Campylobacter species in the Llobregat River and at the most probable sources of pollution in the surrounding area, including poultry wastewater, pig slurries and urban sewage (Hundesa et al., 2009). In this work, strains from those origins, together with some clinical strains, were analyzed in order to see whether a genetic relationship existed between these microorganisms. Multiple methods can be used to determine epidemiological relationships between microbial isolates (Foley et al., 2009). Different typing methods have been described for Campylobacter (Wassenaar and Newell, 2000). The most frequently used are
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pulse-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). MLST is a technique based on sequencing gene fragments of housekeeping loci. This discriminatory procedure has some advantages over other molecular methods, such as the unambiguous nature of the nucleotide sequence data, the employment of reproducible technology (Maiden et al., 1998) and the fact that data can easily be shared electronically between laboratories, facilitating comparisons of Campylobacter sequences from strains isolated worldwide (http://pubmlst.org/campylobacter/) (Dingle et al., 2005; Foley et al., 2009). Thus, using MLST, we addressed the following aims of the investigation: (i) to type and analyze the genetic diversity of the Campylobacter strains collected from environmental waters and some clinical samples; (ii) to compare the Mediterranean strains with those from the international database; and (iii) to add our collection of strains isolated from the Mediterranean area to the existing database.
2. Material and methods 2.1. Campylobacter isolates Campylobacter strains were isolated during a 2-year study of environmental water in a Mediterranean area (Rodríguez and Araujo, 2010). Briefly, appropriate volumes of the water samples were analyzed using the most probable number (MPN). The samples were concentrated and inoculated in tubes containing the Preston enrichment broth (Oxoid, Basingstoke, UK) and incubated at 42 1C for 48 h under microaerobic conditions. The samples were subcultured from the Preston broth tubes to the Karmali agar plates (Scharlab, Barcelona, Spain) and incubated for 48 h at 42 1C under microaerobic conditions using the Oxoid CampyGen™ system (Oxoid, Basingstoke, UK). The plates were examined for the presence of Campylobacter colonies, and the isolates were identified as C. jejuni or C. coli using multiplex PCR according to the methods of Wong et al. (2004). The strains were stored at 80 1C in Brain Heart Infusion broth (Difco, New Jersey, USA) containing 20% glycerol. A total of 26 C. jejuni and 32 C. coli isolates were typed. The samples comprised 21 isolates from Llobregat river water (6 C. jejuni and 15 C. coli), 18 isolates from urban sewage water (7 C. jejuni and 11 C. coli), 8 C. jejuni strains from poultry wastewater and 5 C. coli strains from pig slurry. Six strains (5 C. jejuni and 1 C. coli) from clinical cases of gastroenteritis were also included in the analysis as an outside group not isolated from water. The samples were in-kind donations from Dr. Bartolomé from the Vall d´Hebron Hospital of Barcelona. All strains were cultured on Karmali agar plates (Scharlab, Barcelona, Spain) at 42 1C for 2 days under microaerobic conditions in a jar using the Oxoid CampyGen™ system (Oxoid, Basingstoke, UK). Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega, Madison, Wis.). 2.2. Multilocus sequence typing Multilocus sequence typing (MLST) of seven housekeeping genes (aspA, glnA, gltA, glyA, pgm, tkt and uncA) was performed according to previously described protocols (Devane et al., 2005; Dingle et al., 2001). The DNA was amplified using the Illustra™ PureTaq Ready-to-Go PCR Bead Kit (GE Healthcare, Buckinghamshire, UK). The PCR amplicons were purified using the Invisorb Fragment Clean Up kit (Invitek GmbH, Germany). The nucleotide sequences were determined using the BigDye Terminator v2 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, USA). The reaction products were detected using an ABI Prism 3700 Capillary Sequencer (Applied Biosystems, USA). The sequence files were edited using BioEdit v. 7.1.3 software (Hall, 1999). The allele numbers, sequence types (STs) and clonal complexes were assigned through comparison of the obtained sequences with the Campylobacter MLST database (http://pubmlst.org/campylobacter/) (Jolley and Maiden, 2010). Novel STs were submitted to this MLST database for number designation. Information concerning Campylobacter strains isolated worldwide was also obtained from the same database. Neighbor-joining trees of the concatenated gene sequences of each isolate were constructed using MEGA v. 5.0 (Tamura et al., 2011) with Kimura 2 parameters and 1000 bootstrap simulations.
3. Results 3.1. Sequence typing of Campylobacter strains Strains from five different origins (Llobregat river water, urban sewage, poultry wastewater, pig slurries and clinical samples)
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were analyzed. The results of the MLST analysis are summarized in Table 1. A total of 39 different STs were identified among the 58 strains analyzed. Among these, 20.5% of the STs were identified for the first time and submitted to the database (Table 2). These sequences originated from new combinations of previously described alleles. A total of 22 different STs were detected among the 26 C. jejuni strains. Most of the C. jejuni STs occurred only once in the dataset, except for ST-50, which occurred four times (three clinical strains and one urban sewage strain), and ST-441, which occurred twice (one river water strain and one urban sewage strain). C. coli presented lower ST diversity, with 17 STs among the 32 isolated strains. The predominant C. coli STs were ST-825, which was isolated from river water and pig slurry, and ST-1771, which was isolated from river water. The predominant STs differed depending on the water origin. ST-1771 and ST-1766 were the predominant isolates in river water (5/15 and 4/15 isolates, respectively), ST-825 was the predominant isolate in urban sewage (4/11), and a similar number of STs were isolated from pig slurries (1/5). A comparison of our data with the Campylobacter MLST database resulted in the assignment of STs to a ST-clonal complex (Table 1). ST-clonal complex groups STs that share four or more alleles. Among the 22 C. jejuni STs, eight of them could not be assigned to any complex. Only six STs are grouped in the same two clonal complexes: ST-complex 45 and ST-complex 21. Four STs are grouped in ST-complex 45: ST-45 and ST-5168, isolated from urban sewage, and ST-2663 and ST-3730, both isolated from poultry wastewater. And two STs are grouped in ST-complex 21: ST-50, isolated from urban sewage and clinical samples, and ST-760, isolated from poultry wastewater. Most of the C. coli STs obtained (11/17) were assigned to clonal complex ST-828. These STs were ST-1009, ST-1689 and ST-5169, which were isolated exclusively from urban sewage; ST-890, ST1595 and ST-2713, which were isolated from pig slurries; ST-825 and ST-854, which were isolated from urban sewage and pig slurries; and ST-827, which was isolated from urban sewage and clinical samples. Notably, none of the strains isolated from river water belonged to the 828-complex. The remaining nine STs obtained could not be assigned to any clonal complex.
3.2. Phylogeny and clustering of the isolates A neighbor-joining tree was constructed using the 3309-bp concatenated MLST allele sequences for the two species of Campylobacter analyzed; bootstrapping values of 100% were obtained for the branch separating the two species after 1000 repetitions (data not shown). Therefore, two new neighbor-joining trees were constructed according to species (Fig. 1). We obtained a tree that divided the C. coli isolates into two large groups. The first cluster grouped the isolates obtained from river water and urban sewage. The relationship between these STs was highly variable; remarkably, the ST-4356 obtained from river water was the most genetically divergent sequence. The second major cluster grouped the isolates collected from urban sewage, pig slurries and a clinical sample as well as the reference strain. The bootstrap values within this second cluster were higher than those in the first cluster of STs. The tree obtained for C. jejuni STs showed higher divergence between the STs than the C. coli tree. Although there was no clear division into clusters according to ST origin, various patterns were observed. The tree showed three different associations between poultry wastewater and urban sewage STs: ST-3730 with ST-45, ST760 with ST-50, and ST-305 with ST-969. We also observed a genetic relationship between ST-572 and ST-429, which were isolated from river water and poultry wastewater, respectively.
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Table 1 Distribution of STs among Campylobacter jejuni and Campylobacter coli isolates from environmental waters. N: number of isolates; NI: not isolated from this source; and ND: not defined. In bold, STs isolated in the study submitted for the first time in the dataset. Campylobacter jejuni
Source
River water (N ¼ 21)
Urban sewage wáter (N ¼18)
Campylobacter coli
ST (no. isolates)
ST-clonal complex
Database identity no.
ST (no. isolates/no. isolates with common origin)
ST-clonal complex
Database identity number
177 (1) 441 (1) 572 (1) 823 (1) 1223 (1) 4355 (1)
177 ND 206 ND 1275 ND
8229 5140 5203 12760 12761 5204
1764 (2) 1765 (1) 1766 (4)
ND ND ND
1771 (5)
ND
2272 (1) 4356 (1) 5603 (1)
ND ND ND
5206, 12767 5205 5208, 12769, 12770, 12771 5207, 5209, 5210, 5211, 12768 12772 8212 12773
22 (1) 45 (1) 50 (1) 441 (1) 969 (1) 2236 (1) 5168 (1)
22 45 21 ND 354 353 45
12753 12774 12754 12757 8228 8227 11143
825 (4)
828
827 (1) 854 (2) 1009 (1) 1689 (1) 5169 (1) 5170 (1)
828 828 ND 828 828 ND
Poultry wastewater (N ¼8)
305 (1) 429 (1) 466 (1) 760 (1) 2331 (1) 2663 (1) 3730 (1) 5602 (1)
574 48 ND 21 ND 45 45 ND
12756 5220 12758 12759 5221 12762 12763 12765
NI
NI
Pig slurry (N ¼ 5)
NI
NI
825 (1) 854 (1) 890 (1) 1595 (1) 2713 (1)
828 828 828 828 828
5226 5225 5224 5222 5223
Clinical samples (N¼ 6)
50 (3)
21
827 (1/1)
828
5227
3030 (1) 5601 (1)
ND ND
5228 5230 12755 5229 12764
5212, 5213, 5214, 5215 5218 5216, 5217 12766 5219 11144 11145
Table 2 New Campylobacter STs identified in this study. Clonal complex
C. jejuni
C. coli
Unassigned ST-45 Unassigned Unassigned Unassigned ST-828 Unassigned Unassigned
ST
4355 5168 5601 5602 4356 5169 5170 5603
Allelic profile Asp
Gln
Glt
Gly
Pgm
Tkt
Unc
1 2 2 7 136 33 72 136
172 7 257 1 104 38 39 188
95 1 16 12 88 30 69 69
62 4 243 2 113 82 277 113
43 1 23 2 266 118 143 270
32 7 3 3 238 225 35 277
147 1 12 5 95 17 98 152
3.3. Comparison of the studied strains with the MLST database Fifty-eight isolates, obtained from different origins, were uploaded to the PubMLST database to add our strains to the worldwide statistics (Tables 1 and 3). The information in the database was used to determine whether the Campylobacter
No. of isolates
Source
1 1 1 1 1 1 3 1
River water Urban sewage Clinical Poultry wastewater River Urban sewage Urban sewage River
strains analyzed in the present study are exclusive to the Mediterranean area or share genetic similarities with other isolates. A total of four C. jejuni STs were exclusively isolated in this study: ST4355 (river water), ST-5168 (urban sewage), ST-5601 (clinical samples) and ST-5602 (poultry wastewater). Two C. coli strains isolated from river water, ST-4356 and ST-5603, and two C. coli
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Fig. 1. Radial neighbor-joining trees constructed from 3309-bp concatenated MLST allele sequences of C. jejuni (A) and C. coli (B). Bootstrap of 1000 repetitions. Only the bootstrap values higher than 60% are shown in the trees. RW: river water; UW: urban sewage; PW: poultry wastewater; PS: pig slurries; and CS: clinical samples.
Table 3 International identified strains in PubMLST Campylobacter database. N: number of strains in our study, IN: number of strains described in the international database. C. jejuni
River water
Urban sewage
C. coli
ST (N/IN)
Sources
Countries
177 441 572 823 1223 4355
B, Ch, H, Sd, W Ch, H Ch, H, L No specified B, W
CAN, GBR CAN, ESP, LUX, GBR BEL, DEU, GRC, LUX, NLD, GBR DEU CAN, USA
(1/56) (1/34) (1/127) (1/2) (1/4) (1/1)
22 (1/166) Ch, F, H, Hb, L, M
45 (1/593) B, Ch, H, Hb, L, M, Tk, Sl, W 50 (1/593) Ch, H, Hb, L, Tk, M, dW 441 (1/34) 969 (1/5) H 2236 (1/3) H 5168 (1/1) Clinical
Poultry wastewater
50 (3/593) 3030 (1/3) Ch 5601 (1/1) 305 (1/13) 429 (1/16) 466 760 2331 2663 3730 5602
(1/6) (1/14) (1/3) (1/3) (1/2) (1/1)
River water
Urban AUS, BEL, CAN, CUW, DEU, DNK, GRC, ITA, JPN, NLD, GBR, USA, THA, sewage ZAF AUS, BEL, CAN, CUW, DEU, DNK, FIN, FRA, GRC, JPN, NLD, SWE, GBR, USA AUS, BEL, CAN, CUW, DEU, DNK, FRA, GRC, NLD; CHE, THA, GBR; USA BEL, GBR GBR Clinical
ST (N/IN)
Sources
Countries
1764 1765 1771 1766 2272 4356 5603
W W W W W
GBR GBR GBR LUX, GBR GBR
Ch, H, Pg
NLD, CHE, GBR, USA
(2/4) (1/2) (5/6) (4/6) (1/2) (1/1) (1/1)
825 (4/83)
827 (1/156) Ch, F, H, Pg ESP, LUX, NLD, GBR, USA 854 (2/157) Ch, H, Pg, W 1009 (1/15) Ch, H 1689 (1/7) Ch, H 5169 (1/1) 5170 (1/1)
FRA, LUX, NLD, CHE, GBR, USA NLD, GBR ESP, GBR
827 (1/156) Ch, F, H, Pg ESP, LUX, NLD, GBR, USA
BEL, ITA
Ch, H B, Ch, H
GRC, JPN, NDL, THA, GBR NLD; USA
H H H Ch, H Ch
NLD; GBR DEU, NLD, GBR ESP, GBR LUX, GBR USA
Pig slurries
825 (1/83) Ch, H, Pg 854 (1/157) Ch, H, Pg, W 890 (1/11) Ch, L, Pg 1595 (1/6) H 2713 (1/2) Pg
NLD, CHE, GBR, USA FRA, LUX, NLD, CHE, GBR, USA CAN, USA DNK, GBR, LUX GBR
Sources: B: wild birds; Ch: Chicken; F: farm; H: Human stool; Hb: Human Blood; L: Livestock (beef, cattle, dog, goat, lamb or sheep); M: cow's milk; Sd: Sand (beach); Sl: Soil; Pg: Pig; Tk: Turkey: W: environmental waters; and dW: drinking water. Countries: official codes according to ISO 3166-1 alpha-3 (Anonymous, 1999).
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strains isolated from urban sewage, ST-5169 and ST-5170, were also unique to this study. As the PubMLST Campylobacter database also includes the origin of all uploaded strains, it was possible to analyze any coincidences between the strains isolated in the present studies and other strains. Only two of the seven C. jejuni STs that were isolated from the Llobregat river, ST-177 and ST-1223, were previously isolated from environmental waters. However, all identified C. coli STs were previously identified exclusively from environmental waters, although Campylobacter species are not considered aquatic microorganisms.
4. Discussion Campylobacters are commonly found in aquatic environments including rivers, streams and lakes (Bolton et al., 1987; Carter et al., 1987; Popowski et al., 1997). Although there are several studies on the occurrence of Campylobacter species in water worldwide, few studies have focused on the Mediterranean area (Alonso and Alonso, 1993; Baffone et al., 1995; Moreno et al., 2003). In a previous study, we focused on the occurrence of Campylobacter in the Llobregat River (Rodríguez and Araujo, 2010). The river is located in the northeast of Spain in a highly populated region with problems associated with water availability. We showed that C. jejuni and C. coli are distributed in the surface water of the studied area, with C. coli being the predominant species. However, the highest counts of campylobacter were observed in pig slurry, poultry wastewater and urban sewage, which have been considered the most probable sources of pollution in the area (Hundesa et al., 2009; Rodríguez and Araujo, 2010). This 2-year study facilitated the compilation of a Campylobacter strain collection isolated from different aquatic environments in the same geographical area. To better understand the Campylobacter dissemination in the Mediterranean area, molecular typing was performed using multilocus sequence typing (MLST). MLST has been effectively applied to Campylobacter strains of human and animal origin (Colles et al., 2003; Dingle et al., 2005; Griekspoor et al., 2010; Kinana et al., 2007; Kärenlampi et al., 2007; Oporto et al., 2011). However, only a few studies have included water samples (Clark et al., 2012; Magnússon et al., 2011; McTavish et al., 2009). Indeed, the present study and the study conducted by Hokajarvi et al. (2013) are the first to completely focus on water samples. Our results were consistent with those from previous studies describing high genotypic diversity in Campylobacter populations (Dingle et al., 2002; Manning et al., 2003; Wassenaar and Newell, 2000). In the current study, 39 STs were identified out of 58 strains analyzed, indicating a high ST diversity in the Campylobacter population in the sampled Mediterranean area. Eight of these STs were added to the MLST database for the first time. Four STs were C. jejuni isolates: ST-4355, ST-5601, ST-5168 and ST-5602, which were isolated from river water, clinical samples, urban sewage, and poultry wastewater, respectively. The other four STs were C. coli isolates: ST-4356 and ST-5603, which were isolated from river water, and ST-5169 and ST-5170, which were isolated from urban sewage. These results indicate that some strains could be associated with certain geographical areas (Sheppard, 2010). C. jejuni has a diverse genetic nature and a weak clonal population structure (Dingle et al., 2001; Suerbaum et al., 2001), whereas C. coli has lower allelic diversity (Kinana et al., 2007), which is consistent with the observations reported in the present study. Within the C. jejuni isolates, only two STs were detected twice, ST-441 (isolated from river water and urban sewage) and ST-50 (from urban sewage and clinical samples); the latter ST is one of the most frequently reported STs worldwide (Gripp et al.,
2011). Although some of the C. jejuni STs could be assigned to two ST-complexes, the neighbor-joining tree that was constructed using the concatenated gene sequences showed no strong clusters, confirming the high diversity of this species. In contrast, C. coli presented a more clonal structure. Most of the strains corresponded to the ST-828 clonal complex, in which the most frequent isolate was ST-825. However, ST-1771, the most frequently isolated strain from river water, was not included in the ST-828 clonal complex. Due to the limited number of identified strains and the high genetic diversity of these species, an association between the identified STs of the current study and their isolation origins was not observed; therefore, we compared the Mediterranean dataset obtained in the present study against the international Campylobacter PubMLST database. Colles et al. (2003) and Dingle et al. (2002) previously demonstrated that some C. jejuni STs are widely distributed between different hosts, whereas others STs are specific to a certain host. C. jejuni STs identified from Mediterranean river water were also identified worldwide from wild birds, chickens, human stools, livestock and environmental water. Similarly, the strains identified in urban sewage water had different origins in the international dataset, whereas poultry wastewater strains were more specific to avian hosts, primarily chickens and wild birds and more sporadically from human samples. There is some disagreement regarding the relationship between C. coli STs and the source of pollution or host origin. While Dingle et al. (2005) observed no clear association, Miller et al. (2006), Litrup et al. (2007) and Lang et al. (2010) observed some overlap between the isolates, showing a common origin. In the present study, all C. coli STs identified from the Mediterranean river were previously and exclusively isolated from environmental water. These international isolates have not been associated with any host or source of pollution. The remaining isolates from urban sewage, clinical samples and pig slurries have been isolated from similar origins worldwide. The geographical effects, niche adaptation and host immune selection could determine the clonal structure of the strains (Kärenlampi et al., 2007). These factors could explain why the river water strains differed from the other types of isolates. MLST is a useful tool for studying strain diversity, particularly for comparing geographical areas, using the international database. However, this technique has some disadvantages because a pure culture is required for the application of this method. The fact that all strains typed required a culture step might favor the isolation of some strains over others. Moreover, potential viable but not culturable that were stressed under harsh environmental conditions could not be included. Thus, as Colles et al. (2003) suggested, isolates with STs identified exclusively in river water are most likely strains that have recently entered the river, are better adapted for long-term survival in this habitat or grow better in artificial culture media. In summary, the genetic diversity of Campylobacter environment in the Mediterranean area was consistent with that reported by other authors worldwide. The C. jejuni population showed a highly diverse genetic nature, whereas the C. coli isolates exhibited a more clonal population structure. There was no association between Campylobacter strains and the isolation source in the studied area. Thus, further analyses with more strains are needed. A comparison of the Mediterranean isolates with strains in the international database showed that most of these species have previously been isolated from similar sources. However, in the case of river water isolates, most of the STs (71%) have been exclusively isolated from environmental waters. More studies, focused in different geographical areas, which include a wide range of Campylobacter sources, are required to increase the
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number and diversity of strains in the international database. This collective effort will ultimately improve the global knowledge concerning Campylobacter dissemination in the environment and consequently expand the current understanding of the associated health risks.
Acknowledgments This study was supported through funding from the Generalitat de Catalunya (2005SGR00592), the “Xarxa de Referència en Biotecnologia de la Generalitat de Catalunya (XRB)” and L'Institut de l'aigua (Universitat de Barcelona). Sarah Rodríguez received a fellowship from the Universitat de Barcelona. The authors would like to thank Dr. Bartolomé for the clinical strains, J.A. Oliva for assistance with sampling and J. Balañà, S. Fresno and J.A. López-Portolés for analysis and technical support.
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