3.2.1 Sampling
Untreated drinking water (DW) samples were collected in autoclaved bottles, from fountains, wells, drilled wells and mines, between June and December of 2004, May, June, August and October of 2005 and January of 2007. A total of 93 samples were obtained. Sampling sites were located in North (Districts of Viana do Castelo, Vila Real, Oporto), Centre [Districts of Aveiro, Lisbon, Viseu (Vouzela), Leiria (Pombal)] and South [District of Faro (Lagos)] of Portugal (Fig. 3.1). In some cases, samples were recovered at the same sampling site at different times (Vila Real, sampling sites 47, 48 and 52 in December 2004, July 2005 and January 2007; Lagos, sampling sites 43, 44 and 45 in December 2004 and May 2005). Mineral waters (MW) were collected from two water springs in the Centre. Two samples were collected in the same water spring (WS1 sampling sites 1 and 2) and the other in a different one (WS2 sampling site 3). Samples were examined within 5 h.
Figure 3.1 Map of continental Portugal with sampling districts and municipalities (indicated by arrows).
Aeromonads were isolated by the membrane filter technique. Briefly, 100 ml of each sample were filtered through 0.45 µm pore size cellulose ester filter (Pall Life Sciences, USA), membranes were placed in glutamate starch phenol-red agar (GSP) (Merck, Germany), supplemented with 50 µg/ml of ampicillin (Merck, Germany) and plates were incubated at 30 ºC overnight. Yellow colonies in GSP medium were selected and pure cultures were obtained by repeated isolation of individual colonies in the same medium. Pure cultures were stored in 17% glycerol at -80ºC.
3.2.2 DNA extraction
A single colony was resuspended in 100 µl TE buffer and 200 µl chelex 20% (Bio- Rad, USA) were added; the mixture was vortexed. Cycles of 10 min at 95 ºC followed by 10 min at -20 ºC were repeated three times. The mixtures were centrifuged for 5 min at 13000 rpm in a 5415D – Eppendorf (Germany) centrifuge. The supernatant was transferred to another tube and stored at -20 ºC until use.
3.2.3 Genotyping
Random amplification of polymorphic DNA (RAPD) PCR was performed using the primer OPA 16 (Lockhart et al., 1997). The reaction mixtures (25 µl) contained 1x PCR buffer (buffer with MgCl2), 400 µM dNTPs, 4 µM of primer OPA16, 1 U of Taq
ultratools DNA polymerase (Biotools B&M Labs, S.A., Spain) and 100 – 200 ng of genomic DNA. PCR amplifications were carried out in a PTC-100TM Peltier Thermal Cycler (MJ Research, INC, USA) as follows: initial denaturation (94 ºC for 4 min), 40 cycles of denaturation (94 ºC for 1 min), annealing (36 ºC for 1 min) and extension (72 ºC for 1 min) and a final extension (72 ºC for 5 min). Amplicons were separated by electrophoresis in a 1.3% agarose gel-TAE buffer stained with ethidium bromide at 60 V for 1 h 40 min. Results were observed on a UV transilluminator and images were acquired with the Gel DocMega camera system 5.01 (Biosystematica, UK). Genetic profiles were visually analysed by intra-gel patterns comparison and isolates representative of each RAPD pattern were selected for phylogenetic analysis. Isolates displaying the same gene sequence in phylogenetic analysis were again submitted to RAPD fingerprinting in order to obtain a definitive discrimination of the isolates.
3.2.4 Phylogenetic analysis
The determination of phylogenetic relationships among isolates as well as the taxonomic position of each one relative to described Aeromonas spp. was based on gyrB gene partial sequences. PCR amplification of the gyrB gene was performed with primers gyrB3F and gyrB14R (Yáñez et al., 2003). The final reaction mixture (50 µl) contained 1x PCR buffer (buffer with MgCl2), 200 µM dNTPs, 0.2 µM of each primer, 1 U of Taq DNA
polymerase (Biotools B&M Labs, S.A., Spain) and 50 – 100 ng of genomic DNA. PCR conditions applied were as follows: initial denaturation at 94 ºC for 3 min, 35 cycles of denaturation at 94 ºC for 15 s, annealing at 55 ºC for 30 s and extension at 72 ºC for 45 s, and a final extension at 72 ºC for 3 min. Amplified products were analysed on a 1% agarose gel-TAE buffer stained with ethidium bromide and visualized as described above. Amplicons were purified with QIAquick PCR purification kit (QIAGEN, Germany), following the manufacturer’s instructions. Sequencing analysis using primer gyrB3F (Yañez et al., 2003) were performed as previously described (Saavedra et al., 2006), using
the BigDye Terminator V3.1 cycle sequencing kit and the ABI Prism® 3100 Avant Genetic Analyzer (Applied Biosystems, USA).
Sequences were analysed with the Chromas 1.45 software. Alignments of gyrB partial sequences of the isolates collected in this study and from all Aeromonas species present in the Molecular Diagnostics Center (MDC), Orihuela, Spain, culture collection including type strains, were obtained using the CLUSTALX program (Larkin et al., 2007). Phylogenetic tree was produced by the neighbor-joining method (Saitou and Nei, 1987) with the Kimura’s 2-parameter method (Kimura, 1980), using the MEGA4 program (Tamura et al., 2007).
3.3 Results
3.3.1 Aeromonas isolation from water samples
Water samples were collected from different, either private or public, sources and had in common the fact of being untreated. Most of them were not regularly subjected to monitoring for quality assessment purposes. In all cases the water was used for drinking and other human activities.
A total of 93 untreated drinking water samples were collected from 86 sampling sites (Fig. 3.1). 184 presumptive aeromonads were isolated from 30 samples. Table 3.1 exhibits the geographic distribution and numbers of samples and isolates collected from these sources.
Regarding the sampling sites that were sampled in different occasions, recovery of
Aeromonas spp. was not always achieved (4 cases). For example, in sampling site 45, in Lagos, Aeromonas spp. were recovered in December 2004 but a sample collected at the same site in May 2005 failed to display typical Aeromonas colonies.
Table 3.1 Total numbers of isolates and drinking water samples collected from fountains, wells, drilled wells and mines in different regions of Portugal.
North Centre South
Sampling sites 61 22 3
Samples collected 65 22 6 Samples with Aeromonas spp. 14 12 4 Isolates collected 70 103 11
In the Centre of Portugal, 3 mineral water samples were collected from 3 sampling sites and 22 isolates presumptively members of the genus Aeromonas were obtained.
Physical and chemical characteristics of these waters are on Table 3.2. The mineral water from sampling sites 1 and 2 is hypothermal chloride bicarbonate and contains 903.6 mg/l of dissolved minerals. They were clinically used either by oral consumption or bathing. It was indicated in the treatments of dermatosis, gastrointestinal tract and female genital diseases and rheumatism. The mineral water from sampling site 3 collected in a different water spring, is sulphuric and has the same medical purpose of mineral water from water spring 1.
Table 3.2 Physical and chemical characteristics of mineral waters. Sampling Site Turbidity Temperature
(ºC)
HCO3
(ml)
Conductivity
(µs/cm) pH Hydrogeochemical type Water spring 1 (sampling
sites 1 and 2) clear 24.6 1.1 1148 6 Chloride
Water spring 2 (sampling site 3)
slightly
turbid 17.3 1.8 1338 5.5 Sulphurous
3.3.2 Genotyping
A total of 206 isolates was submitted to RAPD analysis, resulting in 80 different patterns and for each, one representative strain was chosen. 31 patterns were displayed uniquely by one isolate.
The same profile was occasionally obtained from isolates from different sampling sites (10 cases) and from the same sampling site, on different sampling dates (1 case). For instance, isolate DW.126 (representative strain A.126) from sampling site 62 in Porto has the same RAPD profile of isolate DW.A1/2 (representative strain A.126) collected in sampling site 84 in Póvoa de Varzim. Also, isolate DW.096A (representative strain A.096A) has the same RAPD profile of isolates represented by DW.96/2_1 (representative strain A.096A), collected in sampling site 43 in December 2004 and May 2005, respectively. On the other hand, up to 9 different genetic fingerprints were collected from the same sampling site. Sample 136 collected in sampling site 79 in the district of Lisbon harboured 25 isolates with 9 different RAPD profiles, represented by strains A.135/10, A.136/3, A.136/4, A.136/5, A.136/12, A.136/13, A. 136/15, A.136/24, and A.136/26. In Figures 3.2 and 3.3 examples of different RAPD-PCR results are shown.
DW. 129/6 DW. 132/4 DW. 132/8 DW. 132/9 DW. 127/7 DW. 128 DW. 135/7 DW. 129/1 DW. 129/3 g17 g18 R 19 R 19 R 34 R 35 R 20 R 18 R 20 R 44 R 21 R 21 DW. 127/4 DW. 134/8 DW. 134/1 DW. 134/2 DW. 134/3 DW. 134/4 DW. 134/5 DW. 134/6 DW. 134/9 DW. 135/3 DW. 135/1 DW. 135/5 DW. 135/6 DW. 135/7 DW. 135/8 + - DW. 135/9 DW. 135/10 DW. 137/1A DW. 137/1B DW. 137/2 DW. 137/3 DW. 137/4 DW. 137/5 DW. 137/6 DW. 137/7 DW. 137/9 DW. 137/10 DW134 DW135 DW137 R 39 R 39 R 39 R 39 R 39 R 39 R 39 R 39 R 40 R 40 R 42 R 43 R 44 R 40 R 41 R 45 R 54 R 54 R 55 R 55 R 55 R 46 R 46 R 49 R 49 R 46 Figure 3.2 Examples of genotyping results by RAPD-PCR obtained for isolates from the same sample. Legend to figure:
+, positive control; –, negative control.
All isolates (8) recovered from sample DW134 displayed the same RAPD profile (R39). Among 9 isolates from sample DW135 6 distinct profiles were recorded (R40, R41, R42, R43, R44 and R45). Within the 10 isolates collected from sample DW137, four different RAPD profiles were detected (R46, R49, R54 and R55).
Figure 3.3 Examples of RAPD patterns of isolates displaying identical gyrB gene partial sequences.
Legend to figure:
gyrB gene partial sequence g17 (A. bestiarum) was exhibited by 18 isolates, among which 6 different RAPD profiles were detected (R18, R19, R20, R34, R35, R44). gyrB gene partial sequence g18 was exhibited by four A. media isolates (two shown in figure) displaying the same RAPD profile (R21).
3.3.3 Identification and phylogenetic analysis
A representative of each RAPD profile was selected for sequencing analysis of
gyrB gene. Partial sequencing of this gene resulted in an approximately 500 bp high quality nucleotide sequence. The unrooted phylogenetic tree was constructed by using the gyrB gene partial sequence of the isolates and strains from MDC culture collection, including type strains (Fig. 3.4).
The clustering of strains was consistent with all species of the genus described to date as the well characterised strains from MDC culture collection grouped accordingly to their species affiliation, except the unique strain of the newly described species
Figure 3.4 Unrooted phylogenetic tree based on gyrB gene sequences, demonstrating the relationships of the strains collected (A.xxx) to reference and other representative strains from MDC (Orihuela, Spain) Aeromonas culture collection (MDCxxx).
Legend to figure:
Strains collected from mineral water samples are signalized with MW. Isolates displaying the same RAPD profile recovered from different sampling sites and also from the same sampling site at different dates are shown side by side with the respective representative strain. Phylogenetic groups displaying different gyrB gene sequences different from the all known are indicated (Aeromonas. sp. Group I and Aeromonas. sp. Group II). The tree was constructed by the neighbor-joining method with the
A.124/1 A.A2/1 MDC250 A.MW.41/2 A.MW.5/6 A.131/1 MDC219 A.MW.4/6 A.MW.23/1 A.A2/4 A.131/6 A.MW.21/1 A.130/2 A.MW.4/2 A.media CECT4232T A.MW.7/1; MW.9/2 A.130/13 A.129/1 A.133/2 A.MW.22/2 A.MW.30/1 MDC12 MDC98 A.allosaccharophila CECT4199T A.MW.28/2 MDC103 A.sobria CECT4245T MDC175 A.fluvialis CECT7401T A.096A; DW96/2_1; DW.97/2