Phylogenetic affiliation of sequences obtained from DGGE bands from water (W) and feces (F).
High-altitude Andean lakes are exposed to extreme environmental factors like high salinity, ultraviolet radiation, heavy metals, among others. As it was previously shown, these lakes are not only the habitat of a high diversity of bacteria with multiple resistances; they also support an enormous population of flamingos, which migrate among these wetlands, and they could play a role as disseminators and/or reservoirs of pathogenic bacteria.
- Andean Lakes
High-Altitude Andean Lakes (HAAL) are a system of shallow oligotrophic lakes originated in the tertiary age distributed across the
In this chapter, we compare bacterial diversity using Denaturant Gradient Gel Electrophoresis (DGGE) under antibiotic pressure conditions in water and flamingo feces from three HAAL: Laguna Aparejos, Laguna Negra, and Laguna Azul.
2. Materials and methods
2.1. Description of environments and sampling
Aparejos, Negra, and Azul lakes are located in the Andes Mountains in the northwest of Argentina; their physic and chemical characteristics are described by Dib et al. . They are a group of lakes and salar pads called Salar de la Laguna Verde in the Andean region of Catamarca province, Argentina (27º 34´ S; 68º 32´ W). Some of the highest mountains of the Andean system are located in this area: Ojos del Salado (6,885 m) and Nevado Pissis (6,779 m). The water temperature was 5ºC at the sampling time (1 pm local hour) and the maximal UV-B irradiance reached 3.3 Wm-2 for 312 nm (half band with 300–325 nm).
Two types of samples were considered: water and flamingo feces. Surface water samples were collected during summer 2009 (near the beginning of austral spring) in 10 L sterile polyethylene bottles. Water samples were stored at 4ºC until further processing in the laboratory (within 24 h after collection), which is located 600 km away from the sampling site. Flamingo feces were taken near the lake and conserved in sterile bags at 4ºC until processing. Once in a sterile environment in the lab, core feces samples were extracted for cultivation.
2.2. Antibiotics-resistant bacteria-enrichment cultures
To determine bacterial diversity under selective pressure, water samples from Laguna Aparejos, Laguna Negra, Laguna Azul, and four flamingo feces samples from each lake were analyzed. Samples were inoculated in 20 mL of R2A medium (yeast extract 0.5 g L-1, peptone 0.5 g L-1, casamino acids 0.5 g L-1, glucose 0.5 g L-1, soluble starch 0.5 g L-1, sodium pyruvate 0.3 g L-1, K2HPO4 0.3 g L-1, MgSO4×7H2O 0.05 g L-1; pH 7.2), with different ATBs. Control cultures without ATBs were also performed. Five ATBs were used: ampicillin (Amp), 100 µg mL-1; chloramphenicol (Cm), 170 µg mL-1; colistin (Col), 20 µg mL-1; erythromycin (Ery), 50 µg mL-1; and tetracycline (Tet) 50 µg mL-1. After five days of incubation at 30ºC and 150 rpm, the cells were pelleted by centrifugation and total DNA was extracted from the ATB enriched cultures. Afterward, DGGE profiles of total community cultured without or with different ATBs were determined.
2.3. PCR amplification DGGE and sequencing
DNA extraction from total community cultures was performed using a CTAB method . The variable V3 region of 16S rRNA gene was amplified by PCR . The nucleotide sequences of the primers are as follows: primer 1 F341: 5’-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GCC TAC GGG AGG CAG CAG-3’, primer 2 R518: 5’-CGT ATT ACC GCG GCT GCT GG-3’, primer 3 F357: 5’-TTA CTG ATA GAA TGT GGA GC-3’.
PCR amplification was performed with a Biometra Termocycler as follows: 100 ηg of purified genomic DNA, 20 pmol of each primers (Genbiotech), 200 µmol of each deoxyribonucleoside triphosphate, 10 µL of 10× PCR buffer (MgCl2) and 0.25 U of Go Taq polymerase (Promega) were added to a 0.2 mL volume microtube, which was filled up to a volume of 25 µL with sterile Milli-Q-water. PCR was performed using the following conditions: initial denaturing step of 15 min at 95°C, followed by 30 cycles of 95°C for 1 min, 65°C for 1 min, and 72°C for 1 min 30 s. A touchdown program was performed in order to down one grade at each cycle, until 55ºC. At this last temperature, 15 additional cycles were programmed, with a final extension at 72°C for 5 min. DGGE was performed with the Bio-Rad Protean II system, essentially as described previously . PCR products were applied directly onto 8% (wt/vol) polyacrylamide gels in 1X TAE buffer (40 mM Tris base, 20 mM sodium acetate, 1 mM EDTA) and a linear gradient consisting of the denaturants urea and formamide; the concentration of the denaturants increased from 40% at the top of the gel to 60% at the bottom. Electrophoresis was performed at a constant voltage of 120 V and a temperature of 60ºC during 5 h. After electrophoresis, the gel was stained for 10 min with SYBR® Gold (Molecular Probes, Eugene, OR), rinsed with TAE buffer, and visualized with a Bio-Rad UV Gel Doc 2000 transilluminator. Distinguishable bands were excised from the gel; the eluted DNA was reamplified using the primers 2 and 3, and PCR products were sequenced.
2.4. Nucleotide sequence accession numbers and data analysis
Fifty-nine selected 16S rRNA sequences from DGGE bands in this paper have been deposited in GenBank database under the following accession numbers: AM712052–66, AM711573–79, AM711878–90, and AM889064–87.
The similarity in DGGE bands in each lake was assessed by Cluster Analysis using the Jaccard’s index, applying the UPGMA (unweight pair-group method using averages) algorithm with software MVSP 3.2.
3.1. Diversity of ATB-resistant bacteria in water and feces
The affiliation of the prominent reamplified bands from DGGE gels from major bacterial community members obtained from ATB enrichment cultures, from water and feces, in all studied lakes is shown in Table 1. 16S rRNA gene sequence comparisons revealed that most of the water and feces DGGE bands were represented mainly by
Band sequences related to
In Laguna Aparejos, two bands sequences (A15 and A18) were exclusively recovered from water and they presented similarities with members of the genera
The sequence related to
||100||W, F||Amp, Col, Ery Cm, Tet|
|A2; A3; A4A; A16a, A5A||
||96-100||W, F||Amp, Ery, Cm, Tet|
|A9; A10; A14; A8A||
||98-100||W, F||Amp, Ery, Col, Tet|
||95||W, F||Amp, Ery|
||98||W||Amp, Col, Tet|
||98||F||Amp, Ery, Cm, Tet|
||97||W, F||Amp, Col|
||99||W, F||Amp, Col|
|N10; N11; N11a; N12||
||99||W, F||Amp, Col|
||95-99||W, F||Amp, Col, Tet|
||99-100||W||Col, Ery, Tet|
||92-95||W||Amp, Col, Tet|
|Az19; Az20; Az2;||
|Az6; Az15; Az10; Az21; Az8; Az25; Az23; Az7; Az17||
||96-99||F||Amp, Ery, Tet|
|Az13; Az12; Az3||
||98-100||W, F||Ery, Amp, Col|
3.2. Antibiotics resistances
The microbial diversity by DGGE in water and feces after cultivation under antimicrobial pressure could be explained by the presence of ATB-resistant traits or the acquisition of resistant traits by horizontal gene transfer events during cultivation.
In Laguna Aparejos, there was a band sequence detected in the five enrichment cultures conditions. It was the case of a band sequence related to
In Laguna Negra, most of the DGGE-detected bands were found in Col- and Amp-enriched cultures. Two band sequences (N13, N14) matched with
DGGE band sequences matched with
3.3. DGGE analyses
Figure 1 shows the dendrogram resulting from the Cluster Analysis performed among samples taking into account the presence or absence of individual bands obtained by DGGE profiles of Laguna Aparejos. The analysis evidenced that water and flamingo feces without any antimicrobial pressure clustered together conforming a subgroup.
In Laguna Negra, cluster analysis indicates that water, feces, and feces with Amp clustered within the same subgroup (Figure 2).
In Laguna Azul, two clear groups can be observed, one for feces samples and the other for water samples (Figure 3).
It was proposed that landscape ecology, which links the biotic and abiotic factors of an ecosystem, might help to untangle the complexity of antibiotic resistance and improve the interpretation of ecological studies . Continuing that idea, we have previously demonstrated that water in high-irradiated pristine environments was a source for isolating bacteria able to grow in the presence of antibiotics, and that the bacteria were also present in flamingos’ enteric biota, probably taken from the water where they feed . In addition, we have found that several isolated bacteria present giant extra chromosomal linear elements, the so-called linear plasmids [21-23]. We found that the presence of linear plasmids might be related to the antibiotics-resistant dispersion. In this work, we attempt to study the total bacterial community under different selective pressures and the connection between the microbiota associated to lake water and flamingo feces.
4.1. Antibiotic-resistant bacteria is an spread phenomenon in high-altitude lakes
We showed that the ability to grow in ATB or the rapid spread of this ability was abundant, diverse, and widely distributed in the water and feces of the studied high-altitude environments. As it was postulated by our group in previous publications [2, 3], UV radiation would be in connection with ATB resistances since under extreme UV stress, bacteria are known to increase mutational events, through a resistance mechanism named error-prone repair . In many cases, spontaneous resistance to ATB is known to emerge under such mutagenic conditions, as consequence of mutagenesis modified potential target genes. In addition, a possible connection of oxidative stress resistances and an association with ATB resistances were also established . As it was largely established that UV radiation produces high oxidative stress, thus a high-irradiated environment is expected to select oxidative stress-resistant bacteria, and this could also be in connection with ATB resistances found in more irradiated environments.
One the other hand, exposure of wild birds to human-generated wastewater presents a pathway for transfer of bacteria and the antibiotic resistance genes that they carry . Water bodies of Pampean Lakes are threatened by many anthropic activities, resulting from land use, agriculture, and livestock, with the subsequent deposition of a significant amount of organic wastes, fertilizers, and pesticides [27-30]. Therefore, flamingos exposed to such sources could be colonized by microorganisms that are not typical of their natural habitats and are involved in the dissemination of multidrug-resistant bacteria since migration of flamingos, among lakes from Andean lakes in summer to Pampean lakes in winter, is an established phenomenon . Our next challenge is subject to deeper studies the flamingo’s role as disseminators and/or reservoir of multidrug-resistant bacteria.
4.2. Microbiota in water and birds feces
Mostly, band sequences identified in water samples were also found in feces. Thus we observed a connection between the bacterial community’s inhabitant flamingos intestinal and those of the water lake, where these birds obtain their food: community structure harboring similar ATB resistances were similar in both water and feces samples, sampled from the same lake. Special attention should be given to
A band corresponding to
As it was determined by our preview reports [2, 3], we confirm the idea that pathogenic organism resistant to multi-antibiotics are not a phenomena restricted to spoiled environments and that pristine environments could be considered as important reservoirs of bacteria like
HAAL – High Altitude Andean Lakes; ATBs – Antibiotics; DGGE – Denaturant Gradient Gel Electrophoresis
This work was supported by PIE CONICET 6268-6096, Fundación Antorchas Nº 14248-133, PICT-Agencia Nacional de Promoción Científica y Tecnológica Nº14498. María Verónica Fernández-Zenoff is recipient of a CONICET fellowship. Anna Neumann received financial support from DAAD.
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