arsenic and antibiotic resistance in heterotrophic aerobic bacteria from marine

35 Pages·2015·1.07 MB·English

AEM Accepted Manuscript Posted Online 30 January 2015
Appl. Environ. Microbiol. doi:10.1128/AEM.03240-14
Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1 Natural hotspots for multiple resistances gain: arsenic and antibiotic
2 resistance in heterotrophic aerobic bacteria from marine
3 hydrothermal vent fields
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5 Pedro Fariasa,b, Christophe Espírito Santoa, Rita Brancoa, Romeu Franciscoa, o
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6 Susana Santosa†, Lars Hansenc, Soren Sorensenc and Paula V. Moraisa,d# lo
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7 aIMAR-CMA, 3004-517 Coimbra, Portugal e
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8 b Instituto Piaget, Silves, Portugal ro
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9 cDepartment of Biology, Microbiology, University of Copenhagen, Denmark h
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10 dDepartment of Life Sciences, University of Coimbra, 3001-401 Coimbra, Portugal :/
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16 # Corresponding author: m
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17 Paula Vasconcelos Morais r

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18 IMAR-CMA-Department of Life Sciences ,
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19 University of Coimbra 1
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20 3001-401 Coimbra, Portugal. y

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21 [email protected] e
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22 †Present address: Department of Environmental Science (ENVS),University of Aarhus,
23 DK-4000 Roskilde, Denmark
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25
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26 Abstract
27 Microorganisms are responsible for multiple antibiotic resistances that have been
28 associated with resistance/tolerance to heavy-metals, with consequences to public
29 health. Many genes conferring these resistances are located on mobile genetic elements,
30 easily exchanged among phylogenetically distant bacteria. The objective of the present D
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31 work was to isolate arsenic, antimonite and antibiotic resistant strains and to determine n
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32 the existence of plasmids harboring antibiotic/arsenic/antimonite resistance traits in d
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33 phenotypically resistant strains, in a non-anthropogenically impacted environment. The
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34 hydrothermal Lucky Strike field in the Azores archipelago (North Atlantic, between m

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35 11N and 38N), at the Mid-Atlantic Ridge, protected under the OSPAR Convention was tp
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36 sampled as a metal rich pristine environment. A total of 35 strains from 8 different e
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37 species were isolated in the presence of arsenate, arsenite and antimonite. ACR3 and .a
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38 arsB genes were amplified from sediment's total DNA and 4 isolates also carried ACR3 .
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39 genes. Phenotypic multiple resistances were found in all strains, and 7 strains had /

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40 recoverable plasmids. Purified plasmids were sequenced by Illumina, assembled by N
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41 EDENA V3, and contigs annotation was performed using the Rapid Annotation using ve
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42 Subsystems Technology server. Resistance determinants to copper, zinc, cadmim, b
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43 cobalt and chromium as well as to the antibiotics β-lactams and fluoroquinolones were 7
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44 found in the 3 sequenced plasmids. Presence of genes coding for heavy metal resistance 0
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45 and antibiotic resistance in the same mobile element were found, anticipating the b
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46 possibility of horizontal gene transfer and distribution of theses resistances in the g
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47 bacterial population. t
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49 Running title: Resistances in bacteria from marine hydrothermal vents
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51 Keywords: Microbial diversity, Arsenic; Deep sea environment; Heavy metal
52 resistance; Antibiotic resistance
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55 D
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56 n
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57 Introduction
58 The ocean floor near the Azores is constituted in part by the Mid-Atlantic Ridge and
59 series of extensive hydrothermal vents. The largest hydrothermal vent field known until
60 now is located at 37º 18, 5' N, 32º 16, 5' W, average 1700 m depth and is named
61 “Lucky-Strike” (1). Here, 21 active hydrothermal vents are connected to the thermal D
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62 anomalies in the subsea floor at Mid Atlantic Ridge that is spread over 150 km2. The n
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63 field is primarily made of basalt but also spikes interest due to rich deposits of ores. d
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64 Temperatures of the field vary from 170º to 324ºC (2), diminishing with the distance
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65 from the vent outlet due to water circularization with the surrounding cold temperatures m

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66 characteristic of these depths (around 4ºC), therefore harboring a distinct microbial tp
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67 community. Furthermore, hydrothermal activity at Lucky Strike seems to have been e
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68 episodic over a long period of time (2). .a
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69 The nature of the bed rock and the geological events at Lucky-Strike vent sites result in .
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70 smaller concentrations of Rn and heavy metals (Cd, Hg, Cu, Pb, Zn, Fe and Ag) and /

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71 less acidic fluids, making it one of the least extreme environments, in a biological N
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72 perspective of the Mid Atlantic Ridge when compared to neighboring vent sites (3). ve
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73 Antibiotics in the environment may result from adaptive phenotypic and genotypic b
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74 responses of microbiota. Recently, antibiotic-producing bacteria have been found in 7
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75 deep sea environments. Strains belonging to the Streptomyces genus, a genus producer 0
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76 of two thirds of the clinical useful natural antibiotics, were reported as part of the b
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77 microbial community of hydrothermal vents (9) . In very stressful environments as this g
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78 one, where conditions change drastically in millimeters, antibiotic producers, able to kill t
79 competitors and get an ecological advantage, are not so unexpected (10). Resistance to
80 toxic compounds such as antibiotics and metals is commonly a result of resistance
81 determinants located on mobile genetic elements. The mobility of such resistances leads
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82 to their dissemination in a microbial population and to the occurrence of tandem arrays
83 of genetic linked resistance genes, commonly seen as co-resistance, in which genes
84 responsible for two or more resistances are adjacent to one another on a mobile genetic
85 element (5). This tandem arrays manifest many times in the form of multi-drug
86 resistance (MDR) systems (6). Resistance can also be related with cross-resistance, a D
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87 selection process in which heavy metals and antibiotic resistance mechanisms are n
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88 coupled physiologically (7). A resistance pool originated from heavy metal d
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89 contamination pressure will confer resistance to any other contaminant as long as these
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90 are connected through some form of co-selection (5, 8). m

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91 Many heavy-metals have been associated to deep sea hydrothermal vents. Arsenic (As), tp
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92 a well-known environmental toxic (9), is also a significant element in the formation of e
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93 hydrothermal ore deposits. .a
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94 Arsenic (As) and antimony (Sb) are usually co-present in the environment. Both have .
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95 similar geochemical properties and toxicity. The amount and speciation of arsenic in /

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96 hydrothermal deposits is a consequence of source rock composition, redox potential, N
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97 pH, and temperature of water, as well as the concentrations of other elements in solution ve
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98 such as iron and sulfur (10–13). In phosphate-poor systems, such as ocean b
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99 environments, arsenate resistance or detoxification pathways might be essential (14, 7
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100 15). In the case of antimony, little was yet explored in marine environments. 0
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101 Since arsenic is environmentally prevalent, microbes developed mechanisms to handle b
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102 arsenic and avoid its toxicity by several mechanisms, including extrusion mediated by g
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103 arsenite efflux pumps (ArsB and ACR3) These systems also confer resistance to Sb. t
104 Some studies demonstrated that microorganisms from extreme environments with
105 multiple heavy metal resistances, namely arsenate and antimonite, are also resistant to
106 antibiotics that, surprisingly, are not found in the environment from where the
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107 organisms originated (16, 17). Resistances are commonly coupled in genetic elements
108 that, despite being common on plasmids, can occur also in the chromosomes (18, 19).
109 The present work focused on determining the arsenic resistance and antibiotic resistance
110 profile of microorganisms isolated in pristine environments such as hydrothermal vents,
111 contributing to the knowledge of the extent of the microbial diversity that may D
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112 contribute to antibiotic resistance, with the outcome of understanding the arsenic- n
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113 antibiotic co-resistance in microorganisms. d
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115 Materials and Methods
116 Sample collection and isolation procedure
117 Water and sediment samples were collected from the Lucky Strike hydrothermal vent
118 area (37°17 N, 32°16 W, depth 1,695 m) in August/September 2009 during the
119 oceanographic mission EMEPC/Luso/2009 promoted by the Task Group for the D
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120 Extension of the Continental Shelf (EMEPC). Samples were recovered with the remote n
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121 operated vehicle, rated to 6000 m and operated by EMEPC (Figure 1). The procedure d
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122 for sample collection used a robotic harm with a suction device. The suction device is
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123 buried in the sea floor sediment and the sample is drawn. The samples were collected m

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124 from vents by using 0.75 liter titanium bottles and stored at -80ºC for posterior analysis tp
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125 studies. Sediment sample were broken apart, homogenized and partitioned. A quantity e
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126 of 9 mL of sediments from collected sample L09D23S3 was mixed with 10 mL of .a
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127 glycerol (30%). .
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128 /

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129 Bacterial strains and growth media N
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130 In order to create a collection of aerobic heterotrophic bacterial strains from the Lucky ve
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131 Strike vent site, aliquots from sediment samples were inoculated in R2A agar b
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132 (Reasoners 2A agar), in grams/litre, yeast extract 0.5, proteose peptone 0.5, casein 7
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133 hydrolysate 0.5, glucose 0.5, starch 0.5, sodium pyruvate 0.3, di-potassium hydrogen 0
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134 phosphate 0.3, magnesium sulfate anhydrous 0.024, agar-agar 15, (Oxoid, Thermo b
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135 Scientific, UK) prepared with sea water filtrate (SWF) collected from coastal seawater. g
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136 Each dilution was incubated at 15ºC for up to one month. During incubation, as colonies t
137 appeared, all those with different colony morphology were selected, purified by
138 repeated streaking and preserved at -80ºC in Luria Broth containing 15% glycerol.
139 Isolates were also recovered in the same R2A with SWF and in the presence of the
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140 following heavy metals 5 mM Arsenic (V); 2 mM Arsenic (III); 0.5 mM Antimony (III)
141 (Sigma-Aldrich, USA).
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143 DNA extraction and 16S rRNA gene amplification and phylogenetic analyses
144 Total DNA was isolated from hydrothermal vent field sediments using the E.Z.N.A. D
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145 Soil DNA kit D5626-01 (Omega Bio-Tek, USA), according to the manufacturer’s n
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146 instructions, and used for amplification of arsenic genetic determinants. DNA from each d
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147 isolate was obtained using standard freeze-thaw method (20), and used for typing,
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148 plasmid recovery, amplification of resistance genetic determinants and phylogenetic m

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149 identification. The extracted DNA was stored at -20ºC. Isolates were grouped by tp
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150 Random Amplification of Polymorphic DNA (RAPD) typing, using primer OPA-03 e
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151 (5’-AGT CAG CCA C-3’) (Operon Technologies, Inc. Alameda, California, USA). .a
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152 DNA profiles were grouped on the basis of visual similarities of the fragments analyzed .
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153 by electrophoresis in a 2% agarose gel in Tris-acetate EDTA (TAE) buffer stained with /

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154 ethidium bromide. N
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155 Amplification of the near full-length 16S rRNA gene sequence of representative strains ve
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156 selected from each RAPD groups, was performed by PCR with universal primers (21). b
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157 The obtained sequences were matched with the existing sequences using Blast 7
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158 programme in EzTaxon (22, 23). 0
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159 Sequences were aligned using Mega 5 package (24) for construction of phylogenetic b
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160 dendrograms using the Neighbor-Joining algorithm with the following parameters: g
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161 Jukes-Cantor correction model for nucleotides and 1000 bootstraps (25). In order to t
162 identify the isolates that belonged to the genus Sulfitobacter, the 16S rRNA gene
163 sequences were aligned with the sequences from all the type strains of all the species of
164 the genus, using ARB software package (sequences obtained from EZtaxon). A
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165 phylogenetic tree was constructed, using PhyML included in ARB software package the
166 isolates grouped with the type species in phylogenetic clusters and were identified
167 according to the species of the type strain of the cluster (Figure 1 supplementary).
168 Isolates heavy-metal resistance determination
169 Metal resistance of the isolates was determined by incubation at 22ºC for 48h, in D
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170 R2A+SWF agar with a concentration of the following heavy metals: AsO43- (5 mM), n
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171 NaSb(OH)4 (0.5 mM), CdSO4 (0.2 mM) or Na2CrO4 (0.5 mM) (Sigma-Aldrich); AsO33- ad
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172 (1 mM), C4H6O6U.2H2O (1 mM), ZnSO4 (1 mM), CoCl2.2H2O (0.5 mM) or f
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173 CuSO4.5H2O (1 mM) (Merck). m

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174 tp
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175 Antibiotic resistance testing of the isolates e
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176 Strains resistance to antibiotics was determined by evaluating bacterial growth in media .a
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177 containing antibiotics in increasing concentrations. A suspension of each bacterial strain .
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178 in 0.9% sodium chloride, with an optical density of 0.8, was plated and incubated at /

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179 22ºC for 48h. N
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180 For this assay, 12 antibiotics representing different classes and mechanism of action ve
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181 were selected. Two concentration thresholds of the antibiotics were used for b
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182 determining the bacterial antibiotic resistance, which were for each antibiotic, the 7
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183 critical concentration and the limit of solubility of the antibiotic in the medium (26). 0
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184 The antibiotics tested were: gentamycin (16-20 μg/mL), tetracycline (8-12 μg/mL), b
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185 kanamycin (16-20 μg/mL), vancomycin (20-30 μg/mL), rifamycin B (16-32 μg/mL), g
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186 penicillin G (16-20 μg/mL), naladixic acid (16-24 μg/mL), erythromycin (4-8 μg/mL), t
187 cephalosporin C (32-40 μg/mL), trimethoprim (8-16 μg/mL), ampicillin (16-20 μg/mL)
188 and polymyxin B (2-5 μg/mL) (Sigma-Aaldrich).
189
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190 Detection of arsenic genetic resistance
191 Amplification of the gene encoding the ACR3 efflux pump was performed by PCR with
192 the following degenerate primer set: dACR5F (5’-TGA TCT GGG TCA TGA TCT
193 TCC CVA TGM TGV-3’), dACR4R (5’-CGG CCA CGG CCA GYT CRA ARA ART
194 T-3’) (28). PCR reaction included an annealing temperature gradient with 10 cycles D
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195 from 57 to 52ºC (-0.5ºC per cycle) followed by 25 cycles at 52ºC. Each annealing step n
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196 lasted 45 seconds. For the amplification of the arsB encoding gene, the degenerate d
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197 primer set used was: darsB1F (5’-GGT GTG GAA CAT CGT CTG GAA YGC NAC-
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198 3’) and darsB1R (5’-CAG GCC GTA CAC CAC CAG RTA CAT NCC-3’) (28). The m

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199 PCR reaction was performed with a temperature of annealing of 55ºC. Positive PCR tp
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200 reactions were sequenced and the sequences were used in the phylogenetic analysis of e
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201 the clone library for comparison to the data obtained from the community DNA. .a
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202 Clone libraries were generated from the PCR amplicons of the ACR3 and arsB genes. .
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203 Clones were generated using pGEM-T Easy (Promega, USA) and transformed into /

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204 Escherichia coli DH5α. Using the blue/white screening test on LB media supplemented N
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205 with ampicillin, IPTG and X-Gal, clones were randomly selected, checked for inserts by ve
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206 PCR and sequenced (Macrogen Europe, Amsterdam, The Netherlands). Operational b
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207 taxonomic units (OTUs) were taxonomically identified using BLASTx (National Center 7
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208 for Biotechnology Information, Bethesda, MD, USA). Sequences with poor sequence 0
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209 alignment, no BLAST hits, or BLAST hits to genes that did not encode ACR3 or arsB b
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210 were removed from downstream analyses. OTUs and known sequences were aligned g
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211 using BioEdit tool. Mega 5 was used for construction of maximum likelihood t
212 phylogeny dendrograms using Dyhoff model, considering uniform rates and a partial
213 deletion (95% cutoff) on gaps/missing data.
214
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