Potentially Pathogenic Bacteria Isolated from Diverse Habitats in Spitsbergen, Svalbard
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Environmental Earth Sciences (2020) 79:109 https://doi.org/10.1007/s12665-020-8853-4 ORIGINAL ARTICLE Potentially pathogenic bacteria isolated from diverse habitats in Spitsbergen, Svalbard Diana C. Mogrovejo‑Arias1 · Florian H. H. Brill1 · Dirk Wagner2,3 Received: 26 April 2019 / Accepted: 11 February 2020 / Published online: 26 February 2020 © The Author(s) 2020 Abstract The Arctic ecosystem, a reservoir of genetic microbial diversity, represents a virtually unlimited source of microorganisms that could interact with human beings. Despite continuous exploration of Arctic habitats and description of their microbial communities, bacterial phenotypes commonly associated with pathogenicity, such as hemolytic activity, have rarely been reported. In this study, samples of snow, fresh and marine water, soil, and sediment from several habitats in the Arctic archi- pelago of Svalbard were collected during Summer, 2017. Bacterial isolates were obtained after incubation on oligotrophic media at diferent temperatures and their hemolytic potential was assessed on sheep blood agar plates. Partial (α) or true (β) hemolysis was observed in 32 out of 78 bacterial species. Genes expressing cytolytic compounds, such as hemolysins, likely increase the general ftness of the producing microorganisms and confer a competitive advantage over the availability of nutrients in natural habitats. In environmental species, the nutrient-acquisition function of these compounds presumably precedes their function as toxins for mammalian erythrocytes. However, in the light of global warming, the presence of hemolytic bacteria in Arctic environments highlights the possible risks associated with these microorganisms in the event of habitat melting/destruction, ecosystem transition, and re-colonization. Keywords Arctic · Svalbard · Hemolysins · Climate change · Pathogens · Virulence Introduction Bacteria, Archaea, and Eukarya domains (Wagner 2008; Kirby et al. 2012) and constitute major pools of genomic Even though cold environments have long been regarded diversity (Edwards 2015). The Arctic, spanning more than as extreme due to low temperatures, low water and nutri- 7 million km2 across regions of Alaska, Canada, Northern ent availability and high levels of UV radiation, they are Europe, Greenland, Iceland, and Russia (Kirby et al. 2012), actively inhabited by a variety of microorganisms (Maccario has great potential for research focused on the diversity of et al. 2015). Ecosystems such as tundra, permafrost, and microbial communities as well as their interactions with the glaciers are regarded as biomes, harboring species from the environment. However, this ecosystem is highly sensitive to perturbations and environmental changes (Davidson et al. 2011). Due to the Arctic’s low adaptative capacity (Gitay Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1266 5-020-8853-4) contains et al. 2002), the consequences of environmental disturbances supplementary material, which is available to authorized users. are usually amplifed (Doney et al. 2012; Kirby et al. 2012). As a result of climate change, the annual mean temperature * Diana C. Mogrovejo-Arias in the Arctic has increased twice as much as in other parts of [email protected]; [email protected] the world (Davidson et al. 2011; Kirby et al. 2012; Mateos- Rivera et al. 2016) altering a variety of habitats and raising 1 Dr. Brill + Partner GmbH Institut für Hygiene und concerns about diversity loss and the possible (re)emergence Mikrobiologie, Stiegstück 34, 22339 Hamburg, Germany of diseases (Kurane 2010; Davidson et al. 2011; Reich et al. 2 Section Geomicrobiology, GFZ German Research Center 2012; Altizer et al. 2013; Wang et al. 2016). for Geosciences, Telegrafenberg C-425, 14473 Potsdam, Stressful conditions brought about by a changing cli- Germany mate can heavily influence competition within bacte- 3 Institute of Geosciences, University of Potsdam, rial communities and might even play a key role in the Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany Vol.:(0123456789)1 3 109 Page 2 of 9 Environmental Earth Sciences (2020) 79:109 expression of virulence genes (Mekalanos 1992; Liver- implications of their presence in the context of public health, more 2003; D’Amico et al. 2006). Bacterial species can habitat alteration, and destruction caused by climate change. produce extracellular proteins, such as enterotoxins, coag- ulases and cytolysins, aimed at reducing the competition in their environment or at inficting damage in neighboring Materials and methods cells to gain access to their nutrients (Tomita and Kamio 1997; Madigan et al. 2012; Harwood et al. 2018). Among Sampling sites these toxins, hemolysins are of special interest. Since their activity is more easily observed in mammalian erythro- Samples of snow, glacier forefeld soil, sediment, glacier cytes, they are frequently referred to as hemolysins, even meltwater, pond, and marine water were collected in tripli- though red blood cells are rarely their only target in vivo cate from various sites in the High Arctic island of Spitsber- (Tomita and Kamio 1997). Secreted by a variety of organ- gen, Svalbard (Norwegian territory) during summer of 2017 isms, hemolysins are responsible for damaging cellular (Fig. 1; Table 1). During the sampling process, sterile nitrile membranes, causing cell lysis, destroying neighboring gloves were used and sampling was carried out facing the tissues, and are considered a major virulence determinant wind. For the samples of snow and soil, 5 cm of the surface in infection models of laboratory animals (Bhakdi et al. were discarded and a disinfected shovel or sterile spoon was 1994; Bayley 1997; Tomita and Kamio 1997; Madigan used to store the sample in sterile plastic bags (Whirl-Pak et al. 2012). bags; Nasco). Water and sediment samples were collected Bacteria can exhibit three diferent types of hemolytic using sterile collection bottles and a Niskin sampler. All activity (Buxton 2005): Beta (β) hemolysis, when the toxin samples were frozen at −20 °C until processing in the lab. causes the complete lysis of the red blood cells. Often referred to as true lysis, it manifests as a clear, transpar- Cultivation and isolation of bacteria ent area in the blood agar cultures; alpha (α) hemolysis, when lysis does not occur but the hemoglobin of the red Samples were thawed overnight at 4 °C prior to culture. All blood cells is reduced to methemoglobin and a brown/green samples were plated on oligotrophic media R2A (Oxoid) in colored area can be observed in blood agar cultures; gamma duplicate. Serial dilutions were used for all sample types and (γ) hemolysis, or non-hemolysis, when no damage to the the plates were incubated at 5, 17, and 37 °C for as long as cells is caused and no change in the agar plate is observed. 4 months, in sterile plastic bags to conserve the humidity of The distinction between α and β hemolysis is of relevance the agar media. Negative control plates were prepared with in some areas, such as the assessment of the safety of probi- sterile water frozen prior to culturing and thawed follow- otics (Jeon et al. 2017), but it is not generally made in other ing the same procedure as the samples. Two control plates settings because both types of hemolysis are usually consid- were streaked for each testing temperate and incubated in the ered a virulence determinant and one of the many pheno- same conditions and for the same duration as the samples. types studied to determine the clinical relevance of the spe- Liquid and enrichment cultures were not used. cies and/or strains (Bayley 1997; Ramachandran 2013). For Morphologically diferent colonies were selected as soon this reason, in this study, isolates with either α-hemolytic or as they became visible on the plates, and transferred succes- β-hemolytic activity are referred to as hemolytic. sively to new R2A plates to obtain pure cultures. Axenic cul- The expression of hemolysins has infrequently been tures were confrmed by Gram staining and DNA sequencing assessed in bacterial isolates from environmental samples as described below. (Bevivino et al. 2002; González-Rodríguez et al. 2007; Albarral et al. 2016) and much less is known about the 16S rRNA gene sequencing and phylogenetic hemolytic activity of Arctic species. As warming global characterization temperatures are favoring the geographic expansion of pathogens and increasing the risk of human exposure to Genomic DNA was extracted from the isolates using a bac- infectious disease (Revich et al. 2012; IPCC 2014), it is terial DNA extraction kit (OMEGA BIO-TEK) and the 16S increasingly important to study the presence of potential rRNA gene was amplifed via PCR using universal primers pathogens in Arctic environments and the expression of 8F (5′-AGA-GTT-TGA-TCC-TGG-CTC-AG-3′) and 1541R virulent phenotypes. (5′-AAG-GAG-GTG-ATC-CAG-CCG-CA-3′) (Tam et al For this purpose, bacteria from several habitats in Spitz- 2015) and ready-to-use PCR reaction mix (MangoMix, Bio- bergen, the biggest island of the Svalbard archipelago (Nor- line). Two negative controls using nuclease-free water were way) in the High Arctic were isolated and taxonomically prepared for each DNA extraction as well as PCR reaction. classifed. We tested whether or not the isolates displayed The PCR conditions were initial denaturation at 94 °C for hemolytic activity in an efort to highlight the possible 2 min, followed by 30 cycles of denaturation at 94 °C for 1 3 Environmental Earth