Occurrence of integrons and antibiotic resistance genes in cryoconite and ice of Svalbard, Greenland, and the Caucasus glaciers

Journal Pre-proof Occurrence of integrons and antibiotic resistance genes in cryoconite and ice of Svalbard, Greenland, and the Caucasus glaciers

Nicoletta Makowska, Krzysztof Zawierucha, Paulina Nadobna, Kinga Piątek-Bajan, Anna Krajewska, Jagoda Szwedyk, Patryk Iwasieczko, Joanna Mokracka, Ryszard Koczura PII:

S0048-9697(20)30532-5

DOI:

https://doi.org/10.1016/j.scitotenv.2020.137022

Reference:

STOTEN 137022

To appear in:

Science of the Total Environment

Received date:

28 September 2019

Revised date:

29 January 2020

Accepted date:

29 January 2020

Please cite this article as: N. Makowska, K. Zawierucha, P. Nadobna, et al., Occurrence of integrons and antibiotic resistance genes in cryoconite and ice of Svalbard, Greenland, and the Caucasus glaciers, Science of the Total Environment (2018), https://doi.org/10.1016/ j.scitotenv.2020.137022

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© 2018 Published by Elsevier.

Journal Pre-proof Occurrence of integrons and antibiotic resistance genes in cryoconite and ice of Svalbard, Greenland, and the Caucasus glaciers Nicoletta Makowskaa,, Krzysztof Zawieruchab, Paulina Nadobnaa, Kinga Piątek-Bajana, Anna Krajewskaa, Jagoda Szwedyka, Patryk Iwasieczkoa, Joanna Mokrackaa, Ryszard Koczuraa*

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Department of Microbiology, Faculty of Biology, Adam Mickiewicz University in Poznań,

Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz

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Poland

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University in Poznań, Poland

* Corresponding author: Department of Microbiology, Faculty of Biology, Adam Mickiewicz University in Poznań, ul. Uniwersytetu Poznańskiego 6, 61-614 Poznań, Poland. E-mail address: [email protected]

Journal Pre-proof Abstract The prevalence of integrons and antibiotic resistance genes (ARGs) is a serious threat for public health in the new millennium. Although commonly detected in sites affected by strong anthropogenic pressure, in remote areas their occurrence, dissemination, and transfer to other ecosystems is poorly recognized. Remote sites are considered as a benchmark for human-induced contamination on Earth. For years glaciers were considered pristine, now they are regarded as

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reservoirs of contaminants, thus studies on contamination of glaciers, which may be released to

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other ecosystems, are highly needed. Therefore, in this study we evaluated the occurrence and

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frequency of clinically relevant ARGs and resistance integrons in the genomes of culturable

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bacteria and class 1 integron-integrase gene copy number in the metagenome of cryoconite, ice and supraglacial gravel collected on two Arctic (South-West Greenland and Svalbard) and two

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High Mountain (the Caucasus) glaciers. Altogether, 36 strains with intI1 integron-integrase gene

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were isolated. Presence of class 1 integron-integrase gene was also recorded in metagenomic DNA from all sampling localities. The mean values of relative abundance of intI1 gene varied

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among samples and ranged from 0.7% in cryoconite from Adishi Glacier (the Caucasus) to

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16.3% in cryoconite from Greenland. Moreover, antibiotic-resistant strains were isolated from all regions. Genes conferring resistance to β-lactams (blaSHV, blaTEM, blaOXA, blaCMY), fluoroquinolones (qepA, qnrC), and chloramphenicol (cat, cmr) were detected in the genomes of bacterial isolates.

Keywords: Antibiotic-resistant bacteria; Arctic; Contamination; Horizontal gene transfer (HGT); Polar and alpine regions; Supraglacial ecosystems

Journal Pre-proof Introduction

Pristine and remote sites are considered a benchmark for human-induced contamination of Earth. Non-industrialized areas, such as polar and high mountains, free of direct and permanent delivery of contaminants, are target places for tracking human impact in natural ecosystems (Ji et al., 2019; Łokas et al., 2018; Marcelli and Maggi, 2017; McCann et al., 2019; Rafiq et al., 2019;

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Segawa et al., 2013; Subhavana et al., 2019; Tan et al., 2018). Since the last decade, the

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perception of glaciers as sterile have evolved into a view on glaciers as environments inhabited

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by diverse cold-adapted organisms that interact with ice and form extreme ecosystems (Cook et

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al., 2016; Hodson et al., 2008; Kohshima, 1987; Wharton et al., 1985). Owing to the unique climatic conditions and high biological activity, glaciers and ice sheets are currently considered

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the coldest biome on Earth (Anesio and Laybourn-Parry, 2012). Glacier surface (so called

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supraglacial zone) is the most biologically active part of glacial ecosystems, where diverse microbial photoautotrophic communities support food web, including other bacteria, fungi and

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invertebrates (Hodson et al., 2008; Mueller et al., 2001; Stibal et al., 2015; Zawierucha et al.,

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2018). The most prokaryote-diverse and species-rich habitats on glaciers are cryoconite holes: water-filled reservoirs with dark sediments (cryoconite) on the bottom. These sophisticated ecosystems host dynamic microbiomes composed of diverse functional groups changing in time and space (Darcy et al., 2018; Franzetti et al., 2017; Gokul et al., 2016; Pittino et al., 2018). Due to human activity and development of civilization, even remote glacial ecosystems cannot be longer considered as pristine, as they are affected by anthropogenic pressure (Hong et al., 1994; McConnell et al., 2018). Therefore, nowadays the state of a glacier is considered not only as a valuable indicator of climate changes (Thompson et al., 2011; Uemura et al., 2018), but also can be used to assess environmental impact of human activity (Carey, 2007; Łokas et al.,

Journal Pre-proof 2018; McConnell et al., 2018). Contemporary studies on supraglacial ecosystems have brought various, often unexpected results showing that glaciers are contaminated by persistent organic pollutants, heavy metals, insecticides, artificial radionuclides, and black carbon (Ferrario et al., 2017; Hauptmann et al., 2017; Hodson, 2014; Łokas et al., 2018, 2016; Pittino et al., 2018). Among a number of papers devoted to microbial communities and biogeochemistry of supraglacial ecosystems (Cook et al., 2016; Holland et al., 2019; Porazinska et al., 2004; Segawa

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et al., 2017, 2014; Stibal et al., 2015; Zawierucha et al., 2018), the potential threat for human

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health related with glacial bacteria and microbial contamination released downstream from

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glaciers has not been well studied (Edwards, 2015). One of such issues is contamination of

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various ecosystems by bacteria harboring integrons and antibiotic resistance genes (ARGs) (Segawa et al. 2013).

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The emergence and spread of antibiotic resistance among bacteria is the most striking

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example of evolution that has been observed in bacteria over the past several decades (Davies and Davies, 2010; Fair and Tor, 2014). Intensive and uncontrolled use of antibiotics, not only

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therapeutically in human medicine, but also in farming and agriculture, have provoked the spread

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of resistant bacteria and resistance genes by selective pressure and horizontal gene transfer (HGT) between strains of the same or different species (Van Boeckel et al., 2014; BengtssonPalme et al., 2018). The antibiotic-resistant bacteria, integrons and ARGs in the environment are considered biotic pollution and ecological problem (Young, 1993; Martinez, 2009; Gillings, 2017). Integrons are DNA fragments consisting of an integron-integrase gene, a primary recombination site, and a promoter that directs transcription of the integrated genes. They are capable of capturing gene cassettes coding mostly for antibiotic resistance, but also for transport proteins, estherases, phosphatases, transposases, and proteins of unknown function (Stalder et al., 2012). Three classes of integrons, with class 1 being the most ubiquitous, are responsible for

Journal Pre-proof spreading multidrug resistance among bacteria (Cambray et al., 2010). It is essential that, as integrons are embedded in mobile genetic elements like transposons and plasmids, they can be transferred within a microbial population through HGT. Integrons, especially those of class 1, are widely distributed among clinical strains and bacteria isolated from a variety of water environments with different degree of anthropogenic pressure. In the environment, class 1 integron-integrase genes are proposed to serve not only a marker of antibiotic resistance level, but

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also as a proxy for anthropogenic pollution (Gillings et al., 2015; 2017). It is well established that

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class 1 integrons are responsible mainly for the emergence and spread of multidrug antibiotic

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resistance among bacteria (Cambray et al., 2010; Stokes and Gillings, 2011; Gillings, 2014;

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2017). The antibiotic-resistant bacteria, integrons and ARGs have been recently found in many environments, but their distribution and abundance in polar and high alpine regions still remain

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largely unknown (McCann et al., 2019).

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Integrons and ARGs have been reported even in remote areas such as permafrost and soils of High Arctic (McCann et al., 2019). Most likely, they were delivered to remote sites along with

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migratory birds as well as the effect of increasing human activity (Segawa et al., 2013; Tan et al.,

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2018). Despite the fact that glaciers and ice sheets cover 10% of lands on Earth, the distribution of ARGs and integrons on glaciers remains poorly recognized. Segawa et al. (2013) have conducted studies on snow and glacial ice revealing various groups of ARGs and their potential origin. In this study, we focused on integrons and ARGs in cryoconite holes – glacial biodiversity hotspots being also bioreactors producing organic matter, as well as in ice and supraglacial gravel, all together melting and being flushed, then transported to downstream systems from the melting glacier surface (Cameron et al., 2017; Dubnick et al., 2017). Since cryoconite holes have not been extensively studied with regard to antibiotic resistance determinants, the main aim of

Journal Pre-proof our study was to evaluate the occurrence of clinically relevant ARGs and resistance integrons in the genomes of culturable bacteria and class 1 integron-integrase gene in the metagenome of cryoconite and ice collected in remote places, on two Arctic (South-West Greenland and Svalbard) and two High Mountain (the Caucasus) glaciers. Identification and understanding mechanisms of ARG dissemination, especially in natural environments is critical for developing effective strategies for tracking antibiotic resistance. Such studies seem to be crucial for glaciers

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which are a source of water that maintains downstream ecosystems, as well as a source of

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freshwater for domestic use for more than one billion people (Bradley, 2006; Mark et al., 2015;

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Milner et al., 2017).

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2. Material and methods

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2.1. Sample collection

Samples were collected from glaciers located in alpine and Arctic regions (Figure 1).

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Sampling sites were chosen to encompass diversity of glaciers and regions. Glaciers were

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characterized by different latitudes, altitudes, daily and annual temperature, and light fluctuations, representing different thermal regimes (cold base, temperate, polythermal) and various supraglacial topography, which may influence bacterial abundance and diversity. Cryoconite samples were collected from Adishi (the Caucasus, six samples) in 2014, South-West Greenland (area of Kangerlussuaq, eight samples) in 2015, and from Spitsbergen (Longyearbreen, Svalbard Archipelago, three samples) in 2013. Additionally, four samples (two from ice and two from gravel from water cavities) were collected from Chalaati (also known as Chalaadi) Glacier (the Caucasus). For detailed description of glaciers see Supplementary data 1. The samples were collected aseptically to sterile Falcon tubes. For each sample, independent sterile gloves were

Journal Pre-proof used. Samples were collected in each locality always in a manner to avoid self-contamination of samples – when a cryoconite hole was passed during hiking, it was not sampled due to the probability of contamination from boots, etc. Immediately after collection, the samples were placed in an icebox packed with ice and transported to the laboratory at the Adam Mickiewicz University in Poznań, Poland and maintained at -20°.

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2.2. Bacterial cultures

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Total number of culturable heterotrophic bacteria was determined by pour-plate method on Brain Heart Infusion agar (bioMérieux). Bacteria resistant to sulfonamides, glycopeptides,

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tetracyclines, fluoroquinolones, β-lactams or chloramphenicol were selected on BHI agar

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supplemented with sulfamethoxazole (350 μg/ml), vancomycin (4 μg/ml), tetracycline (20

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μg/ml), ciprofloxacin (2 μg/ml), cefotaxime (2 μg/ml), oxacillin (6 μg/ml), or chloramphenicol (10 μg/ml), respectively. Series of logarithmic dilutions of cryoconite samples were prepared in

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sterile saline, inoculated onto the media and the plates were then incubated at 8°C for 21 days.

(vol/vol) glycerol.

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The bacterial isolates were maintained at -80°C in Brain Heart Infusion broth containing 50%

2.3. DNA template preparation

For genomic DNA templates from bacterial isolates, bacterial colonies were suspended in sterile H2O and lysed by heating (95°C for 2 min) (Grape et al., 2005). The lysates were stored at -20°C.

Journal Pre-proof For isolation of metagenomic DNA, 1 gram (wet weight) of each sample was centrifuged and total DNA was extracted from the pellet by heat lysis and Genomic Mini kit (A&A Biotechnology). The quality of DNA was assessed spectrophotometrically (A260/280 ratio 1.72.0) and by agarose gel electrophoresis.

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2.4. Detection of integron-integrase genes and analysis of the variable regions of integrons

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The presence of class 1, 2 and 3 integrons among culturable bacteria was determined by

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multiplex PCR assay for the detection of intI1, intI2 and intI3 integron-integrase genes. Integronharboring bacteria samples from Caucasus and Greenland were pre-selected on BHI agar

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supplemented with streptomycin (40 μg/ml), an antimicrobial to which ca. 99% of integron-

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bearing strain are resistant (Koczura et al., 2012), whereas in Spitsbergen samples the integrons

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were identified among all heterotrophic bacteria with the use of multiplex PCR assay according

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Lévesque et al. (1995).

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to Dillon et al. (2005). The variable regions of the integrons were amplified according to

2.5. Identification of bacteria by sequencing of 16S rRNA gene

Integron-bearing bacteria were identified by partial sequencing of the 16S rRNA gene. A 456-bp-long fragment of 16S rRNA gene was PCR-amplified with primers 343F and 798R according to Nossa et al. (2010). The partial 16S rRNA gene sequences were subjected to BLASTn sequence similarity search (Altschul et al., 1990).

2.6. Detection of ARGs in bacterial isolates

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To determine the frequency and type of genes determining resistance among sulfamethoxazole-, vancomycin-, tetracycline-, ciprofloxacin-, cefotaxime-, chloramphenicoland oxacillin-resistant isolates, a series of PCR assays were carried out. Amplification of two genes conferring resistance to sulfonamides (sul1, sul2) was determined among 18 sulfamethoxazole-resistant isolates, vanA gene conferring resistance to vancomycin among 88

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vancomycin-resistant isolates, 14 tetracycline-resistance genes: tet(A), tet(B), tet(C), tet(D),

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tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), tet(AP), tet(Q), and tet(X) among 31

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tetracycline-resistant isolates, six genes determining fluoroquinolone resistance (qnrA, qnrB, qnrC, qnrS, qnrD, and qepA) among 40 ciprofloxacin-resistant isolates, eight genes determining

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β-lactam resistance (blaCTX-M, blaSHV, blaTEM, blaGES, blaVEB, blaOXA, blaCMY, and blaDHA) among

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138 cefotaxime- and 187 oxacillin-resistant isolates, cat and cmr genes among 68

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chloramphenicol-resistant isolates, and mecA gene conferring resistance to methicillin among 187 oxacillin-resistant isolates. The PCR conditions comprised initial denaturation at 94°C, 5

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min, followed by a 30 cycles of denaturation 94°C for 1 min, annealing (varied; 45-64°C) for 45

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s, extension 72°C for 90 s, with a final extension of 72°C for 7 min. Primer sequences are shown in Supplementary Table S1.

All PCR amplifications were done in a C1000 Touch thermal cycler (Bio-Rad). The amplicons were separated in 1.5% agarose gel. Molecular weight of PCR products was determined with GelCompar II 3.5 (Applied Maths). The amplicons were purified and sequenced in a 3130xl Genetic Analyzer (Applied Biosystems). Variants of ARGs were confirmed upon comparing the sequences with available GenBank sequence data by using ClustalW and the neighbor-joining method.

Journal Pre-proof 2.7. Quantification of class 1 integron-integrase gene in cryoconite metagenomes

Quantitative real-time PCR (qPCR) was used for determination of the copy number and relative abundance of class 1 integron-integrase gene, intI1 in the total DNA from the samples. The sequences of primers targeting intI1 have been recommended by Barraud et al. (2010). The gene quantities were normalized to the 16S rRNA gene copy number and the relative abundance

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values were expressed as percentages and calculated using the formula: [(intI/16S)×4×100], with

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four being the average number of copies of the gene encoding 16S rRNA per bacterial cell,

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according to the ribosomal RNA database (Stalder et al., 2012). Standard curves for qPCR were constructed from serial dilutions of purified PCR products ranging from 101 to 108 gene copies

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per μl. Reactions were carried out in 96-well plates in a final volume of 20 μl with Luminaris

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Color HiGreen qPCR Master Mix (Thermo Scientific). Specificity of amplification was

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determined by melt curve analysis and gel electrophoresis. Reactions were carried out in a

3. Results

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(Bio-Rad).

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CFX96 Touch Real-Time PCR Detection System and analyzed with CFX Manager 3.1 software

3.1. Isolation of bacteria and detection of integrons in bacterial isolates

The highest average total number of culturable heterotrophic bacteria in the samples was found in cryoconite from Adishi Glacier (8.4×102 CFU/g), then in cryoconite from Spitsbergen (2.5×102 CFU/g), from gravel on Chalaati Glacier (1.0×102 CFU/g), followed by cryoconite from

Journal Pre-proof Greenland (5.4×101 CFU/g), and the lowest in the samples of ice from Chalaati in the Caucasus (1.1×101 CFU/g) (Figure 2). Screening for integron-integrase genes showed the presence of intI1 gene in the genomes of 36 isolates: 22 of them originated from the samples from Spitsbergen, 10 from Adishi and Chalaati glaciers, and four from Greenland. Samples of ice did not contain bacteria with intI1.

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We did not find class 2 and 3 integrons in any sample.

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3.2. Analysis of the variable regions of integrons

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The analysis of the variable region of integrons yielded amplicons for eight isolates (two from Greenland and 6 from Spitsbergen). Integrons present in the bacteria from Greenland had

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dfrA12-orfF-aadA2 cassette array containing aadA2 gene coding for aminoglycoside

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adenylyltransferase conferring resistance to streptomycin and spectinomycin, dfrA12 gene encoding dihydrofolate reductase conferring resistance to trimethoprim, and orfF coding for an

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unknown protein. One strain from Spitsbergen had a variable region of 180 bp, which indicates a

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lack of integrated gene cassettes, whereas the other had variable regions ranging from 0.7 to 2.5 kbp. In the variable region of intI1-positive strains from Spitsbergen, genes directly determining resistance to antibiotics were not detected, instead genes encoding other proteins, such as chitinbinding protein, LytD beta-N-acetylglucosaminidase, two-component response regulator, sensor histidine kinase, chemotaxis proteins, and proteins of unknown functions were found. The bacteria with established variable regions of integrons were identified as Enterobacter sp. from samples of Greenland and Bacillus licheniformis, Caulobacter crescentus, and Brevundimonas subvibrioides

from

Spitsbergen.

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3.3. Quantification of class 1 integron-integrase gene in cryoconite samples by using qPCR

The average number of 16S rDNA gene copies ranged between sampling sites: it amounted to 1.9×107/g in Georgia (2.4×107/g in cryoconite from Adishi, 1.6×107/g in ice and 7.2×106/g in gravel from Chalaati Glacier) (Makowska et al., 2016), 1.4×107/g in cryoconite from

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Greenland (Zawierucha et al., 2018), and 8.9×106/g in cryoconite from Spitsbergen. The

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occurrence of class 1 integron-integrase gene was recorded in all metagenome samples, with the

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exception of one sample from a cryoconite from Adishi Glacier. The average intI1 copy number per gram was the highest in cryoconite from Greenland (2.3×105), then from Chalaati in ice

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(8.4×104) and gravel (6.0×104), in cryoconite from Adishi (4.4×104), and the lowest in cryoconite

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from Longyearbreen (2.9×103). To determine the relative abundance of class 1 integron-integrase

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gene in cryoconite metagenomes, the copy number of the intI1 genes in each site was normalized to the copy number of bacterial 16S rRNA gene (Figure 3). The highest mean value of relative

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abundance of intI1 genes was observed in Greenland (16.3%), then in the Caucasus (12.1% for

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gravel and 2.2% for ice from Chalaati Glacier), in Spitsbergen (1.1%), and the lowest in cryoconite from Adishi (0.7%). The relative abundance of class 1 integron-integrase in the metagenome ranged from 1.6% to 30.3% in Greenland, from 0% (Adishi) to 23.8% (Chalaati) in the Caucasus, and from 0.03% to 3.2% in Spitsbergen (Figure 3).

3.4. PCR detection of ARGs

Antibiotic-resistant bacteria were isolated from each region. Among 570 resistant isolates, 109 cultured from cryoconite samples in Greenland were resistant to β-lactams, 392 isolates from

Journal Pre-proof Georgia samples were resistant to sulfonamides (18 strains), glycopeptides (72), tetracyclines (21), fluoroquinolones (36), β-lactams (177) and chloramphenicol (68). Among 69 isolates from Spitsbergen, there were 16 glycopeptide-, 10 tetracycline-, four fluoroquinolone-, and 39 βlactam-resistant isolates. Among isolates from Greenland, blaCMY gene was found in 4.6% of βlactam-resistant strains. Among antibiotic-resistant isolates from Georgia, qnrC gene was found among 8.3% of ciprofloxacin-resistant strains, blaOXA among 4.6% of oxacillin-resistant isolates,

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cmr and cat among 4.4%, 2.9%, respectively, of chloramphenicol-resistant isolates, qepA among

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2.8% of ciprofloxacin-resistant isolates, and blaSHV and blaTEM among 2.2% (each) of cefotaxime-

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resistant strains (Figure 4). Among isolates from cryoconite in Spitsbergen, blaOXA gene was

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found in 1.2% of oxacillin-resistant strains.

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Discussion

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Regardless of geographical region, type and setting of glaciers we detected bacteria harboring

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integrons considered to be biotic pollution in cryoconite. We determined the presence of integrons and genes conferring resistance to fluoroquinolones, β-lactams and chloramphenicol in the genomes of bacteria and class 1 integron-integrase gene in the metagenomic DNA isolated from, at first glance, pristine environments: Alpine (the Caucasus) and Arctic (South-West Greenland, Spitsbergen) glaciers. Water and hydrological systems in the supraglacial zone, as well as wind, are main drivers in passive dispersal of glacial organisms and finally shape their communities (Franzetti et al., 2017; Liu et al., 2017; Zawierucha et al., 2019), which also may affect the emergence of ARGs on glaciers. Transport of integron- and ARG-harboring bacteria by wind, tourists or feces

Journal Pre-proof of mammals or migrating birds (Figure 5) may trigger appearance of antibiotic resistance in glacier microbiome (Segawa et al. 2013). Mueller et al. (2001) suggested inter-hole water sediment mixing as a common process in Arctic cryoconite holes. A system of streams, cryoconite holes, water mixing due to ice melting or rain, and diverse glacier topography (see Supplementary data 1), may influence the dispersal of bacteria on glacier surface and facilitate HGT, which in turn promotes the spread of antibiotic resistance.

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Integrons embedded in mobile genetic elements are responsible for the spread of

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multidrug resistance among bacteria by HGT. At first, they were detected predominantly in

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clinical strains, whereas many recent studies indicated their widespread presence in

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environmental bacteria (Cambray et al., 2010; Lupo et al., 2012; Gillings et al., 2014; 2015). Their spread is mainly related to selection pressure caused by the presence of antibiotics, heavy

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metals and other pollutions which facilitate transfer and acquisition of resistance (Gillings, 2017).

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In areas of low anthropopressure, the presence of integrons is also associated with the action of natural secondary metabolites of microorganisms, such as antibiotics. On glaciers, selection

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pressure comprises permanently low temperature, high doses of UV irradiation, daily freezing as

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well as most probably heavy metals and radionuclides commonly accumulated and stored in cryoconite holes, all of which may enhance HGT. The total number of culturable heterotrophic bacteria was four to six orders lower than the estimated number of bacteria calculated on the basis of bacterial 16S rRNA gene copies number (Figure 2, Figure 3). It indicates that, as in the case of many environments, most of microorganisms originating from the polar or high mountain regions are non-culturable under laboratory conditions (Amann et al., 1995). The relative abundance of intI1 gene, calculated by normalization to the copy number of 16S rRNA gene, was the highest in Greenland (16.3%) compared to Georgia (12.1% for gravel and 2.2% for ice from Chalaati, 0.7% for cryoconite from

Journal Pre-proof Adishi) and Spitsbergen (1.1%). Literature data lack information on the abundance of class 1 integron-integrase gene collected by metagenomic approach in glacial habitats. In aquatic habitats, like river waters, the intI1 relative abundance can vary from 0.04% in sites not impacted by human activity (Stalder et al., 2012) to 0.65% in sites located downstream the discharge of wastewater treatment plant effluents (Koczura et al., 2016; Makowska et al., 2016) and 4% in sites under intense anthropogenic pressure (Stalder et al., 2012) (Figure 6).

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The origin of integrons on glaciers might be the effect of direct delivery of guano on ice

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surface by migratory birds (Figure 5) which feed on farmlands in Europe or North America,

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reindeers crossing through small glaciers, cooling muskox on the ice, and other vertebrates

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accidentally visiting glaciers (Rosvold, 2016) or human activity (field camps). Feces of vertebrates (including humans) may contain integron-bearing and antibiotic resistant bacteria.

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This may lead to the spread of ARGs in the environment through HGT and integrons (Gillings et

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al., 2015, 2017), which on glaciers may be facilitated by systems of streams and water mixing. Although it seems extraordinary, storing of fecal material on glaciers by climbers was estimated

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in Alaska and constitutes a serious problem during melting, especially for natural downstream

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ecosystems (Goodwin et al., 2012). Such depositions of wastes by tourists undoubtedly may be a source of integrons and ARGs. Such a high abundance of intI1 gene on Greenland may be related to strong human activity on ice in this area (mainly touristic and scientific camps). Integrons of strains isolated from cryoconite samples collected in Greenland contained gene cassettes conferring resistance to aminoglycosides and trimethoprim were detected. Previous studies have shown that integrons accumulating new gene cassettes were present also in commensal bacteria of wild animals in remote areas. Such evidence has been shown for the class 1 integron in E. coli strain isolated from a wild reindeer (Rangifer tarandus tarandus) in Norwegian mountains. Bacteria from the reindeer harbored a gene cassette determining resistance

Journal Pre-proof to aminoglycosides (ant(3’')-Ia), and acquired a new one encoding a protein of unknown function, previously described in Xanthomonas sp. (Sunde, 2005). Similar gene cassettes have been noted also in Gram-negative bacteria isolated from areas of low anthropogenic pressure, like water bodies in national parks (Koczura et al., 2014) and a boar living in the neighborhood of national parks (Mokracka et al., 2012). Wild animals accidentally visiting glaciers and perennial snow (Rosvold, 2016) may be a source of clinical ARGs. Another possible source of integrons in

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Arctic/Antarctic regions is human activity. Power et al. (2016) have found that 21% of

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Escherichia coli strains isolated from marine sediments and seawater samples collected near the

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discharge point of the wastewater from Davis Station, Antarctica, carried a class 1 integrons, with

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gene cassettes coding for aminoglycoside and trimethoprim resistance. The phenomenon of transferring resistance from clinical to glacier bacteria poses a danger due to generation of new

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resistance phenotypes in the crucial freshwater ecosystem on Earth – glaciers and snow patches.

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Class 1 integrons of bacteria from Spitsbergen did not contain gene cassettes determining resistance to antibiotics. Instead, they carried genes coding for proteins rather of physiological

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significance that may increase the adaptation of bacteria to the extreme environment (Gillings,

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2017). Within the variable parts of class 1 integrons, genes encoding LytD beta-Nacetylglucosaminidase, chitin binding protein, short chain dehydrogenase, two-component response regulator, sensor histidine kinase, and hypothetical proteins were identified. Most of them may increase adaptation to the environment, yet some might have impact on bacterial resistance. Hypothetical proteins which are proposed to be of phage origin, in some cases may constitute as much as 15% prokaryotic genome and have versatile presumptive functions (Dziewit

et

al.

2013).

One of the gene cassetes found in this study coded for a chitin-binding protein (CBP). This protein exhibits an affinity toward chitin and chitooligosaccharide which can be utilized as a

Journal Pre-proof source of carbon and nitrogen. Such adaptation might be especially important on glaciers, where availability of nutrients is poor or they are highly diluted. It is believed that CBP, previously described in clinical bacteria, plays a critical role in adhering to the chitinous surfaces of host cells and (Vaaje-Kolstad et al., 2005; Bhattacharya et al., 2007; Tran et al., 2011). The source of chitin in cryoconite holes might be rigid structures (buccal apparatus, claws, trophi) of invertebrates: water bears (Tardigrada) and rotifers (Rotifera) (Klausseman et al., 1990; Guidetti

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et al., 2015), which commonly inhabit cryoconite holes in Svalbard archipelago, with high

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abundance on Longyearbreen (Zawierucha et al., 2019). Another source may constitute yeasts,

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common in Arctic cryoconite holes (Singh & Singh, 2012).

Ushida et al. (2010) studied the occurrence of ARGs in cryoconite from the northern

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hemisphere – Alaska and Central Asia, and from the southern hemisphere – James Ross Island

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(Antarctica). The authors identified tetracycline resistance genes, mecA, ampC, blaIMP, blaSHV,

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blaOXA, blaCTX-M, blaCMY, and cmrA. They were detected only in samples from glaciers of the northern hemisphere (Ushida et al. 2010). In our study, we also demonstrated the presence of

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genes conferring resistance to β-lactams and chloramphenicol. Segawa et al. (2013) conducted

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research on the occurrence of ARGs in places of low anthropopressure (perennial snow patches and glacier ice) located in the Arctic, Antarctica, Central Asia, North and South America, and in Africa. The authors found the same genes encoding resistance to β-lactams and chloramphenicol (blaCMY, blaOXA, cat and cmr) as we found in this study. Although Segawa et al. (2013) studied material from glacial and snow ecosystems, they did not investigate glacial “bioreactors” like cryoconite holes. Our studies provided also data from the Caucasus, important mountain area covered by glaciers which have not been investigated so far with regard to integrons and ARGs. In our study, we found genes conferring resistance to fluoroquinolones (qepA and qnrC), βlactams (blaSHV, blaTEM, blaOXA, and blaCMY) and chloramphenicol (cat and cmr) in the genomes

Journal Pre-proof of bacteria isolated from cryoconite collected in all sampling regions. Segawa et al. (2013) showed also the presence of genes conferring resistance to tetracyclines: tet(D), tet(G), tet(L), tet(M), tet(O), tet(S), tet(X), tet(W). We did not detect any of them. Ball et al. (2014) conducted studies on a glacier in Venezuela and showed the presence of bacteria resistant to chloramphenicol, which is consistent with the results of our work. In previous studies, intestinal bacteria with ARGs encoding resistance to β-lactams (ampC) and bacteria resistant to ampicillin,

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sulfamethoxazole, trimethoprim, chloramphenicol, and tetracyclines were detected in cloacal

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samples and feces of birds from the Arctic region, which strongly suggests the spread of resistant

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bacteria through migratory birds (Sjölund et al., 2008; Literak et al. 2014).

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Although antibiotic resistance can be intrinsic, the source of resistance genes on glaciers can be introduced through human or animals (human activity on glaciers, feces of migratory

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birds). Bacteria harbouring these genes can also be transported to glaciers with wind (Segawa et

et al., 2017).

na

al., 2013). Therefore, melting glaciers are becoming secondary sources of contaminants (Baccolo

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Summing up, we showed that cryoconite hole can be inhabited by bacteria carrying

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resistance integrons and clinically relevant ARBs. The relative abundance of class 1 integrons among bacteria can reach even 30%. Therefore, we should carefully monitor water originated from glaciers as a potential source of this hazard – antibiotic-resistant bacteria, integrons and ARGs. Special attention should be given to areas where water from glaciers is the main source of freshwater in farms and domestic use.

Declaration of competing interest

The authors declare no competing interests.

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Acknowledgements Material in Svalbard was collected within Polish Ministry of Science and Higher Education Diamond Grant programme no. DIA 2011035241 and from Greenland within National Science Center grant no. NCN 2013/11/N/NZ8/00597 to K.Z. Authors would like to thanks Mr Maciej Wilk and PhD Adam Nawrot (from Science Fundation) for logistical support during

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sampling.

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141–150. https://doi.org/10.1007/s10201-017-0528-9

Journal Pre-proof Figure 1. Map indicating sampling sites.

Figure 2. Total number of heterotrophic bacteria in samples collected from glaciers in the Caucasus (C), Greenland (G) and Spitsbergen (S). C Cryoconite, G Gravel, I Ice.

Figure 3. The copy number of 16S rRNA and intI1 genes and relative abundance of intI1 genes

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in the metagenome of samples from the Caucasus, Greenland and Spitsbergen.

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Figure 4. Frequency of ARGs in antibiotic-resistant bacteria isolated from cryoconite collected on Adishi and Chalaati Glacier (the Caucasus). Data for Spitsbergen and Greenland are not

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presented due to low abundance: on Greenland blaCMY gene was found in β-lactams-resistant

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strains and on Spitsbergen blaOXA gene was found in oxacillin-resistant strains.

Figure 5. A) Guano of reindeer on Longyearbreen (Svalbard) in cryoconite hole observed in 2016, B-D)

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Guano of geese on edge of the South-West Greenland Ice Sheet in 2015.

Figure 6. Relative abundance of class 1 integron integrase gene in the metagenome of samples from the Caucasus, Greenland and Spitsbergen (gray bars) and samples from diverse water environments (black bars; data upon Stalder et al., 2012).

Supplementary Data 1. Description of glaciers.

Supplementary Table S1. Primer sets used to detect antibiotic resistance genes by PCR.

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Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Journal Pre-proof Graphical abstract

Highlights Integron-bearing antibiotic-resistant bacteria are present on glaciers

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Relative abundance of integron-carrying bacteria on glaciers can reach 30%

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Clinically relevant resistance genes are found in the genomes of glacial microbes

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