First report on antibiotic resistance and antimicrobial activity of bacterial isolates from 13,000-year old cave ice core

Despite the unique physiology and metabolic pathways of microbiomes from cold environments providing key evolutionary insights and promising leads for discovering new bioactive compounds, cultivable bacteria entrapped in perennial ice from caves remained a largely unexplored life system. In this context, we obtained and characterized bacterial strains from 13,000-years old ice core of Scarisoara Ice Cave, providing first isolates from perennial ice accumulated in caves since Late Glacial, and first culture-based evidences of bacterial resistome and antimicrobial compounds production. The 68 bacterial isolates belonged to 4 phyla, 34 genera and 56 species, with 17 strains representing putative new taxa. The Gram-negative cave bacteria (Proteobacteria and Bacteroidetes) were more resistant to the great majority of antibiotic classes than the Gram-positive ones (Actinobacteria, Firmicutes). More than 50% of the strains exhibited high resistance to 17 classes of antibiotics. Some of the isolates inhibited the growth of clinically important Gram-positive and Gram-negative resistant strains and revealed metabolic features with applicative potential. The current report on bacterial strains from millennia-old cave ice revealed promising candidates for studying the evolution of environmental resistome and for obtaining new active biomolecules for fighting the antibiotics crisis, and valuable cold-active biocatalysts.


Results
Bacterial strains isolated from the ice chronosequence of Scarisoara Ice Cave. A total of 68 distinct bacterial strains were isolated from the 13,000-years old ice core of Scarisoara cave by cultivation at 4 °C and 15 °C on R2A medium (Supplementary Table S1). These isolates retrieved from 19 cave ice samples of the ice chronosequence at an interval of ~ 300 years covered all ice layers, with a higher number originating from the 10,000-years old (9 strains), and 400-, 1000-and 7000-years old ice (7 strains) ice strata (Supplementary  Table S2). Amongst strains with identical 16S rRNA amplicon sequence, SC51B.2 and SC14F.2 exhibited different growth temperature intervals (Supplementary Table S1), and Microbacterium hydrocarbonoxydans, Microbacterium pygmaeum and Chryseobacterium hominis isolates differed by their antibiotic susceptibility profiles (Supplementary Table S3).
Among the 56 identified species, a high number belonged to Actinobacteria (24) and Proteobacteria (19), with a lower representation of Firmicutes (7) and Bacteroidetes (6) (Supplementary Table S1). Most species were singular, only 9 being found in different ice strata. The widest distribution along the ice core was observed for Microbacterium hydrocarbonoxydans present in 100 www.nature.com/scientificreports/ Table S1), and M. pygmaeum originating from 700, 4000, 5000 and 9000-years old ice strata. Chryseobacterium hominis strains were isolated from old ice layers (7000, 10,000, and 11,000-years old ice). In addition, two different Bacillus safensis homologs were retrieved from old strata (12,000 and 13,000-years BP), and different B. thuringiensis strains from 400-years BP ice. The isolates from the oldest cave ice layers also comprised Aeromicrobium panaciterrae, Paenibacillus amylolyticus, Pseudomonas brenneri, and P. grimontii homologs among the Late Glacial cave ice bacterial strains (Supplementary Table S1). The growth temperature interval of the cave isolates varied, with a minimum of 4 °C or 10 °C, and upper values ranging from 15 to 37 °C (Supplementary Table S1). The majority (58) of the strains had a minimum growth temperature of 4 °C, while 10 strains grew only above 10 °C (Supplementary Table S1 Antibiotic susceptibility profile of cave isolates. Antimicrobial susceptibility of the cave isolates to 28 antibiotics of 17 classes having broad-spectrum, specificity for Gram-positive bacteria and for anaerobes revealed various resistance profiles (Supplementary Table S3). Gram-negative bacteria (Proteobacteria and Bacteroidetes) were more resistant to the great majority of antibiotic classes than the Gram-positive (Actinobacteria, Firmicutes) ones (Table 1). Both Gram-positive and Gram-negative isolates were highly resistant (> 77%) to metronidazole, mostly belonging to Bacteroidetes (100%), Proteobacteria (94.7%), and Actinobacteria (93.8%),  www.nature.com/scientificreports/ while Firmicutes showed the lowest AR rate of 77.8%. Over 70% of phyla representatives were resistant to lincosamides and fatty acyls antibiotic classes ( Table 1). The AR profile of the cave isolates indicated the presence of multi-drug (MDR) (25), extended-drug (XDR) (8), and pan-drug (PDR) (2) resistance phenotypes, according to the definitions proposed by Magiorakos et al. 51 , described below in "Methods" section. Among these, Arthrobacter psychrolactophilus SC7AB.1 isolated from the 400-years old ice layer was resistant only to nalidixic acid and cefixime (Supplementary Table S3). At the opposite pole, the SC97A.1 and SC97A.2 Pseudomonas strains isolated from the 13,000-years old ice layer showed a PDR phenotype to all antibiotics.
Relative to the total number of drugs tested (Fig. 2), the antibiotic susceptibility of cave bacteria belonging to different phyla showed large taxonomic-related variations. Gram-positive isolates displayed the highest resistance to metronidazole, fatty acyls, lincosamides, cephalosporins and aminoglycosides, and to nitrofurantoin and nalidixic acid in the case of Actinobacteria (Table 1) More than 50% of the Gram-negative cave ice bacteria were resistant to all tested antibiotic classes, excepting rifampin, tetracyclines and carbapenems in the case of Proteobacteria ( Table 1). The majority of Proteobacteria (16 isolates) showed a MDR phenotype, while E. caeni SC41AB.2, Caulobacter sp. SC61A.5 and P. glaciei SC80A.2 were only resistant to 14, 18 and 16 antibiotics, respectively (Fig. 2C). Noticeably, Pseudomonas spp. SC97A.1 and SC97A.2 strains presented a PDR profile (Fig. 2C). Gram-negative Bacteroidetes strains were also characterized by a high AR with all 8 strains resistant to > 50% of the tested antibiotics (Fig. 2D). Among these, M. phyllosphaerae SC21E.2 showed the broadest (27 antibiotics) and P. bambusae SC1A.4 the most limited AR (16 antibiotics).
Among the retrieved cave isolates, Actinobacteria showed a variable resistance profile, 93.8% being resistant to metronidazole and only 9.4% to carbapenems. Firmicutes were highly resistant to lincosamides (83.3%) while only 11.1% to nitrofurantoin, and completely susceptible to carbapenems and tetracyclines (Table 1). Gram-negative Proteobacteria strains had a variable resistance profile, ranging from 31.6% for carbapenems and tetracyclines, to 100% for fatty acyls, and for Bacteroidetes from 25% (rifampin) to 100% (aminoglycosides, glycopeptides and metronidazole), with total susceptibility to tetracyclines (Table 1).   Table S3) and age of the ice substrate (Supplementary Table S2 Table S1). Screening of enzymatic activities using API ZYM test system 52 showed a distinct enzymatic profile of the tested cave bacteria (Table 2). Although none of the isolates hydrolyzed all 19 substrates, A. psychrolactophilus SC7AB.1, M. ginsengiterrae SC1A.5 and M. pygmaeum SC65A.2 tested positive for 74% of activities. According to evaluation codes, high leucine arylamidase and naphthol-AS-BI-phosphohydrolase activities were identified for all isolates, while no α-chymotrypsin and α-fucosidase activities were detected ( Table 2) Complementary API 20NE tests revealed a distinct substrate utilization profile of the selected cave strains ( Table 3). All 11 isolates could hydrolyze β-glucosidase (esculin), while none of the strains were able to reduce nitrates and tested negative for indole production, glucose fermentation and arginine dihydrolysis. Nitrate reduction was observed for most of the strains except for Caulobacter sp. SC61A.5, M. ginsengiterrae SC1A.5 and P. brenneri SC97A.1. Only four isolates were able to metabolize urea. Gelatin hydrolysis was limited to B. toyonensis SC86E.3 and M. ginsengiterrae SC1A.5. A. psychrolactophilus SC7AB.1, M. ginsengiterrae SC1A.5, M. pygmaeum SC65A.2 and Pseudarthrobacter sp. SC86E. Four cave bacteria tested positive for β-galactosidase, and cytochrome oxidase activity was measured for five strains (Table 3).
Antimicrobial activity of isolates from Scarisoara cave ice core. Eleven cave isolates showing optimal growth parameters and diverse resistance profiles have been selected for their antimicrobial activity against   (Table 4).
A broad antimicrobial spectrum was determined for the 13,000 cal BP P. grimontii SC97A.2 strain, active against most tested pathogens (Table 4). Interestingly, the methicillin susceptible and resistant S. aureus strains, and the P. aeruginosa pathogens were inhibited by all 11 cave isolates. M. pygmaeum SC65A.2 and Dietzia sp. SC61A.5B isolated from 5000 cal BP Scarisoara ice inhibited 11 and 10 of the tested pathogens, respectively, and M. ginsengiterrae SC1A.5 (100 cal BP) showed activity against 9 pathogens. No antimicrobial effect was recorded against one Enterobacter, two methicillin resistant S. aureus, one Klebsiella, and all Acinetobacter sp. strains (Table 4). Table 2. API ZYM enzymatic activity of cave ice isolated strains. The color intensity decreased with the level of enzymatic activity from 5 (high) to 0 (no activity). The numbers correspond to the amount of hydrolyzed substrate (nmol), as indicated in "Methods" section.

Discussion
This study represents the first characterization of 68 distinct cold-active strains isolated from a 13,000-years old underground ice core of Scarisoara ice cave (Romania) focused on their growth temperature, antibiotic resistance, enzymatic and antimicrobial activity profiles.
Cold-adapted bacterial isolates from 13,000-years old cave ice. Despite the lower microbial density found in Scarisoara ice strata 18,22 as compared to glacier ice 53 , the cave isolates exhibited a large diversity, covering the main phyla commonly found in frozen habitats, i.e. Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes 1,7,43 . Among these, an important fraction (17 strains) showed a low 16S rRNA gene identity with previously reported bacteria (Supplementary Table S1), suggesting the existence of possible new species in this habitat. Also, the Aeromicrobium sp. and Chryseobacterium sp. strains isolated from the ice strata accumulated during the Late Glacial Period (LGP, > 12,000-years BP) appeared unrelated to other representatives of these genera. Interestingly, all of these LGP-originating bacteria were psychrotrophs able to grow above 30 °C. From Actinobacteria, a prevalent microbial taxa in cold environments 7,54,55 , the Cryobacterium spp. Scarisoara isolates are, to our knowledge, the first retrieved from ice accumulated in caves 53,56-60 and Salinibacterium xinjiangens [61][62][63] was cultivated for the first time from ice deposits. Amongst Proteobacteria, Pseudomonas (13,000 years old ice) and Psychrobacter glaciei (8000 years old ice) strains were isolated for the first time from the ice core of this cave, adding this habitat to other frozen environments colonized by these bacteria [64][65][66][67] . Interestingly, the Phyllobacterium cave isolates from 700 and 7000-years old ice homologous to plants-associated species 68,69 could originate from the abundant vegetation above the cave 70 . Firmicutes strains belonging to Bacillus and Sporosarcina genera were retrieved from different Scarisoara ice strata, confirming the resilience of these genera in recent and old icy habitats 8,9,53,54,[71][72][73][74][75] . The presence of Flavobacterium glaciei in 100-years old Scarisoara ice, previously reported in China No.1 glacier 76 , highlighted a wide geographical distribution of this psychrophilic species. In addition, the recovery from 100-years old ice of Pedobacter strains closely related to oil and aquatic strains 77,78 confirmed their adaptation to frozen habitats 9,79 . Meanwhile, the scarce recovery of Bacteroidetes strains from Scarisoara ice suggested the high sensitivity of this phylum 67 to environmental variations.
Antibiotic resistance in Late Glacial cave ice core. Scarisoara ice cave isolates showed high resistance to different antibiotic classes including the ones drastically affected by the emergence of resistant strains in the clinical sector. Most Scarisoara isolates exhibiting MDR and XDR phenotypes thrived in ice strata formed at least 1000-years ago. Bacterial strains with the broadest (PDR) resistance belonged to Pseudomonas species originating from 13,000-years old ice, similar to Arctic 9 and Antarctic 80 homologs.
Most of the cave isolates with broad AR were Gram-negative belonging to Proteobacteria (6) and Bacteroidetes (1), while Arthrobacter psychrolactophilus SC7AB.1 isolated from 400-years old ice was resistant only to nalidixic acid and cefixime, (Supplementary Table S3). Although not specific for cold-habitats 81,82 , a high tetracycline susceptibility rate of the cave isolates was observed, also reported in Arctic bacteria 9 .
A different response to antibiotics of the cave isolates was observed for the 5000-years old ice Dietzia sp. SC61A.5B (XDR) as compared to their homologs D. maris 83 and D. papillomatosis 84 susceptible to most antibiotics. The two XDR Paralcaligenes cave strains SC18B.3 (900-years BP) and SC71.3 (7000-years BP) appeared to have an opposite phenotype as their homologs isolated from soil resistant to only lincomycin, oleandomycin, rifampicin, and vancomycin 85 . Other four XDR cave isolates belonging to Flavobacterium, Paralcaligenes, Phenylobacterium and Microbacterium genera isolated from up to 9000-years old ice (Supplementary Table S1) revealed a broader AR than the corresponding species from clinical isolates 86 , but also Antarctic 80 and Arctic 9,54 cold habitats.
Similar susceptibility patterns were observed for Brevundimonas SC86E.5 isolated from 10,000-years old Scarisoara ice and Antarctic lake homologous species 80 . Some cave isolates showed a species specific antibiotic resistance profile independent on the age and type of habitat, as in the case of the MDR P. trifolii SC14F.1 (700years BP) and XDR P. loti SC71.2 (7000-years BP) susceptible to different antibiotics (Supplementary Table S3), although with similar response as their best match strains originating from plants 68,69 . Comparable AR profile was also observed for Mucilaginibacter species from frozen environments 87,88 and Scarisoara SC21E.2 strain (XDR). Interestingly, Delftia sp. SC71.5 from 7000-years BP cave ice shared a similar resistance profile with clinical homologs 89 , although the low 16S rRNA gene identity (95%) could indicate a more distal cave strain ancestor.
The prevalence of MDR found in the 68 isolated strains revealed the existence of highly diverse resistome in old samples (5000-30,000-years old) from permafrost, Arctic soil, and a deeper cave site disconnected from the surface for over 4 Myr 33,81 , suggesting a correlation between AR and cold-adaptation mechanisms via horizontal gene transfer (HGT) 90 . In our study, the high incidence of XDR and PDR phenotypes recovered from layers of old ice sealed between 1000-13,000 years could be associated with the high relative abundance of phyla Actinobacteria and Proteobateria 18 . Actinobacteria are known for their ability to produce bioactive compounds and for their multiple resistance to antibiotics 91 , and Proteobacteria constitute a recognized group susceptible to acquire AR genes by HGT 35,92,93 . In some cases, MDR could be conferred by cross-resistance as observed for cave isolates resistant to tetracycline, various β-lactams, all aminoglycosides and almost all macrolides. A similar pattern of cross-resistance was observed in methicillin-resistant S. aureus strains 94 . Climate impact on the AR reservoir of cave ice bacteria. Climate variation during ice deposition appeared to model the composition of bacterial 18,22,27,28 and fungal 24,29 communities entrapped in perennial ice accumulated in Scarisoara ice cave, in particular during the cold and dry Little Ice Age (LIA, AD 1250-AD 1860), and the warm and wet intervals Medieval Warm Period (MWP) (AD 800-AD 1250) and Mid-Holocene Warm Period (MHWP; 5500-6500 years BP) 16 96 . The current data (Fig. 3A) revealed a variable AR profile of cave isolates along the 13,000-years BP ice core, with broader resistance (MDR and PDR phenotypes) in recently formed ice, the warmer periods MWP, MHWP, and 7000-years old ice, alternating with higher susceptibility during LIA, 3000-4000-years BP, 5500-years BP, and 9000-years BP periods. An increased resistance was also observed for isolates from older ice extended to Late Glacial period (12,000-13,000-years BP) (Fig. 3A).
Applicative potential of Scarisoara cave ice bacterial isolates. To overcome the MDR in microorganisms with medical and industrial relevance, screening of microbiomes from unexplored old habitats is crucial for understanding their evolution and discovery of new antibiotics. Microorganisms from cold environments, mostly belonging to Actinobacteria, are able to inhibit opportunistic human pathogens, constituting largely unexplored reservoirs of new natural antimicrobials 49,[97][98][99] . Cold-adapted bacteria were also reported as a source of enzymes with enhanced stability and novel characteristics 23,100 .
The corroborated characterization of the 11 bacterial isolates from Scarisoara ice cave revealed new psychrotolerant strains showing high AR and antimicrobial activity against at least one or more clinical Gram-negative and Gram-positive pathogens (Table 4), in addition to a series of enzymatic activities as valuable catalysts candidates for low temperature industrial processes. To our knowledge, these data on microbial isolates from the 13,000-years old underground ice deposits of Scarisoara cave provided the first glimpse on the ice caves' potential as reservoirs of new antimicrobial biomolecules.  www.nature.com/scientificreports/ Among cold habitats bacteria, Pseudomonas species presented a broad antimicrobial activity 43,49 . In the case of Scarisoara isolates, the two Pseudomonas strains from 13,000 cal BP ice strata (P. brenneri SC97A.1 and P. grimontii SC97A) showed a high antimicrobial activity against 7 and 14 pathogens, respectively, the later one with extensive preference for Gram-negative clinical isolates. Considering their PDR phenotype, these strains could constitute important candidates for studying the resistome of millennia-old bacteria and AR evolution. Moreover, their positive testing for acid phosphatase and leucine arylamidase activities with applications in food industry 101 and alkaline phosphatase, commonly used for clinical diagnostics and dairy industry 102 constitute promising leads for new biocatalysts.
The 5000-years old Dietzia sp. SC61A.5B showing a XDR phenotype also inhibited the growth of Gramnegative pathogens. Similar to Pseudomonas cave isolates, this Scarisoara strain showed high leucine/valine arylamidase and acid phosphatase activities, important catalysts for food processing 101 . Moreover, M. pygmaeum SC83A.2 isolated from 9000-years BP cave ice displayed a high antimicrobial activity mainly against Gram negative pathogens, associated with XDR phenotype. The broader AR of this strain as compared to Artic isolates 9 uncovered a valuable bacterial candidate for exploring new antimicrobial mechanisms.
Caulobacter sp. SC61A.5 cave isolate (5000-years old ice) was also one of the strains with broader antimicrobial activity against Gram-negative pathogens and broad AR phenotype, unlike other representatives of this genus 41 . This psychrotrophic ice bacterium also presented α-and β-glucosidase activities with putative extensive applications 103 . Similar to Pseudomonas and Dietzia cave isolates, a high Leu/Val arylamidase and acid phosphatase activity was measured for this strain.
Arthrobacter psychrolactophilus SC7AB.1 showed a particular AR to fluoroquinolone and cephalosporins, and antimicrobial activity against Gram-negative and Gram-positive pathogens, in addition to a variety of enzymatic activities as putative source of cold-active catalysts. The XDR phenotype of Delftia sp. SC71.5 isolated from 7000-old cave ice, unlike the homologous soil strain susceptible to broad spectrum antibiotics 104,105 , and the broad antimicrobial activity against Gram-negative pathogens recommend theses strains as novel candidates for studying the environmental resistome and for isolation of putative active biomolecules.
Most of the cave isolates tested positive for acid phosphatase activity used in food industry, constituting valuable catalyst candidates for low temperature food processing 101 . Cold-active lipase activity for a wide range of applications in biofuels and pharmaceutical industry 106 , detergents 107 , and food processing 108 was also identified in most of the cave ice isolates.

Conclusion
In addition to our previous reports on cultivable bacteria from ice layers formed during the last 900-years in Scarisoara ice cave 21,22 , the current study adds to the knowledge on functional characteristics of isolated bacteria from this underground perennial ice core. The isolated bacterial strains belonging to the four major phyla ubiquitous for frozen environments confirmed their viability along the 13,000 cal BP cave ice, and revealed their extended antibiotic resistance profiles, as a pioneering survey for this type of habitat for understanding the environmental resistome evolution.
Although with a lower microbial density as compared to glacier ice, our cave isolates belonging to Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes exhibited a large diversity encompassing putative new representatives for this habitat or worldwide. Many of these strains preserved in millennia-old ice from a secluded type of icy habitat exhibited a large spectrum of antimicrobial resistance, highlighting their important contribution to the environmental resistome, and the potential risk of releasing antibiotic resistant bacteria in the water and soil due to temperature increase and ice melting. However, further studies are needed to identify the associated AR genes, their localization and transferability potential. In addition, the psychrotrophic isolates highlighted significant antimicrobial and catalytic activities, being thus valuable candidates for biomedical and biotechnological applications. Overall, due to their extended AR profile as compared to that of the reported homologs and the antimicrobial activity of all isolates, in particular against the Gram-negative pathogens raising global treatment limitations, these cave bacterial strains retrieved from up to 13,000-years old ice could provide important clues for understanding the evolution of natural and clinical resistance and for developing novel antimicrobial strategies.

Methods
Ice samples. Ice sampling was performed by vertical drilling to a depth of 25.33 m into the perennial ice block of Scarisoara Ice Cave resulting in 97 ice core fragments covering a chronology of up to 13,000 calibrated years before present (cal BP) ice, as previously described 18 . All ice samples were collected under aseptic conditions by flaming the drilling equipment and using sterile 1-L plastic bags, transported to the laboratory under permanent frozen conditions where they were stored at − 20 °C until processed 18  www.nature.com/scientificreports/ by PCR as previously described 21 . After purification (Invitrogen, USA), the amplicons were sequenced using the amplification primers (Macrogen, Netherlands). Chimera analysis of the sequences and low-quality aligned regions were eliminated using CodonCode Aligner version 8.0.1 (CodonCode Corporation, www.codon code.com), and the closest phylotype was assigned by BLAST alignment 109  Antibiotic susceptibility test. The antimicrobial susceptibility analysis was carried out using the disk diffusion method 110 . The isolates were cultivated on R2A agar and TSA agar plates at 15 °C for 2-5 days. The antibiotic susceptibility profile was determined after incubation at 37 °C for 24 h, using Stahylococcus aureus ATCC 25923 (Thermo Scientific, USA) as control strain 110 . Based on the number of antibiotics with inhibitory effect, the strains were assigned as MDR, defined as non-susceptible to at least one agent in three or more antimicrobial categories, XDR as non-susceptible to at least one agent in all but two or fewer antimicrobial categories, and PDR, defined as non-susceptible to all agents in all antimicrobial categories. According to these definitions, our strains exhibiting a MDR phenotype were resistant to 20-25 antibiotics, from at least three different classes, the XDR ones were resistant to 26-27 antibiotics, and those exhibiting a PDR were resistant to all tested 28 antibiotics 51 .  Table S4), using the Kirby-Bauer method 112 . The cave isolates were cultivated in 20 mL Tryptic Soy Broth (TBS) medium (Merck Millipore, Germany) at 15 °C, under agitation (160 rpm), for up to 14 days, and the cells were harvested by centrifugation at 7500 rpm for 20 min. The supernatant was filtered-sterilized using 0.22 µm syringe filters (Merck Millipore, Germany), and concentrated to 1 mL with a vacuum Concentrator Plus Complete system (Eppendorf, USA). The pathogen (test) strains were inoculated on Mueller-Hinton agar (MHA) solid medium (2 g L −1 beef extract, 17.5 g L −1 casein acid hydrolysate, 1.5 g L −1 starch, and 17 g L −1 agar) and grown at 37 °C for 18 h. One colony from each test strain was resuspended in physiological saline solution to 0.5 McFarland turbidity, and further plated on MHA solid medium covering uniformly the plate surface. Immediately after pathogen plating, 5 µL samples of concentrated extracts of cave bacteria were added as single drops in separated quadrants (6 per plate). After incubation at 37 °C for 18 h, the presence/absence (±) of antimicrobial activity was estimated based on the presence of an inhibition zone inside each quadrant. The assay was performed in duplicate. A negative control comprising 20 mL of sterilized TSB medium concentrated to 1 mL was used for each pathogen test strains following the same protocol.
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