Abstract
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.
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Introduction
Earth is a primarily cold planet, with more than 85% of the biosphere being exposed to permanent temperatures below 5 °C and with frozen habitats covering up 20% of our planet surface1,2. Thus, cold-adapted microbiomes are very important for the global ecology, while their biological activities maintain the nutrient flux in the environment and contribute to the global biogeochemical cycles, being also sensitive indicators of climate changes3. Moreover, their unique physiology and metabolic pathways provide essential knowledge for understanding the adaptation mechanisms to low temperatures and a valuable source of biomolecules for biotechnological applications. To overcome the challenges of life in harsh conditions (nutrients scarcity, high salinity, dryness, and low water activity, high UV irradiation and oxidative stress at high altitudes, and high pressure in the deep sea)4, cold-adapted microorganisms have developed a wide range of adaptations, from cellular envelope (incorporation of unsaturated membrane fatty acids and carotenoid pigments, cell wall peptidoglycan layer, modified lipopolysaccharides of the outer cell membrane) and metabolic adaptation (antifreeze and ice-nucleating proteins, cold-active enzymes with increased structural flexibility, cold-inducible promotors, increased variety and number of tRNA species, multiple stress-responsive genes), to cryoprotectants (extracellular polymeric substances, biosurfactants, compatible solutes) and increased variety and number of chaperons production, and novel metabolic capabilities (microbial growth and multiplication possible at 0–30 °C, vitrification, storage materials such as polyhydroxyalkanoates and cyanophycins), that can be exploited to develop novel biotechnological perspectives5. Despite the constantly increasing data on the microbial composition and diversity in Polar ice sheets and glaciers6,7,8,9, Polar soil and permafrost10,11, and aquatic environments12,13, microbiomes entrapped in perennial ice from caves still remain a largely undiscovered life system14.
Among the explored ice caves, Scarisoara Ice Cave (Romania) constitutes a unique cold environment, its 100,000 m3 perennial ice block dated about 13,000-years before present (BP) being one of the oldest and largest in the world15,16,17,18.
Recent investigations revealed the presence of cultivable bacteria19,20,21,22 and fungi23,24 in cave ice deposits. The microbial diversity and geochemical dependence of uncultured microbiome from underground frozen habitats highlighted complex bacterial communities in sediments from Antarctic volcanic ice caves from Mount Erebus25, and both sediments and ice from Hawaiian lava tubes26. A rich uncultured prokaryotic diversity was also found in Scarisoara cave ice strata of different ages18,27,28, in addition to a complex fungal community29. The distribution of the total and putatively active bacterial and fungal communities appeared to be modeled by the climatic characteristics during ice deposition and the organic carbon content of the ice substrate18,22,24,28,29.
Antimicrobial resistance (AR) is nowadays in top ten of global threats, concerning the animal and human health, food security and affecting the entire societal development (WHO 2020, www.who.int)30. AR is an old natural phenomenon, the antibiotic biosynthetic pathways and the resistance genes evolving over millions of years31,32,33,34,35. More evidence indicated environmental resistome as the reservoir for resistance genes found in clinical pathogens36,37. Due to their multiple physiological roles in natural environments, antibiotics acted as selecting and accelerating factors of AR evolution32,38,39. Bacterial strains highly resistant to known antimicrobial compounds usually have the potential for antimicrobial activity against other bacteria9.
Taking into account that extreme environments mirror a very remote moment from our planet past, prospecting their microbiome could provide a reference time point and new data for understanding the AR occurrence and evolution, and promising leads for new biologically active compounds including antimicrobials40,41,42,43. Cold-adapted microbes isolated from Polar habitats, mostly Actinobacteria and Proteobacteria from various Antarctic and Arctic habitats demonstrated their ability to produce antimicrobial compounds and activity against human pathogens9,44,45,46,47,48. However, due to the rather scarce recovery of psychrophilic and psychrotrophic antibiotic producers from cold environments43, limited information on the antimicrobial susceptibility is available as compared to that of mesophiles49.
Most studies on bacterial communities found in ice caves—unique, secluded, light-deprived and low content nutrients icy habitats—mainly focused on the identification and characterization of their diversity and composition and their response to climatic and environmental parameters during ice deposition14. So far, viable bacterial communities from Scarisoara cave ice were reported in distinct ice strata formed during the last 900 years by PCR-DGGE screening of the ice microbial cultures22. In this context, our investigation focused on isolating bacterial strains from up to 13,000-years old cave ice core, providing first cave isolates from perennial ice deposited since the Late Glacial period, and first culture-based evidences of cave ice bacteria resistome and their ability to produce antimicrobial compounds, representing putative novel tools to fight antibiotic resistance. In addition, screening for various biochemical features of cave ice isolates revealed a new reservoir of cold-active enzymes as valuable candidates for industrial applications.
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).
The isolates were assigned to 4 phyla, 34 genera and 56 species, based on a 16S rRNA gene sequence identity of 89–100%, with 535–1299 bp amplicon coverage (Supplementary Table S1). Molecular identification revealed various phylogenetic groups belonging to phyla Actinobacteria (32 strains), Proteobacteria (19 strains), Firmicutes (9 strains) and Bacteroidetes (8 strains) (Supplementary Table S1). Actinobacteria were present in all ice layers except for 11,000-years old ice, Proteobacteria species in 11 out of 19 strata, while Firmicutes and Bacteroidetes in only 6 and 5 layers, respectively (Fig. 1A).
Phyla and growth temperature distribution of cave ice bacterial isolates along Scarisoara ice core. (A) Number of phyla in ice core strata up to 13,000 years BP; (B) Growth temperature interval distribution of cave isolates belonging to Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes phyla.
Most of Actinobacteria isolates belonged to family Microbacteriaceae (22 strains), in addition to Micrococcaceae (6 strains), Nocardioidaceae (3 strains) and Dietziaceae (1 strain) (Supplementary Table S1). Proteobacteria isolates mainly belonged to class Aphaproteobacteria (10 strains), with species of famillies Caulobacteraceae (6), Phyllobacteriaceae (2) and Sphingomonadaceae (2). Betaproteobacteria isolated strains were assigned to Alcaligenaceae (5) and Comamonadaceae (1) families. The Gammaproteobacteria strains (3) belonged to order Pseudomonadales. Phylum Firmicutes was represented by Bacillaceae (5 strains), Planococcaceae (3 strains) and Paenibacillaceae (1 strain) families. Bacteroidetes isolates were assigned to families Flavobacteriaceae (5 strains) and Sphingobacteriaceae (3 strains).
At genus level, a high majority of cave strains were singular, except for Microbacterium (13), Bacillus (5), Arthrobacter (4), Brevundimonas (4), Chryseobacterium (4), Cryobacterium (3), and 2 isolates of Aeromicrobium, Paralcaligenes, Pedobacter, Phyllobacterium, Pseudarthrobacter, Pseudomonas and Sporosarcina genera (Supplementary Table S1).
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, 400, 900, 5000, 8000 and 12,000-years old ice (Supplementary 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; Fig. 1B). Based on this parameter50, four cave isolates were classified as psychrophiles, growing in the 4–15 °C interval (S. xinjiangense SC80A.3 and Cryobacterium levicorallinum SC21E.1), and 4–20 °C range (Flavobacterium glaciei SC1A.2 and Arthrobacter sp. SC41AB.3). The remaining 64 strains were psychrotrophs, with minimum growth temperatures of 4 °C and 10 °C. Among these, a variable number of Actinobacteria (10), Proteobacteria (7), Firmicutes (2) and Bacteroidetes (1) strains were cultivated between 4 and 25 °C, Phenylobacterium sp. SC71.1B and Eoetvoesia caeni SC41AB.2 could grow between 10 and 25 °C, and Pedobacter bambusae SC1A.4 within the 4–28 °C temperature interval (Supplementary Table S1; Fig. 1B). An upper limit for growth was accepted by the majority of isolates growing within 4–30 °C (19), 4–37 °C (14), 10–37 °C (7), most of them belonging to Bacillus spp., and 10–30 °C (1) intervals (Supplementary Table S1; Fig. 1B).
Blast analysis of 16S rRNA gene fragments indicated that 11 of the isolates were homologous to cold-environments bacteria (Supplementary Table S1), including Arthrobacter (2 strains), Cryobacterium (3 strains), Leifsonia (1 strain), Glaciihabitans (1 strain), Salinibacterium (1 strain), Psychrobacter (1 strain), Paenisporosarcina (1 strain) and Flavobacterium (1 strain). Among these, F. glaciei SC1A.2, S. xinjiangense SC80A.3, and C. levicorallinum SC21E.1 were psychrophiles. The remaining 8 isolates (A. psychrochitiniphilus SC21C.1, A. alpinus SC86E.1, Cryobacterium sp. SC14E.1, Leifsonia antarctica SC7AB.2, Glaciihabitans tibetensis SC21E.3, Psychrobacter glaciei SC80A.2, Cryobacterium flavum SC83A.1 and Paenisporosarcina macmurdoensis SC80A.5) were classified as psychrotrophs based on their growth temperature range (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%), 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). Among these, Microbacterium pygmaeum SC83A.2, Pseudarthrobacter oxydans SC70B.1 and Dietzia sp. SC61A.5B displayed a XDR phenotype, while G. tibetensis SC21E.3, Mycetocola manganoxydans SC51A.2 and A. psychrolactophilus SC7AB.1 were susceptible to most of the tested antibiotics (Fig. 2A). The highest AR of Firmicutes was determined for Sporosarcina globispora SC21C.2 (MDR) and Bacillus sp. SC8A.7 (19 antibiotics) (Fig. 2B, Supplementary Table S3), while Sporosarcina sp. SC21C.3, Bacillus safensis SC93A.3 and P. macmurdoensis SC80A.5 were resistant to only 6, 13 and 6 antibiotics, respectively (Fig. 2B).
Antimicrobial susceptibility of cave ice bacterial isolates. The percentage of susceptible under standard dose (green), and resistant (blue) strains to the 28 tested antibiotics was indicated for the strains belonging to phyla Actinobacteria (A), Firmicutes (B), Proteobacteria (C), and Bacteroidetes (D).
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).
The highest resistance rate to broad-spectrum aminoglycosides was displayed by Bacteroidetes (100%), Proteobacteria (96.5%) and Actinobacteria (88.5%) strains, while only 62.9% of Firmicutes were resistant to this class (Table 1). A similar resistance rate against cephalosporins was observed for Proteobacteria and Bacteroidetes isolates (92.1%, 95.8%), unlike Actinobacteria and Firmicutes (76%, 79.6%) (Table 1). All Firmicutes and Bacteroidetes strains were susceptible to tetracyclines, while 28.1% Actinobacteria and 31.58% Proteobacteria were resistant to this antibiotic. Firmicutes strains were completely susceptible to carbapenems, unlike Actinobacteria (9.4%) and Proteobacteria (31.6%) isolates (Table 1).
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).
Functional variability of ice core isolates
Based on the antimicrobial susceptibility profile (Supplementary Table S3) and age of the ice substrate (Supplementary Table S2), 11 cave isolates were selected along the ice core chronosequence for further characterization comprising five Gram-positive Actinobacteria (A. psychrolactophilus SC7AB.1, Dietzia sp. SC61A.5B, M. ginsengiterrae SC1A.5, M. pygmaeum SC65A.2 and Pseudarthrobacter sp. SC86E.4), five Gram-negative Proteobacteria (Candidimonas bauzanensis SC8A.3, Caulobacter sp. SC61A.5, Delftia sp. SC71.5, P. brenneri SC97A.1 and P. grimontii SC97A.2), and the Firmicutes B. toyonensis SC86E.3 strain (Supplementary Table S1).
Screening of enzymatic activities using API ZYM test system52 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). The only strain showing a weak N-acetyl-β-glucosaminidase activity was M. pygmaeum SC65A.2. P. brenneri SC97A.1, A. psychrolactophilus SC7AB.1, C. bauzanensis SC8A.3, Delftia sp. SC71.5 and Dietzia sp. SC61A.5B had a high alkaline phosphatase activity, unlike B. toyonensis SC86E.3 and M. pygmaeum SC65A.2 showing no activity. Hydrolysis of 2-naphthyl-αd-glucopyranoside was prominent for A. psychrolactophilus SC7AB.1, B. toyonensis SC86E.3, Caulobacter sp. SC61A.5, Dietzia sp. SC61A.5B, M. ginsengiterrae SC1A.5 and M. pygmaeum SC65A.2. The lowest number of hydrolyzed substrates (5 out of 19) was recorded for Delftia sp. SC71.5.
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).
In addition, a variable substrate assimilation pattern was observed (Table 3). All strains were able to use malate, and 10 of the isolates could metabolize glucose. P. brenneri SC97A.1 used all tested substrates except for maltose, followed by C. bauzanensis SC8A.3 and M. pygmaeum SC65A.2 (10 substrates). Meanwhile, a reduced assimilation capacity was observed for Delftia sp. SC71.5 and B. toyonensis SC86E.3 testing positive for 4 and 5 substrates, respectively (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 2 ATCC strains and 20 clinical pathogenic bacteria, exhibiting epidemiologically important resistance phenotypes (Supplementary Table S4).
All selected cave isolates showed antimicrobial activity against Staphylococcus aureus ATCC 25923, P. aeruginosa CN11 and MRSA 19081 F1 (Table 4). Ten cave strains inhibited Enterobacter cloacae 19069 ONE2, E. cloacae 19069 ONE3, and P. aeruginosa 19053 CNE5, while nine isolates had activity against Escherichia coli ATCC 25922, with a low inhibition of clinical Klebsiella 19094 strains CK1, KC2, and CK3, and of three Enterococcus faecium strains (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).
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 strata18,22 as compared to glacier ice53, the cave isolates exhibited a large diversity, covering the main phyla commonly found in frozen habitats, i.e. Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes1,7,43. 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 environments7,54,55, the Cryobacterium spp. Scarisoara isolates are, to our knowledge, the first retrieved from ice accumulated in caves53,56,57,58,59,60 and Salinibacterium xinjiangens61,62,63 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 bacteria64,65,66,67. Interestingly, the Phyllobacterium cave isolates from 700 and 7000-years old ice homologous to plants-associated species68,69 could originate from the abundant vegetation above the cave70. 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 habitats8,9,53,54,71,72,73,74,75. The presence of Flavobacterium glaciei in 100-years old Scarisoara ice, previously reported in China No.1 glacier76, 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 strains77,78 confirmed their adaptation to frozen habitats9,79. Meanwhile, the scarce recovery of Bacteroidetes strains from Scarisoara ice suggested the high sensitivity of this phylum67 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 Arctic9 and Antarctic80 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-habitats81,82, a high tetracycline susceptibility rate of the cave isolates was observed, also reported in Arctic bacteria9.
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. maris83 and D. papillomatosis84 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 vancomycin85. 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 isolates86, but also Antarctic80 and Arctic9,54 cold habitats.
Similar susceptibility patterns were observed for Brevundimonas SC86E.5 isolated from 10,000-years old Scarisoara ice and Antarctic lake homologous species80. 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 (700-years 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 plants68,69. Comparable AR profile was also observed for Mucilaginibacter species from frozen environments87,88 and Scarisoara SC21E.2 strain (XDR). Interestingly, Delftia sp. SC71.5 from 7000-years BP cave ice shared a similar resistance profile with clinical homologs89, 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 Myr33,81, 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 Proteobateria18. Actinobacteria are known for their ability to produce bioactive compounds and for their multiple resistance to antibiotics91, and Proteobacteria constitute a recognized group susceptible to acquire AR genes by HGT35,92,93. 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 strains94.
Climate impact on the AR reservoir of cave ice bacteria
Climate variation during ice deposition appeared to model the composition of bacterial18,22,27,28 and fungal24,29 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)16,17,95. In this respect, uncultured prokaryotic communities from LIA and MWP formed ice strata of this cave appeared to be dominated by Firmicutes or Actinobacteria28, while Proteobacteria dominated both the total and potentially active bacterial communities contained in MHWP ice18 accumulated during to Sahara Desert formation period96.
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).
Impact of substrate age and climate during ice deposition on the antimicrobial susceptibility of bacterial isolates from Scarisoara Ice Cave. (A) Resistance profile of the cave ice chronosequence. (B) Antibiotic resistance (blue) and susceptibility at standard dose (green) to 28 antibiotics (Supplementary Table S3) of cave isolates from ice layers accumulated during Little Ice Age (LIA; 430–475 years BP), Medieval Warm Period (MWP; 953–1124 years BP) and Mid Holocene Warm Period (MHWP; 4751–5335 years BP) (Supplementary Table S2).
Bacterial isolates from cave ice deposits accumulated during LIA, MWP and MHWP intervals (Fig. 3B) showed a variable AR. Among LIA isolates, a broad resistance (13–25 antibiotics) was observed for C. bauzanensis SC8A.3, R. argentea SC7AB.4 and M. hydrocarbonoxydans SC8A.4, Bacillus spp. SC8A.7, SC8A.1 and L. antarctica SC7AB.2, while A. psychrolactophilus SC7AB.1 (Actinobacteria) was highly sensitive to most of the antibiotics (Fig. 3B).
Most of the isolates from MWP and MHWP ice had broad AR, including 11 MDR strains mostly belonging to Proteobacteria and the XDR Dietzia sp. SC61A.5B (Fig. 3B). For these warm and wet climate intervals, the most susceptible bacterial strains belonged to Actinobacteria (G. tibetensis SC21E.3, M. hydrocarbonoxydans SC18B.1, A. psychrochitiniphilus SC21C.1 and M. pygmaeum SC65A.2 and SC14F.2), and Firmicutes (Sporosarcina sp. SC21C.3) species (Fig. 3B). Therefore, the broad resistance of resilient Proteobacteria representatives from cave ice could be a hint in searching new candidates for untangling environmental AR mechanisms.
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 antimicrobials49,97,98,99. Cold-adapted bacteria were also reported as a source of enzymes with enhanced stability and novel characteristics23,100.
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.
Among cold habitats bacteria, Pseudomonas species presented a broad antimicrobial activity43,49. 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 industry101 and alkaline phosphatase, commonly used for clinical diagnostics and dairy industry102 constitute promising leads for new biocatalysts.
The 5000-years old Dietzia sp. SC61A.5B showing a XDR phenotype also inhibited the growth of Gram-negative pathogens. Similar to Pseudomonas cave isolates, this Scarisoara strain showed high leucine/valine arylamidase and acid phosphatase activities, important catalysts for food processing101. 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 isolates9 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 genus41. This psychrotrophic ice bacterium also presented α- and β-glucosidase activities with putative extensive applications103. 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 antibiotics104,105, 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 processing101. Cold-active lipase activity for a wide range of applications in biofuels and pharmaceutical industry106, detergents107, and food processing108 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 cave21,22, 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 described18. 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 processed18.
Isolation of bacterial strains
A series of 28 core ice samples covering an age span of 92 ± 26 cal BP to 13,098 ± 29 cal BP at 100–300 years old interval18 were thawed at 4 °C in the absence of light, and inoculated in 10 mL R2B liquid media (Reasoner’s 2B broth, Melford Biolaboratories Ltd., UK). The enriched samples were incubated at 4 °C and 15 °C in shaker incubators (160 rpm) for up to 120 days. The enriched cultures (100 µL) were used to inoculate R2A Agar (Reasoner’s 2A agar, Merck Millipore, Germany) plates using a 100–10–10 serial dilution, and incubated at 4 °C and 15 °C for 2 to 4 weeks. Morphologically different colonies were isolated and purified under the same growth conditions.
Isolates identification
Bacterial isolates were identified by 16S rRNA gene sequencing. Total DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, USA) and the 16S rRNA gene fragments were amplified by PCR as previously described21. 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.codoncode.com), and the closest phylotype was assigned by BLAST alignment109.
Bacterial strains characterization
Growth temperature
The minimum and maximum growth temperatures of the isolated strains were determined by cultivation at 4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C and 37 °C up to 14 days on Tryptic Soy Broth-agar (TSA; Scharlab, Spain) solid medium.
Antibiotic susceptibility test
The antimicrobial susceptibility analysis was carried out using the disk diffusion method110. 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 strain110. 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 antibiotics51.
Biochemical characterization
Functional characterization of selected bacterial isolates was performed using API ZYM and API 20NE strips (BioMérieux, France) according to the manufacturer protocol, with incubation at 15 °C. The enzymatic activity was evaluated based on the number of nmol of hydrolyzed substrate as (0): no activity, (1 and 2): low activity (5 nmol and 10 nmol, respectively), (3): moderate activity (20 nmol), (4 and 5): high activity (30 nmol and ≥ 40 nmol, respectively)111.
Antimicrobial activity
The antimicrobial activity of the ice cave isolates was tested against two reference strains, i.e. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 (Thermo Scientific, USA), and 20 clinical isolates from the Research Institute of The University of Bucharest Microbial Collection (Supplementary Table S4), using the Kirby–Bauer method112. 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.
Data availability
The partial 16S rRNA gene sequences of the bacterial isolates from cave ice core were deposited in GenBank database under accession numbers MG642093-MG642131, MG642137-MG642141, MG680928, MG680933-MG680934, MH321580-MH321599, MH321601-MH321603, MN577392-MN577395, MN577397, MN577399, MN577401, MN577406, MN577408.
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Acknowledgements
We thank Aurel Persoiu, Christian A. Ciubotarescu, Vlad Murariu, Alexandra Hillebrand-Voiculescu, Catalina Haidau, Carmen Badaluta and Antonio Mondini for ice sampling technical support. This study was financially supported by the H2020 EraNet-LAC ELAC2014/DCC0178 Joint Program and PN-III-P2-2.1-PED2019-2461.
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This study was conceived and coordinated by C.P.; the manuscript was written by V.I.P and C.P., with substantial input from M.C.C. and P.L.; V.I.P. performed the microbiology and molecular biology experiments; M.C.C and P.L. had an important contribution to data interpretation. All of the authors reviewed the manuscript and approved the final version.
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Paun, V.I., Lavin, P., Chifiriuc, M.C. et al. First report on antibiotic resistance and antimicrobial activity of bacterial isolates from 13,000-year old cave ice core. Sci Rep 11, 514 (2021). https://doi.org/10.1038/s41598-020-79754-5
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DOI: https://doi.org/10.1038/s41598-020-79754-5
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