Characterization of Culturable Bacterial Communities in Permafrost, Moraine, and Rhizosphere Soils near the Ecology Glacier (King George Island, Maritime Antarctica): Patterns of Antibiotic Resistance and Virulence Factors in Isolated Bacteria

Characterization of Culturable Bacterial Communities in Permafrost, Moraine, and Rhizosphere Soils near the Ecology Glacier (King George Island, Maritime Antarctica): Patterns of Antibiotic Resistance and Virulence Factors in Isolated Bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Characterization of Culturable Bacterial Communities in Permafrost, Moraine, and Rhizosphere Soils near the Ecology Glacier (King George Island, Maritime Antarctica): Patterns of Antibiotic Resistance and Virulence Factors in Isolated Bacteria Jacquelinne J. Acuña, Constanza Venegas, Marco A. Campos, Nicole Huerta, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6667169/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Glacier forelands in the Antarctic Peninsula are increasingly affected by climate change. However, the impact on the composition of culturable soil bacteria communities remains unclear. Here, we explored the culturable bacterial communities from permafrost (P), moraine (M), and Deschampsia antartica rhizosphere (R) soil samples collected near the Ecology Glacier, Antarctica. Using traditional plating-on agar (PM) and ' in situ ' cultivation (ISC) methods, bacterial counts were significantly higher in R (8.2×10 5 CFU g − 1 soil) than in M and P (~ 3.9 ×10 3 CFU g − 1 soil). Culturable lawn bacteria communities and 158 genotypically different isolated strains (76 by ISC and 82 by PM) were identified, purified. And their antibiotics multiresistance (AMR) and virulence factors (VFs) were also screened. Our results revealed phyla Pseudomonadota (55–75%), Actinomicetota (20–35%), and Bacteroidota (5–10%) as the most abundant bacterial taxa in culturable bacteria lawn communities. The isolated strains belonged to 24 different bacteria genera, where Pseudomonadota (76%), Actinomicetota (18%), Bacteroidota (4.6%), and Bacillota (3.2%) were the most dominant phyla. Using ISC, a wider genera diversity (e.g., Bosea , Rathayibacter , and Rugamonas ) was isolated. On the other hand, Bacillus exclusively grew on PM. Among these isolates, 86% were resistant to beta-lactams, 77% to cephalosporins, and 71% to oxazolidines. Interestingly, some Flavobacterium , Pseudomonas , and Curtobacterium strains showed AMR to > 18 different antibiotics. For VFs assays, we also observed > 35% lecithinase and hemolytic activity, 20% pyocyanin production, and 7% DNAse activity among all isolates. A high diversity of AMR and VFs was observed in culturable bacteria inhabiting the surrounding soils of the Ecology Glacier. Biological sciences/Microbiology Earth and environmental sciences/Environmental sciences Bacterial diversity Antibiotic resistance virulence factors Antarctica iChip Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Climate change significantly contributes to the melting of glaciers and permafrost around the globe [ 1 , 2 ]. Antarctica is the fastest warming area [ 3 ], exhibiting accelerated deglaciation in its northeast areas, such as the Antarctic Peninsula [ 4 ]. Due to ice mass shrinkage, the newly formed terrain becomes the habitat for microorganisms [ 5 ] brought from other sites in addition to autochthonous microorganisms [ 6 ]. Bacterial communities are determinants of glacier foreland, which play crucial roles in biogeochemical cycles and accelerating soil formation [ 7 ]. Krauze et al. [ 8 ] found that microbial communities near glaciers influence soil formation and nutrient speciation, which can modulate and promote the colonization and establishment of the unique native vascular plants Deschampsia antarctica and Colobanthus quitensis . Several studies in the Antarctic have focused on unraveling the structure and functions of soil and plant microbiomes [ 9 ]. However, the consequences of climate change, such as ice melting on culturable indigenous soil bacteria communities, as well as their functions, structures, and interactions in the plant-soil-microbe continuum in Antarctica, remain poorly understood and difficult to predict. Climate change can also lead to the emergence or reemergence of infectious diseases by altering the distribution of microbial pathogens and their vectors, posing a threat to humans, animals, and plants [ 5 ]. Bacteria community studies showed the presence of a natural resistome and virulome from Antarctic soils, as well as evidence of anthropogenic introduction of antibiotics multiresistance (AMR), highlighting the impact of human activities in this less intervened environment [ 10 , 11 , 12 ]. However, antimicrobial resistance profiles are strain-specific and environment-dependent, reflecting unique evolutionary adaptation processes rather than solely a recent adaptation [ 13 ]. The Antarctic ice-free zone expansion could lead to the discovery of previously unknown microorganisms carrying novel virulence factors (VFs) and multidrug-resistant (MDR) strains. Furthermore, new vegetation and animals could facilitate the proliferation of pathogens, promoting gene horizontal transfer among microbes and increasing the risk of disease emergence [ 14 ]. Cultivation-independent techniques have increased our current knowledge about the bacteria diversity of Antarctic soil habitats [ 15 ], strongly contrasting with the narrow diversity recovery using the traditional plating-on agar method [ 16 ]. The 'in situ' cultivation approach aims to isolate previously uncultured bacteria with rich biodiversity and novelty [ 17 ]. For this, microporous membrane-based microwell well chamber devices (MWC) installed in soils allow the exchange of chemical components from the surrounding soil into each well. The environment provides unknown but essential metabolites, signaling molecules and nutrients for growth, as co-culture with other microorganisms can increase bacteria culturability, particularly in moraine and permafrost soils where the frozen conditions pose additional challenges [ 18 ]. This ' in situ ' cultivation on sub-Antarctic and Arctic soil using i-Tip and cryo-iPlate, respectively, allowed the isolation of priorly unculturable bacteria [ 19 , 20 ]. Although glacier retreatment has been known for a long time and represents one of the most damaging effects of climate change, the underlying culturable microbiome and microorganisms containing AMR and VFs remain largely unexplored in Antarctica. We isolated culturable bacteria by traditional plating-on agar (PM) and ' in situ ' cultivation (ISC). The latter employed an MWC along the expansion of frozen to ice-free soil zones of the Ecology Glacier located on King George Island (South Shetland Islands, Maritime Antarctic). Culturable bacteria lawns and their respective isolated strain collections were characterized by 16S rRNA gene partial sequencing. Isolated strains were also screened by their AMR and VFs. Our results provide insights into the culturable bacterial diversity during Ecology Glacier's frozen expansion to ice-free soil zones and their potential risk by AMR and VFs. 2. Materials and methods 2.1. Sampling Samples were collected from three distinct types of soil environments near the Ecology Glacier, inside the Antarctic specially protected area (ASPA, N°128: Occidental coast of Admiralty Bay), Polish Henryk Arctowski Station. The samples were from frozen permafrost (P) and two ice-free soil types, moraine (M) and rhizosphere (R) (Fig. 1 A). The samples were taken during the 58th Scientific Antarctic Expedition (ECA58) in the summer season (November 2021 - March 2022), following the protocols and supervision by the Chilean Antarctic Institute (INACH; https://www.inach.cl/ ). Six samples of both P (62°10'15.4 "S 58°28'21.6 "W) and M (62°10'14.4 "S 58°28'20.5 "W) were collected by removing a 10 cm surface layer and then introducing ~ 1 kg of the underlying soil into Whirl-Pak sterile bags (Sigma-Aldrich, Saint Louis, MO, USA) using an aseptic spade and storing them at 4°C until processing. In parallel, the R samples were collected in an area with a notable presence of vegetation. Then, six specimens of D. antarctica (62° 09 '48. 5" S 58° 27' 48.3" W) were randomly taken in a 500 m transect from the M sampling site, in the direction of Henryk Arctowski Polish Antarctic Station by using a clean spade to remove intact roots from the soil (Fig. 1 ). Collected rhizosphere samples were placed within Whirl-Pak sterile bags (Sigma-Aldrich, Saint Louis, MO, USA) and stored at 4°C. All samples were transported on ice to the Applied Microbial Ecology Laboratory (EMALAB) at Universidad de La Frontera, Temuco, Chile, for further cultivation experiments (Fig. 1 B). 2.2 Soil physicochemical characterization Air-dried soil samples were used to determine soil pH, electrical conductivity (EC), organic matter (OM), total phosphorus (TP), available P (Olsen), total carbon (TC), total nitrogen (TN), and exchangeable cations (Ca²⁺, Mg²⁺, Na⁺, K⁺, and Fe⁺). After air drying, the soil was macerated using a mortar and pestle to remove large constituents such as rocks and organic debris. The pH and EC were measured in 1:2:5 and 1:5 soil/deionized water suspension with a high-grade benchtop meter (model H15522; Hanna Instrument Ltda., Leighton Buzzard, UK). The OM contents were estimated by wet digestion [ 21 , 22 ]. The TP was extracted from the soil samples using NaOBr [ 23 ] and quantified as described by Murphy and Riley [ 24 ]. Inorganic phosphorus (POlsen) was extracted using the bicarbonate method (pH 8.5 in 0.5 M NaHCO 3 ) and analyzed using the molybdate-blue method [ 24 ]. For the TC and TN content, soil samples were firstly homogenized to under 0.1 mm and freeze-dried. Then, 1.5–2.5 mg aliquots of the samples were weighed into an EA 3000 automated elemental analyzer (Eurovector, Italy). The elemental composition was determined by interpolation using a suitable calibration curve (R² = 0.98) with EDTA as a standard (99.4% purity; LECO®, USA). The results were expressed in mg of TP, TC, or TN per kg of dry-weight soil sample (mg kg⁻¹ DW). Exchangeable cations were extracted using 1 M CH₃COONH₄ at a pH of 7.0 and then analyzed using flame atomic absorption spectrophotometry [ 25 ]. 2.3 Selection of medium and determination of total culturable bacteria Three types of non-selective media were used in this study in order to determine the plate count of culturable bacteria from P, M, and R soil samples and to test the best media for following used in culture experiments for both traditional direct plating-on agar method and as well sub-cultivation for in situ method. One gram of each sample (in triplicates) was suspended in 25 ml of sterile distilled water (SDW) and sonicated for 30 s at 130 W (20 kHz). Appropriate serial dilutions were then spread onto three media used in this study: (1) Reasoner's 2A broth (R2A) medium (Sigma-Aldrich, Saint Louis, MO, USA), (2) NM-1 oligotrophic agar medium [ 26 ], and (3) 1:10 diluted LB media (10% of the manufacturer's suggested concentration, Sigma-Aldrich, Saint Louis, MO, USA). All media contained 1.5% agar and were supplemented with 10 µg ml − 1 of cycloheximide (Sigma-Aldrich, Saint Louis, MO, USA) to prevent fungal growth. Colonies grown on agar plates were counted after 14 days of incubation at 15°C. 2.4 Culturable bacteria lawns and isolation bacterial strains by in situ cultivation method The ' in situ ' cultivation (ISC) method considered using a MWC to obtain isolates from all P, M, and R soil samples. The MWC was prepared as described previously by Acuña et al. [ 27 ] and Berdy et al. [ 18 ]. (Fig. 1 C). To construct an MWC, two rectangular pieces of polyamide plastic and one central piece of polytetrafluoroethylene (PLASTIGEN, Chile) with 48 through-holes (w = 3 cm, l = 1.2 cm, d = 0.3 cm) in each piece were used. In addition, four rectangular pieces of polycarbonate membrane (Midland Scientific Inc., CO, USA) with a 0.03-µm pore size, silicone glue, and screws were used to assemble and seal the MWCs for sub-cultivation assays. To prepare soil samples inoculum, 1 g of fresh soil collected from P, M, and R were serially diluted (10 − 3 ) in SDW mixed with 1.5% gellan gum at 40°C (Sigma-Aldrich, Saint Louis, MO, USA) and then inoculated into an MWC by dipping the central plates into each soil sample cell suspension (P, M, and R). The assembly of MWC was as follows: two membranes were adhered to both sides of the central plate using hot silicon glue; the top and bottom plates were aligned and adjusted, and the screws were tightened to provide pressure (Berdy et al. 2017). Finally, the screws and all edges of the MWC were sealed using hot silicone glue to prevent external contamination. Two MWC devices were inoculated and assembled using the same method for each P, M, and R soil subsample. Subsequently, the MWCs were incubated in a sterilized plastic box containing each soil sample (P, M, and R) and maintained for 21 days inside refrigerators at 4°C (Fig. 1 D). After incubation, the MWCs were washed vigorously in particle-free DNA-grade water (Fisher Scientific, Hampton, NH) and disassembled under laminar flow. The first MWC was disassembled, and their samples (P, M, and R soils suspension in gellan gum) contained inside of the device were transferred entirely to Petri dishes containing R2A in order to obtain culturable bacteria lawn for further metabarcoding analysis from pre-incubated assays by ISC. On the second MWC, R2A plates were inoculated with five wells and incubated at 15°C until colonies were observed. Experimental procedures are summarized in Fig. 1 E. Colonies with different phenotypes (color, size, shape, texture, brightness, and elevation) were randomly chosen, transferred, and grown in fresh R2A plates. Three hundred ninety (390) isolates were purified by streaking on agar plates. Isolates were stored at − 80°C in R2A:glycerol (7:3) and used for further assays. 2.5 Culturable bacteria lawns and isolation bacterial strains by plating-on agar method Traditional direct plating-on agar (PM) methods were carried out to obtain bacteria lawns and isolate pure culture bacteria colonies, then compared with those obtained using the ISC approach (Fig. 1 E). For the PM bacteria lawns, the same soil inoculum used for ISC (1:1000 dilution) was plated directly (in triplicates) onto the R2A agar medium (1.5%) and incubated at 15°C until complete bacteria lawns were observed on the plate. In parallel, another set of was also plated following the above-mentioned steps and incubated until single colonies were visible. Those were streaked, incubated, and purified onto R2A medium. Colonies grown on agar plates were counted after 4 days of incubation at 15°C. Colonies with different phenotypes (color, size, shape, texture, brightness, and elevation) were randomly chosen, transferred, and grown in fresh plates. Three hundred twenty-eight (328) isolates were purified by streaking on agar plates. Isolates were stored at − 80°C in R2A:glycerol (7:3) and used for further assays. 2.6 Metabarcoding 16S rRNA of culturable bacteria lawns community from in situ cultivation and plating-on agar methods The genomic DNA (gDNA) of both bacteria lawns (ISC and PM) was extracted via DNeasy PowerSoil Kit (Qiagen, Inc.). Two ml of SDW was added to the bacteria lawn, and bacteria were scratched out from the lawn and suspended in the SDW with a spreader. An aliquot of 200 µL of the obtained suspension was used for DNA extraction of "total culturable bacteria" samples. DNA sample integrity and concentration were fluorometrically determined and adjusted to ~ 20 ng uL-1. Bacteria communities from each extraction were explored by Illumina MiSeq, following the instructions of Yarimizu et al. [ 28 ]. Briefly, the v4-v5 region of the 16S rRNA gene was amplified with the 341F (5'-CCT ACG GGN GGC WGC AG-'3) and 805R (5'-GAC TAC HVG GGT ATC TAA TCC-'3) primer set coupled in the 5'-end to Illumina overhang sequences. Libraries were indexed using Nextera XT v2 indexes, and paired-end sequenced (2x300 bp) in Illumina MiSeq (Illumina Inc.). The resulting reads were processed with SHI7 to keep only QC > 35 reads() [ 29 ]., Trimmed sequences were rarefied to 25,000 reads in QIIME2, denoised and analyzed as amplicon sequence variants (ASVs) via DADA2. In parallel, biodiversity analysis for richness (observed ASVs, abundance-based coverage estimates, Chao1), diversity (coverage, Shannon, and Simpson indexes), and taxonomic assignation of the bacteria lawn communities were also performed with the same tool [ 30 ]. The community taxonomic compositions were plotted in R ( https://www.r-project.org/ ). Principal component (PCA) and Redundancy (RDA) analyses were used to ordinate the samples, with and without soil physicochemical parameters as constraints, respectively. In parallel, differences in beta diversity among the community were evaluated by analysis of similarity (PERMANOVA) using Bray-Curtis dissimilarity matrices [ 31 , 32 ]. 2.6 Genotyping and molecular identification of cultured bacterial strains of in situ and plating-on agar methods Genomic DNA was extracted from the 390 and 328 bacterial strains isolated using ISC and PM methods, respectively. The DNA purification was done using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). The Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction (ERIC–PCR) was performed to avoid isolates genotypic redundancy as described by Cid et al. [ 33 ]. Non-redundant isolates from ISC and PM culture collections were characterized by partial sequencing of their 16S rRNA genes. Partial amplification of the 16S rRNA gene was performed by endpoint PCR using the 27f (5'‒AGA GTT TGA TCC TGG CTC AG‒3') and 1492r (5'‒TAC GGY TAC CTT GTT ACG ACT T‒3') primer set [ 34 ] and sanger-sequenced. The sequences were compared with the available NCBI GenBank database using the BLASTn tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). 2.7 Antibiotic susceptibility testing Bacterial culture collection obtained from ISC and PM antibiotic susceptibility was evaluated by the Kirby–Bauer disc diffusion method in triplicates on Mueller–Hinton agar (Merck, Darmstadt, Germany). The bacteria culture collections obtained from ISC and PM were tested. Susceptibility (or resistance) was evaluated for 24 antibiotic classes and tested in the bacterial culture collections using commercial disks for susceptibility testing (Bio-Rad, Hercules, CA, USA). The tested antibiotic were grouped per family as follow: aminoglycosides (amikacin 30 µg; gentamicin 10 µg; kanamycin 30 µg), fluoroquinolones (levofloxacin 5 µg; ciprofloxacin 5 µg), glycopeptides (vancomycin 30 µg), glycylcyclines (tigecycline 30 µg; fosfomycin 200 µg), amphenicols derivatives (nitrofurantoin 300 µg); oxazolidinones (linezolid 30 µg), lincosamides (lincomycin 2 µg), lipopeptides (daptomycin 30 µg), rifamycins (rifampicin 5 µg), sulfonamide-trimethoprim-combinations (sulfamethoxazole/trimethoprim 25 µg), fifth-generation cephalosporins (cefadroxil 30 µg; cefazolin 30 µg), second-generation-cephalosporins (cefoxitin 30 µg; cefaclor 30 µg), third-generation-cephalosporins (ceftazidime 50 µg; ceftazidime/avibactam 30 µg), fourth-generation-cephalosporins (cefepime 30 µg), fifth-generation cephalosporins (ceftolozane/tazobactam 40 µg), betalactams (amoxicillin/clavulanic-acid 30 µg; cefoxitin 30 µg; ampicillin/sulbactam 20 µg; oxacillin 1 µg), macrolide (erythromycin 15 µg), polymyxins (colistin 4 µg), bacitracin 10 µg, carbapenems (ertapenem 10 µg), monobactams (aztreonam 30 µg), and penicillins (piperacillin 100 µg). All plates were incubated at 15 ºC for 2 days. Then, the inhibition halo diameter was measured considering "resistant" the determined antibiotic when the diameter was ≤ 10 mm and "sensible" when the halo measured ≥ 11 mm, as described by Marcoleta et al. [ 12 ]. 2.8 Potential Virulence Activity Isolates obtained from both ISC and PM were phenotypically assessed for the presence of VFs, meaning, such as deoxyribonuclease (DNase), hemolytic activity lecithinase and pyocyanin production using agar plate methods with some modifications described by Marcoleta et al. [ 12 ]. The DNAse activity was detected in DNAse agar (1% NaCl) containing 0.01% (w/v) of toluidine blue, and the incubation was performed at 15°C for 48h. For hemolytic activity, the bacterial strains were cultured on tryptone soy agar (TSA) plates containing 1% NaCl and supplemented with 5% sheep red cells (bioMériux SA, Marcy-l'Étoile, France) incubated at 35°C for 48h. For lecithinase and pyocyanin production tests were assays used TSA supplemented with 5% (v/v) egg yolk emulsion and Pseudomonas agar medium, respectively (Winkler Ltda. Santiago, Chile), with incubation at 15°C for 48h. Pseudomonas aeruginosa strain ATCC 27853 and Escherichia coli strain ATCC 25922 were used as positive and negative controls, respectively. All clear halo-producing bacteria in the agar media were considered positive. 3. Results 3.1 Chemical properties of frozen and ice-free soils Soil chemical properties were analyzed (Tukey's post-hoc test, P ≤ 0.05) from P, M, and R soil samples (Table 1 ). The low pH values were observed in R samples (pH 5.3) related to P and M, which had neutral pH values (7.8 and 7.6, respectively). A higher EC was also observed in the R (51.2 µs cm − 1 ), compared with M and P with values 31.6 51.2 µs cm − 1 and 29.951.2 µs cm − 1 , respectively. Similarly, TP and available P (Olsen) were ~ 3-fold higher in R samples (366 mg kg − 1 and 119 mg kg − 1 , respectively) compared to M and P samples. Moreover, TC and OM content were low in P and M (~ 3.5 mg kg − 1 and 0.4, respectively) samples, while R obtained 79.7 mg kg − 1 and 10.6 mg kg − 1 , respectively. Similarly, TN was higher in R samples (8.5 mg kg − 1 ) than in M and P with values ~ 1 mg kg − 1 samples. Similarly, exchangeable cations content differed among frozen and ice-free soils, with the R containing the highest amounts of Mg + Na + , Ca + , and Fe 2+ (Table 1 ). Table 1 Physicochemical properties of permafrost (P), moraine (M), and rhizosphere (R) soils from Ecology Glacier. Sample pH EC (µs cm − 1 ) OM (mg kg − 1 ) TP (mg kg − 1 ) P olsen (mg kg − 1 ) TC (mg kg − 1 ) TN (mg kg − 1 ) Ca 2+ (mg kg − 1 ) Mg + (mg kg − 1 ) Na + (mg kg − 1 ) K + (mg kg − 1 ) Fe 2+ (mg kg − 1 ) P 7.6 ± 0.3 a 29.9 ± 8.3 b 0.3 ± 0.1 b 109 ± 6.8 b 13.1 ± 5 b 3.1 ± 0.3 b 1 ± 0.2 b 2357 ± 158 b 402.6 ± 42.c 645.3 ± 64 a 645.3 ± 11 b 11.6 ± 0.5 b M 7.8 ± 0.2 a 31.6 ± 7.3 b 0.5 ± 0.1 b 108.7 ± 14.4 b 11.3 ± 3 b 4.1 ± 0.5 b 1.4 ± 0.3 b 3303.3 ± 365 a 678.3 ± 102 b 460 ± 26 b 552.7 ± 43 b 15.5 ± 2.9 b R 5.3 ± 0.4 b 51.2 ± 10.9 a 10.6 ± 2.9 a 366 ± 38.2 a 119 ± 36.1 a 79.7 ± 11.8 a 8.5 ± 1.1 a 1402.5 ± 232 c 1013.3 ± 175 a 557.8 ± 88 ab 554.4 ± 51 a 152.1 ± 32.7 a P = Permafrost; M = Moraine; R = Rhizosphere; EC = electro conductivity; TP = total phosphorus; P Olsen = available phosphorus; TC = total carbon; OM = organic matter; TN = total nitrogen; Ca 2+ = exchangeable calcium ion; Mg + = exchangeable magnesium ion; Na + = exchangeable sodium ion; K + = exchangeable potassium ion; Fe 2+ = exchangeable iron ion. † All deviation values represent mean ± standard error from n = 6. ‡ Different lower letters represent significant differences (one-way ANOVA, Tukey HSD test, P ≤ 0.05) among samples. 3.2 Plate count of total culturable bacteria from frozen and ice-free soils Total bacterial counts on agar plates revealed a significant difference (P ≤ 0.05) in the abundance of culturable heterotrophic bacteria among frozen and ice-free soil samples from Ecology Glacier. The abundance of culturable bacteria was greater in rhizosphere samples (from 2.94×10 5 to 8.21×10 5 CFU g − 1 dw) than in the moraine and permafrost samples (from 1.39×10 3 to 6.35×10 3 CFU g − 1 dw) (Fig. 2 ). Higher diversity in bacterial phenotypes was observed across samples using R2A media (2.86×10 3 to 8.21×10 5 CFU g − 1 dw) than LB 1:10 (1.95×10 3 to 4.97×10 5 CFU g − 1 dw), and NM1 (1.39×10 3 to 2.96×10 5 CFU g − 1 dw) during the 30 d of incubation (Fig. 1 A). Pearson correlation showed significant (P ≤ 0.05) positive correlations among bacterial abundance with most nutrients but negatively correlated with pH and Ca + 2 (Supplementary, Table S1 ). 3.3 Metabarcoding of culturable bacteria lawns 3.3.1 Alpha and beta diversity The 16S rRNA gene metabarcoding of culturable bacteria lawns from frozen and frozen-free soil recovery by using PM and ISC revealed differences in ASVs between R (164) and M (125) samples but not with P (146) in PM methods (P ≤ 0.05). Alpha diversity showed that the Chao1 index was influenced by the culturing method and significantly higher in the R (PM: 165.3 and ISC: 134.8) compared to M (PM: 125.3 and ISC: 125.5) and P (P: 146.8 and ISC: 119.5). Concerning culture methods, the highest ASV and richness values were found using PM compared to ISC methods (Fig. 3 ). However, Shannon and Inverse Simpson indexes were contrasting, with higher values in the R using PM (3.3 and 14.7, respectively). In contrast, by using ISC, the diversity index was higher in M samples (2.9 and 11.6, respectively). In addition, the P communities were identified as the less diverse (P ≤ 0.05) in this study, independently of isolation methods (Fig. 3 a). The beta diversity showed noticeable differences in the bacteria community lawn structure between both soil sample type and isolation methods, as revealed by PCA (Fig. 3 b). In this analysis, both PC1 and PC2 axes explained 54% of the variability of the bacterial communities (P ≤ 0.05), with the culturing method appearing more relevant than the soil sample type. The PERMANOVA analysis confirmed the differences in the bacterial community structure in lawns associated with the method (R 2 = 0.30; P ≤ 0.001) and as well sample type (frozen and ice-free soils; R 2 = 0.34; P ≤ 0.001). In this sense, a clear differentiation in bacterial community structure was observed according to the recovery isolation methods used. Regarding soil type, the bacteria communities showed more similarity between the M and P samples than those in the R samples, independently of cultivation methods. Once physicochemical variables were introduced into the structure analysis, the redundancy analysis (RDA) showed that pH (R 2 = 0.12, P ≤ 0.003) and electroconductivity (R 2 = 0.13, P ≤ 0.001) were the main soil chemical factors with a significant effect on bacterial community lawns. However, their inclusion reduced the variance explanation to 44.1%, revealing that underlying variables not measured in this study are significant for each bacteria community structure (Fig. 3 c). 3.3.2 Taxonomic assignation of bacterial community lawns The taxonomic assignment of 16S rRNA sequences of bacterial composition from lawns revealed phyla Pseudomonadota (55–75%), Actinomycetota (20–35%), and Bacteroidota (5–10%) as the most abundant bacterial taxa in culturable lawn bacteria lawn communities. In addition, members of Gammaproteobacteria were the most abundant class in frozen and ice-free soils, with an average relative abundance varied from 50.1–51.6%, from 40.6–74.4%, and from 51.4–37.8% for P, M, and R using PM and ISC, respectively (Fig. 4 a). On average, the Actinobacteria class was also the most dominant phyla in the R using ISC with 33.2% relative abundance, while M and P samples ranged from 18.7–14.8%, respectively. In contrast, when using PM, a higher abundance of Actinobacteria was observed in M (29.9%) and P (31.8%) than those obtained in R samples (13.8%). Similarly, Bacteroidia was the second dominant class in P (30.6%) under the ISC method, but their abundances were less observed in M and R samples under PM varied from 14.0–11.6%. Remarkably, this class was less than 2% abundant in P samples using PM (Fig. 4 a). Noteworthy, the Bacilli class was only observed in samples subjected to the PM method, with values of 12.2%, 7.9%, and 7.3% in M, P, and R samples, respectively (Supplementary, Table S2 ). Despite the dominance of the same classes for both methods and all soil samples, differences were observed across samples at the genus level (Fig. 4 b). Pseudomonas was the most abundant genus across soil samples, with values ranging from 32.1–16.3% for PM and 35.8–23% for ISC (Fig. 3 b). Moreover, the Janthinobacterium genus was only observed in M and P using both PM (3.5–5.1%) and ISC (7.4% and 16.4%) culturing methods. Notably, members of the Stenotrophomonas genus were abundant across all samples, varying from 7.6–15.9% using PM. In addition, Sphingobacterium (11.8–13.4%) genera were only observed in bacteria lawns from moraine samples obtained exclusively using PM. It is important to mention that R samples showed a significantly different pattern in bacterial taxonomic assignations compared to M and P, independently of the culture method used (Fig. 4 b). Pedobacter genus was only observed in rhizosphere samples, ranging from 12.9–13.7% by PM and from 10.9–11.3% via ISC methods. Additionally, rhizosphere bacteria lawns composition under PM was presented members of Bacillus (4.3–12.9%), Rhodanobacter (3.6–4.0%) and Lysobacter (1.6–2.2%) genera, while Flavobacterium (~ 4.9%) Microbacterium (~ 30.9%), Sphingomonadaceae family and Arthrobacter (2.3–6.4%) genera were dominant in bacteria lawns only recovery by ISC (Fig. 4 b). 3.4 Bacterial culture collections and isolate strain identification In total, 390 and 328 bacterial strains were isolated from iced and iced-free soil samples in Ecology Glacier using PM and ISC, respectively. Based on ERIC-PCR assays, 21% (82/390) of isolates recovered with PM methods showed distinct genetic variability (24, 33, and 25 from M, P, and R-associated isolates, respectively). In addition, using ISC methods, 76/328 (23%) genotypes were classified as non-redundant isolates, which included 19, 27, and 30 for M, P, and R, respectively (Fig. 5 ). Despite the isolation method used, M samples showed the highest percentage of genetic variability than those observed in R and P samples, respectively (26,4% ISC and 28.2% PM). Alpha diversity analysis showed higher richness values in R samples using both isolation methods (PM:9 and ISC: 8). Moreover, ISC methods also revealed a higher richness in M (8) and P (7) samples compared to those obtained via PM in the M and P samples (6 and 4, respectively). Similarly, the Shannon index reveals the higher diversity in M (1.590) and P (1.120) samples using ISC methods compared to values obtained via PM (1.516 and 0.495, respectively). Based on the Simpson index, the culturable community by PM was more diverse in M and R samples (0.722 and 0.725, respectively) than the measured by ISC (0.687 and 0.609, respectively). Despite this, the ISC method (0.488) was able to recover more diverse bacterial isolates compared to PM (0.222) in P samples, as shown in the Simpson index (Fig. 5 ). The taxonomic characterization of isolated strains showed that the most abundant phyla were Pseudomonadota (76%), followed by Actinomicetota (17,7%), Bacteroidota (3,15%) and Bacillota (3,15%) (Fig. 5 ). A total of 24 different bacterial genera were isolated from all soil samples using both ISC (13 genera) and PM (15 genera) approaches (Fig. 5 ). In detail, Pseudomonas was the most isolated genus across soil samples using PM (51 isolates) and ISC (47 isolates) methods (48, 29, and 21 isolates from P, R, and M, respectively). Other isolates within the Pseudomonadota phylum were assigned to the Stenotrophomonas (5 isolates) obtained by both methods. Moreover, the Brucella genus (3 isolates) was isolated by PM and the Janthinobacterium genus (3 isolates) by ISC. Finally, using both methods, Burkholderia and Sphingomonas genera (2 isolates) were isolated from the R soil samples. The next dominant phylum was Actinomycetota, represented by the Arthrobacter (10 isolated by PM and 6 by ISC), distributed as 6 isolates from M, 5 isolates from R, and 4 isolates from P soil samples (Fig. 5 ). Moreover, members of the Microbacterium (6 isolates) and Curtobacterium (2 isolates) genera were only isolated by the ISC method. In contrast, PM exclusively obtained isolates belonging to the Pseudarthrobacter genus (3 isolates). Isolates belonging to Bacteroidota phylum were distributed among the Flavobacterium (3 isolates) and Pedobacter (2 isolates) genera obtained by PM and ISC methods, respectively (Fig. 5 ). In particular, members of the Bacillota phyla were obtained exclusively using the PM method, coincidentally with the high-throughput sequencing (HTS) approach results. Members of rare taxa (unique colonies observed in culture collection) Curtobacterium , Achromobacter , Bosea , Massilia , Rhodococcus , Sporosarcina , Staphylococcus , Phyllobacterium , and Polaromonas genera were also isolated by the ISC method. Surprisingly, poorly studied bacterial taxa from Antarctic soils, such as Rathayibacter and Rugamonas , were isolated using ISC. 3.5 Antibiotic resistance and virulence patterns of plate and in situ cultured bacterial strains 3.5.1 Qualitative antibiotic resistance The resistance (or sensibility) to 35 types (24 classes) of antimicrobial agents was tested in the bacterial collection obtained by the disk diffusion susceptibility test (Kirby-Bauer technique). Antibiotic pattern distribution was higher in isolated strains from rhizosphere samples than those obtained from isolates of permafrost and moraine samples (Fig. 6 ). Based on the method, a higher count of resistances was observed in strains isolated by the ISC approach compared to PM, mainly in bacterial culture collections obtained from M and P soil samples. A higher number of bacteria (86%) were resistant to beta-lactams, followed by Cephalosporins (77%), Oxazolidines (71%), and Liconsamids (70%), among others, across all soil samples. Bacterial strains belonging to the Flavobacterium genus resisted more than 22 different antibiotics, while Pseudomonas and Curtobacterium genera showed multi-resistance over 18 different antibiotics independently of isolated methods used. Remarkably, we found 42 isolates resistant to Carbapenems, the majority members of Pseudomonas (35 isolates), Pedobacter , and Curtobacterium (2 isolates, respectively). Additionally, we found resistance to some antibiotics defined as "last-line," such as Colistin, in members of Pseudomonas (30 isolates), Arthrobacter (4 isolates), Pseudoarthrobacter , and Flavobacterium (2 isolates). 3.5.2 Potential virulence activity The cultured bacteria collections (76 isolates by ISC and 82 by PM) were subjected to virulence potential assessment, including deoxyribonuclease (DNase), hemolytic activity lecithinase, and pyocyanin production. A total of 12 of 23 genera (52.2%) carried out at least one virulence factor evaluated, representing 41 isolates and 58 isolates (99 isolates strains) from ISC and PM, respectively. A higher number of virulence factors was observed in isolates from P compared to R and M samples, independently of the culture method (Fig. 7 ). Based on those, higher virulence was observed among bacteria isolated by PM compared to ISC. Overall, virulence factor assays revealed a prevalence of > 35% for both lecithinase activity (75 isolates) and hemolytic activity (68 isolates) among isolates from all the soil samples, while pyocyanin production was positive to 39 isolates (20%) from 158 strains evaluated Additionally, DNAse activity was observed with low frequency (~ 7%; 13 isolates) among the isolates tested. In detail, members of the Pseudomonas genus were positive for all virulence factors evaluated independently of isolated methods used. In contrast, isolated strains belonging to Microbacterium , Brucella , and Sporosarcina genera carried out only the DNAse activity (Fig. 7 ), while pyocyanin production was the main activity found in isolated Sphingomonas and Staphylococcus genera. 4. Discussion This study represents the first effort to investigate the culturable bacterial communities in the soils surrounding the Ecology Glacier, Antarctica, using traditional and ‘ in situ ’ cultivation methods, focusing on characterizing the antibiotic resistance profiles and virulence factors exhibited by the isolated bacterial strains. A higher abundance of bacteria counts and diversity was found in R compared to M and P samples. The rhizosphere is the primary hotspot for microbial colonization and activity in soils, highlighting the influence of plant-microbe interactions on shaping soil microbial communities [ 9 ]. Root exudates and decomposing plant material provide a rich source of nutrients and create micro-niches that support a greater abundance and diversity of bacteria [ 35 ]. These results concord with those published by Purcell et al. [ 36 ], in which they observed higher bacterial counts in early terrestrial zones with apparent vegetation on King George Island. The differences in bacterial counts could be attributed to the freezing soil, such as moraine and permafrost, which are considered a restricted niche where microbial colonization depends on limited water and nutrient availability [ 37 ]. The use of both traditional plating-on agar method (PM) and " in situ' ' cultivation (ISC) proved crucial in capturing a broader spectrum of bacterial diversity. The ISC approach, utilizing MWC devices, allowed the recovery of similar ASVs but a significantly more diverse bacterial community lawn than traditional culturing across all soil samples. This observation contrasts with several studies where similar "in situ" techniques have demonstrated increased ASV recovery from various environments, such as marine sponges [ 38 ], corals [ 39 ], and rhizosphere [ 27 ]. This discrepancy could be attributed to inherent characteristics of the soil and water availability required for the ISC methods, which might have limited the growth of some bacterial taxa. Despite this, differences in the diversity of lawn bacteria communities were found in the R compared with the M and P soil samples independently of isolation methods. Previous studies on culturable bacterial communities of M samples from Ecology Glacier pointed out that the chemical changes in soils modulated the taxonomic diversity of culturable bacterial communities [ 40 ]. Based on the RDA analysis, significant differences were observed in the structure of bacteria lawn obtained by using ISC and PM, modulated by the electronic conductance and pH. Several studies have shown that soil salinity and pH can modify the composition of the bacterial community in Antarctic soils [ 41 , 42 ]. The observed differentiation in diversity and composition of the bacterial community could be attributed to the type of samples, where the nutrient content of R was higher than M and P, given the plant influence, as evidenced by our analytical findings (Table 1 ). Independent of the isolation method, the Illumina-based sequencing analyses revealed the dominance of members of the phyla Gammaproteobacteria in all studied bacteria lawn communities. Members of Proteobacteria are the most abundant phyla in Antarctic soils [ 43 ]. Additionally, using both methods, Actinobacteria and Bacteroidetes phyla were prevalent in bacteria lawns recovery from soil samples. The Actinobacteria and Bacteroidetes are the dominant bacterial groups in soils in general, as well as in the Antarctic soil microbiome [ 44 , 45 , 12 ]. At the genus level, differences were observed between bacteria lawns from all soil samples, as members of Pseudomonas , Micrococcaceae family, and Flavobacterium were most abundant in moraine and permafrost by ISC methods. These genera are common glacier-ice lineages present in Tibetan Plateau ice cores [ 46 ], China glaciers [ 47 ], and Antarctica [ 48 ]. Similarly, bacteria lawn communities from R were dominated by Pseudomonas , Microbacterium , Stenotrophomas , and Pedobacter genera. This differentiation concerning alpha and beta diversity analysis in bacteria lawns from frozen and iced-free Antarctic soils is known. The highest diversity found in R samples suggests that heterotrophic bacteria are abundant in Antarctic vascular plants rhizosphere. These differences may result from the significant influence of birds and animals around vegetation, which could exert selective pressure on the rhizosphere microbiome [ 49 ]. Based on the methods, the R bacteria lawn composition under PM was dominated by members of the Bacillus genus. In contrast, the Arthrobacter genus was dominant in bacteria lawns, only recovered by ISC. Members of the bacterial Arthrobacter and Bacillus genera are both readily cultured and commonly identified in Antarctic soil communities [ 50 , 51 ]. Regarding isolated bacterial strains, our study showed affiliations to 24 different bacterial genera, where Pseudomonas members were the most dominant taxa for both PM and ISC methods. After Pseudomonas , Stenotrophomonas , Arthrobacter , and Microbacterium (Actinomicetota) were the most dominant taxa. The isolated strains belonging to Bacillota phyla were mainly represented by the Bacillus genus, only recovered by PM. These results are coincident, as observed from the bacteria lawns community analysis. Using ISC, isolating poorly studied genera (such as Bosea , Rathayibacter , and Rugamonas ) was possible. Studies have shown that up to 80% of bacteria from Antarctic soils are "yet-to-be cultured", including phylotypes from well-studied phyla such as the Actinobacteria [ 52 , 53 , 54 ]. However, only a few taxa were unique in culture collections between ISC and PM culture methods, as revealed by the diversity index, suggesting there are still considerable culturomic challenges associated with bacteria domestication [ 55 ]. Despite this, P and M showed higher diversity when pre-culture incubation by ISC was applied compared to R samples. This finding underscores the limitations of relying solely on traditional cultivation techniques, which often favor fast-growing and readily culturable microorganisms. Employing diverse cultivation strategies is essential for a more comprehensive understanding of soil bacterial diversity and ecological function in Antarctic soils. The widespread antibiotic resistance patterns observed among the isolates recovered by ISC and PM revealed resistance to several antibiotics, including beta-lactams, tetracyclines, and aminoglycosides, indicating a broad resistance profile in culturable bacteria communities. Numerous studies have demonstrated the occurrence and abundance of a natural resistome in iced soils as a response to microbial competition and environmental pressures in the Antarctic ecosystem [ 43 , 56 , 57 ]. Despite this, animal and human influence are often described as an important source of bacteria-resistant dissemination [ 58 ]. Our results revealed differences in the distribution of antibiotic patterns among isolate strains, where the ones from the R samples were resistant against a broad spectrum of antimicrobial drugs compared to those recovering from M and P samples. Calisto et al. [ 59 ] evidenced that 63% of isolates (47 strains) from R of D. antartica were multidrug-resistant to 14 over 21 antibiotics tested. However, Pseudomonas resistance phenotypes observed in P samples significantly differ between the R and M isolates, emphasizing the AMR role as a competition mechanism in driving species survival. Based on the method, ISC yielded more resistant bacterial strains than PM, particularly in bacterial cultures obtained from M and P samples. Although most carbapenem-resistant isolates belonged to Pseudomonas (35 isolates), the ISC also facilitated the isolation of carbapenem-resistant Pedobacter and Curtobacterium . These results conflict with those published by Wong et al. [ 60 ], in which they reported that Pedobacter sp. strains BG5 isolated from soils of King George Island, Antarctica, were sensitive to imipenem, indicating its lack of resistance to this carbapenem antibiotic. Moreover, Curtobacterium species are cosmopolitan plant pathogens exhibiting distinct diverse antibiotic resistance patterns [ 61 ], but studies based on AMR in Antarctic isolates are still scarce. Notably, 86% and 76% of the isolated strains resisted beta-lactams and cephalosporins, respectively. Previous studies have detected multidrug-resistant bacteria collected from soils, wildlife, freshwater, and glacier environments, among other habitats in Antarctica [ 12 , 62 , 58 ]. These studies reported bacterial isolates with high levels of antibiotic resistance, including aminoglycosides, β-lactams, and trimethoprim, consistent with our findings. Additionally, isolates express multidrug-resistant phenotypes with susceptibility to 'last-line' antibiotics, such as Colistin, mainly in Pseudomonas , Arthrobacter , Pseudoarthrobacter , and Flavobacterium . Pseudomonas are common resistant-bacteria taxa whose resistome compromised more than 170 genes (ancestral genes) that could confer resistance to natural or synthetic antibiotics [ 56 ]. Remarkably, Flavobacterium strains in this study resisted more than 22 clinical antibiotics. These results are in agreement with a previous report of Flavobacterium identified from iced and iced-free soils in the Arctic and Antarctica exhibiting distinct resistance mechanisms [ 12 , 63 ]. Although the genera Arthrobacter and Pseudoarthrobacter comprise numerous species isolated from the Antarctic region [ 16 ], data on antibiotic susceptibility of environmental isolates from iced soils is scarce [ 64 ]. On the other hand, detecting VFs in many isolates suggests potential pathogenicity within these culturable bacterial communities. It is known that VFs and antibiotic resistance are recognized as crucial mechanisms for adaptation, competition, and colonization processes under harsh conditions [ 65 ]. The results also revealed that the isolates had positive percentages of > 40% lecithinase activity, 35% hemolytic activity, 21% pyocyanin production, and 5% DNAse activity. Several VFs have been reported from the Arctic and Antarctic bacterial strains, mainly proteinase and DNase enzymes [ 66 , 67 ]. In addition, previous studies reported that 11 virulence factors amongst the Antarctic isolates were characteristic of E. coli strains more commonly associated with humans [ 65 ]. The ability to produce enzymes like lecithinase and DNase, exhibit hemolytic activity, and produce toxins like pyocyanin indicates the potential for these bacteria to cause harm to plants or animals. In addition, metagenomic analyses revealed the occurrence of VFs genes (VFGs) in soil from the Fildes peninsula in Antarctica [ 12 ], indicating that resistance mechanisms would result from thousands of years of evolution [ 68 , 69 ]. While the data on antibiotic resistance and VFs in Antarctic Glaciar are scarce, we have previously published a study that reported the resistome and virulome in Union Glaciar using metagenomic and culture-dependent approaches [ 70 ], in which the bacterial isolate was resistant to up to 24 clinical antibiotics. In addition, some isolates produced putative VFs, including siderophores, pyocyanins, and exoenzymes with hemolytic activity, lecithinase, protease, and DNase. It is relevant to mention that these resistant bacterial isolates mainly belonged to the Pseudomonas , Arthrobacter , Plantibacter , and Flavobacterium genera, similar to our findings. Despite the importance of horizontal transference of antibiotic resistance genes and VFs to microbial communities, no previous reports have explored potential VFGs or the production of VFs among bacteria from Antarctica. While additional research is needed to assess the actual risk posed by these microorganisms, their presence warrants careful monitoring. These results highlight the importance of the One Health approach, emphasizing the need for multidisciplinary efforts to understand the pathways of resistant bacteria and virulence factors, especially in the current climate emergency scenario. 5. Conclusions This study revealed a remarkably diverse culturable bacterial community inhabiting the soils surrounding the Ecology Glacier, with Pseudomonadota, Actinomicetota, and Bacteroidota as the dominant phyla. Our selected " in situ " cultivation method successfully recovered a wider range of bacteria genera than the traditional plating-on agar method, including less studied genera such as Bosea , Rathayibacter , and Rugamonas . This finding emphasizes the importance of comprehensively employing diverse culture techniques to represent soil bacterial diversity. Many isolated bacterial strains exhibited multidrug resistance, particularly against beta-lactams, cephalosporins, oxazolidinones, and lincosamides. In many isolates, virulence factors, including lecithinase, hemolytic activity, pyocyanin production, and DNase activity, suggest potential pathogenicity within these bacteria communities. These approaches to Antarctic bacteria must be considered in the near future to properly understand the effects of ice melting in Antarctica and possibly in other environments. Declarations Author Contributions: Conceptualization, A.E.M, F.P.C., V.C., M.A.J. and J.J.A.; formal analysis, C.V., N.H., M.A.C. and J.J.A.; data curation, N.H. and J.R.; investigation, C.V., N.H., A.E.M., F.P.C., L.A.B., V.C., M.A.J. and J.J.A.; writing—original draft preparation, C.V. and J.J.A.; writing—review and editing, C.V., M.A.C., J.R., A.E.M., F.P.C., L.A.B., V.C., H.S., M.A.J and J.J.A.; funding acquisition, A.E.M., F.P.C., V.C., M.A.J. and J.J.A. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by The Regular Research Team Projects in Science and Technology & Thematic Research Team from The Chilean National Agency for Research and Development (ANID), code ACT210044. Partial support was also provided by The National Fund for Scientific and Technological Development (FONDECYT), Project no.1240602 and 1221228 (to M.A.J and J.J.A.), and by the Millennium Science Initiative Program, code ICN2021_044 (to V.C. and J.J.A.). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All sequences obtained in this study were deposited in the NCBI GenBank Nucleotide database (https://www.ncbi.nlm.nih.gov/genbank/) under bioproject PRJNA1095903. Additional raw data from this study is available under request to the corresponding author (J.J.A). 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Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil. ISME J. 3 , 243–251 (2009). D'Costa, V. M. et al. Antibiotic resistance is ancient. Nature 477 , 457–461 (2011). Arros, P. et al. Life on the edge: Microbial diversity, resistome, and virulome in soils from the Union Glacier cold desert. Sci. Total Environ. 957 , 177594 (2024). Additional Declarations No competing interests reported. Supplementary Files TableS1.docx TableS2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6667169","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":473557137,"identity":"e546726d-4522-438d-a5d6-92d94b71229f","order_by":0,"name":"Jacquelinne J. Acuña","email":"data:image/png;base64,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","orcid":"","institution":"Universidad de La Frontera","correspondingAuthor":true,"prefix":"","firstName":"Jacquelinne","middleName":"J.","lastName":"Acuña","suffix":""},{"id":473557138,"identity":"a61648c5-b9c9-4444-b928-cf3ecc452fc3","order_by":1,"name":"Constanza Venegas","email":"","orcid":"","institution":"Universidad de La Frontera","correspondingAuthor":false,"prefix":"","firstName":"Constanza","middleName":"","lastName":"Venegas","suffix":""},{"id":473557144,"identity":"59fe99ea-44b2-437e-bb1b-219e799bbcd6","order_by":2,"name":"Marco A. Campos","email":"","orcid":"","institution":"Universidad Católica de Temuco","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"A.","lastName":"Campos","suffix":""},{"id":473557147,"identity":"d4c1cb66-87b2-4e84-a683-3f7001854afd","order_by":3,"name":"Nicole Huerta","email":"","orcid":"","institution":"Universidad de La Frontera","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Huerta","suffix":""},{"id":473557151,"identity":"a90a9a3d-286c-41bd-824b-ee066ade15aa","order_by":4,"name":"Joaquin Rilling","email":"","orcid":"","institution":"Universidad de La Frontera","correspondingAuthor":false,"prefix":"","firstName":"Joaquin","middleName":"","lastName":"Rilling","suffix":""},{"id":473557153,"identity":"ad6d312c-73d8-4c3d-a2c3-233e19154f68","order_by":5,"name":"Francisco P. Chávez","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"P.","lastName":"Chávez","suffix":""},{"id":473557154,"identity":"adbdcaab-39e1-481d-96f6-4cb71ed5955a","order_by":6,"name":"Andrés E. Marcoleta","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Andrés","middleName":"E.","lastName":"Marcoleta","suffix":""},{"id":473557155,"identity":"517e0b68-3ab5-4169-b7e4-da7e616af09c","order_by":7,"name":"Verónica Cambiazo","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Verónica","middleName":"","lastName":"Cambiazo","suffix":""},{"id":473557156,"identity":"44f8681b-c368-48fd-9aac-892e542b9ed9","order_by":8,"name":"León Bravo","email":"","orcid":"","institution":"Universidad de La Frontera","correspondingAuthor":false,"prefix":"","firstName":"León","middleName":"","lastName":"Bravo","suffix":""},{"id":473557158,"identity":"418271cf-42b9-48d1-9890-2d77d56ff283","order_by":9,"name":"He Shan","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Shan","suffix":""},{"id":473557159,"identity":"b5841de8-85c6-48f6-8a5d-c2385ca2b5cd","order_by":10,"name":"Milko A. Jorquera","email":"","orcid":"","institution":"Universidad de La Frontera","correspondingAuthor":false,"prefix":"","firstName":"Milko","middleName":"A.","lastName":"Jorquera","suffix":""}],"badges":[],"createdAt":"2025-05-14 20:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6667169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6667169/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85084592,"identity":"7253ccce-3a62-439b-8283-9f2b0dbb2655","added_by":"auto","created_at":"2025-06-20 18:53:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":628420,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667169/v1/f164898200baf74e7b5e9272.jpg"},{"id":85084363,"identity":"22a7c2f8-7e62-4d18-9c17-7214c791a68b","added_by":"auto","created_at":"2025-06-20 18:45:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":342694,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667169/v1/346b1629531b51d06681fee9.jpg"},{"id":85085053,"identity":"0f3d4e5f-af9c-4e69-b443-659cd8ac89c1","added_by":"auto","created_at":"2025-06-20 19:01:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":501836,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667169/v1/0689bf9685237e9cd530d47e.jpg"},{"id":85085054,"identity":"b8a5a776-3643-4b96-93fc-339a048d8e92","added_by":"auto","created_at":"2025-06-20 19:01:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":806950,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667169/v1/31ba2e7dd7d5ff46e8d1e3bc.jpg"},{"id":85084367,"identity":"f4c2f78b-b7c3-4020-af88-a9d3a6d26fa4","added_by":"auto","created_at":"2025-06-20 18:45:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":490392,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure 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legend.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667169/v1/1821f9e6636c141d83c8c928.jpg"},{"id":100796934,"identity":"6706193d-3a31-41eb-b592-74410ce009c4","added_by":"auto","created_at":"2026-01-21 13:46:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6064924,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6667169/v1/24ac6bba-62cc-4091-8204-b4f3b8fa703d.pdf"},{"id":85084595,"identity":"84e78d8d-525b-419d-a691-a0b70c7196b0","added_by":"auto","created_at":"2025-06-20 18:53:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":44818,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6667169/v1/cc25d3751bc7a07010ef27e1.docx"},{"id":85084359,"identity":"0e5a8b09-8e4a-4cb9-9806-ad37f8af8fda","added_by":"auto","created_at":"2025-06-20 18:45:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15778,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6667169/v1/50c1cec8691b379bcfafd485.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of Culturable Bacterial Communities in Permafrost, Moraine, and Rhizosphere Soils near the Ecology Glacier (King George Island, Maritime Antarctica): Patterns of Antibiotic Resistance and Virulence Factors in Isolated Bacteria","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eClimate change significantly contributes to the melting of glaciers and permafrost around the globe [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Antarctica is the fastest warming area [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], exhibiting accelerated deglaciation in its northeast areas, such as the Antarctic Peninsula [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Due to ice mass shrinkage, the newly formed terrain becomes the habitat for microorganisms [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] brought from other sites in addition to autochthonous microorganisms [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Bacterial communities are determinants of glacier foreland, which play crucial roles in biogeochemical cycles and accelerating soil formation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Krauze et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] found that microbial communities near glaciers influence soil formation and nutrient speciation, which can modulate and promote the colonization and establishment of the unique native vascular plants \u003cem\u003eDeschampsia antarctica\u003c/em\u003e and \u003cem\u003eColobanthus quitensis\u003c/em\u003e. Several studies in the Antarctic have focused on unraveling the structure and functions of soil and plant microbiomes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the consequences of climate change, such as ice melting on culturable indigenous soil bacteria communities, as well as their functions, structures, and interactions in the plant-soil-microbe continuum in Antarctica, remain poorly understood and difficult to predict.\u003c/p\u003e \u003cp\u003eClimate change can also lead to the emergence or reemergence of infectious diseases by altering the distribution of microbial pathogens and their vectors, posing a threat to humans, animals, and plants [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Bacteria community studies showed the presence of a natural resistome and virulome from Antarctic soils, as well as evidence of anthropogenic introduction of antibiotics multiresistance (AMR), highlighting the impact of human activities in this less intervened environment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, antimicrobial resistance profiles are strain-specific and environment-dependent, reflecting unique evolutionary adaptation processes rather than solely a recent adaptation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The Antarctic ice-free zone expansion could lead to the discovery of previously unknown microorganisms carrying novel virulence factors (VFs) and multidrug-resistant (MDR) strains. Furthermore, new vegetation and animals could facilitate the proliferation of pathogens, promoting gene horizontal transfer among microbes and increasing the risk of disease emergence [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCultivation-independent techniques have increased our current knowledge about the bacteria diversity of Antarctic soil habitats [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], strongly contrasting with the narrow diversity recovery using the traditional plating-on agar method [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The 'in situ' cultivation approach aims to isolate previously uncultured bacteria with rich biodiversity and novelty [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For this, microporous membrane-based microwell well chamber devices (MWC) installed in soils allow the exchange of chemical components from the surrounding soil into each well. The environment provides unknown but essential metabolites, signaling molecules and nutrients for growth, as co-culture with other microorganisms can increase bacteria culturability, particularly in moraine and permafrost soils where the frozen conditions pose additional challenges [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This '\u003cem\u003ein situ\u003c/em\u003e' cultivation on sub-Antarctic and Arctic soil using i-Tip and cryo-iPlate, respectively, allowed the isolation of priorly unculturable bacteria [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although glacier retreatment has been known for a long time and represents one of the most damaging effects of climate change, the underlying culturable microbiome and microorganisms containing AMR and VFs remain largely unexplored in Antarctica. We isolated culturable bacteria by traditional plating-on agar (PM) and '\u003cem\u003ein situ\u003c/em\u003e' cultivation (ISC). The latter employed an MWC along the expansion of frozen to ice-free soil zones of the Ecology Glacier located on King George Island (South Shetland Islands, Maritime Antarctic). Culturable bacteria lawns and their respective isolated strain collections were characterized by 16S rRNA gene partial sequencing. Isolated strains were also screened by their AMR and VFs. Our results provide insights into the culturable bacterial diversity during Ecology Glacier's frozen expansion to ice-free soil zones and their potential risk by AMR and VFs.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sampling\u003c/h2\u003e \u003cp\u003eSamples were collected from three distinct types of soil environments near the Ecology Glacier, inside the Antarctic specially protected area (ASPA, N\u0026deg;128: Occidental coast of Admiralty Bay), Polish Henryk Arctowski Station. The samples were from frozen permafrost (P) and two ice-free soil types, moraine (M) and rhizosphere (R) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The samples were taken during the 58th Scientific Antarctic Expedition (ECA58) in the summer season (November 2021 - March 2022), following the protocols and supervision by the Chilean Antarctic Institute (INACH; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.inach.cl/\u003c/span\u003e\u003cspan address=\"https://www.inach.cl/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Six samples of both P (62\u0026deg;10'15.4 \"S 58\u0026deg;28'21.6 \"W) and M (62\u0026deg;10'14.4 \"S 58\u0026deg;28'20.5 \"W) were collected by removing a 10 cm surface layer and then introducing\u0026thinsp;~\u0026thinsp;1 kg of the underlying soil into Whirl-Pak sterile bags (Sigma-Aldrich, Saint Louis, MO, USA) using an aseptic spade and storing them at 4\u0026deg;C until processing. In parallel, the R samples were collected in an area with a notable presence of vegetation. Then, six specimens of \u003cem\u003eD. antarctica\u003c/em\u003e (62\u0026deg; 09 '48. 5\" S 58\u0026deg; 27' 48.3\" W) were randomly taken in a 500 m transect from the M sampling site, in the direction of Henryk Arctowski Polish Antarctic Station by using a clean spade to remove intact roots from the soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Collected rhizosphere samples were placed within Whirl-Pak sterile bags (Sigma-Aldrich, Saint Louis, MO, USA) and stored at 4\u0026deg;C. All samples were transported on ice to the Applied Microbial Ecology Laboratory (EMALAB) at Universidad de La Frontera, Temuco, Chile, for further cultivation experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Soil physicochemical characterization\u003c/h2\u003e \u003cp\u003eAir-dried soil samples were used to determine soil pH, electrical conductivity (EC), organic matter (OM), total phosphorus (TP), available P (Olsen), total carbon (TC), total nitrogen (TN), and exchangeable cations (Ca\u0026sup2;⁺, Mg\u0026sup2;⁺, Na⁺, K⁺, and Fe⁺). After air drying, the soil was macerated using a mortar and pestle to remove large constituents such as rocks and organic debris. The pH and EC were measured in 1:2:5 and 1:5 soil/deionized water suspension with a high-grade benchtop meter (model H15522; Hanna Instrument Ltda., Leighton Buzzard, UK). The OM contents were estimated by wet digestion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The TP was extracted from the soil samples using NaOBr [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and quantified as described by Murphy and Riley [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Inorganic phosphorus (POlsen) was extracted using the bicarbonate method (pH 8.5 in 0.5 M NaHCO\u003csub\u003e3\u003c/sub\u003e) and analyzed using the molybdate-blue method [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For the TC and TN content, soil samples were firstly homogenized to under 0.1 mm and freeze-dried. Then, 1.5\u0026ndash;2.5 mg aliquots of the samples were weighed into an EA 3000 automated elemental analyzer (Eurovector, Italy). The elemental composition was determined by interpolation using a suitable calibration curve (R\u0026sup2; = 0.98) with EDTA as a standard (99.4% purity; LECO\u0026reg;, USA). The results were expressed in mg of TP, TC, or TN per kg of dry-weight soil sample (mg kg⁻\u0026sup1; DW). Exchangeable cations were extracted using 1 M CH₃COONH₄ at a pH of 7.0 and then analyzed using flame atomic absorption spectrophotometry [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.3 Selection of medium and determination of total culturable bacteria\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThree types of non-selective media were used in this study in order to determine the plate count of culturable bacteria from P, M, and R soil samples and to test the best media for following used in culture experiments for both traditional direct plating-on agar method and as well sub-cultivation for in situ method. One gram of each sample (in triplicates) was suspended in 25 ml of sterile distilled water (SDW) and sonicated for 30 s at 130 W (20 kHz). Appropriate serial dilutions were then spread onto three media used in this study: (1) Reasoner's 2A broth (R2A) medium (Sigma-Aldrich, Saint Louis, MO, USA), (2) NM-1 oligotrophic agar medium [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and (3) 1:10 diluted LB media (10% of the manufacturer's suggested concentration, Sigma-Aldrich, Saint Louis, MO, USA). All media contained 1.5% agar and were supplemented with 10 \u0026micro;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of cycloheximide (Sigma-Aldrich, Saint Louis, MO, USA) to prevent fungal growth. Colonies grown on agar plates were counted after 14 days of incubation at 15\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Culturable bacteria lawns and isolation bacterial strains by in situ cultivation method\u003c/h2\u003e \u003cp\u003eThe '\u003cem\u003ein situ\u003c/em\u003e' cultivation (ISC) method considered using a MWC to obtain isolates from all P, M, and R soil samples. The MWC was prepared as described previously by Acu\u0026ntilde;a et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and Berdy et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To construct an MWC, two rectangular pieces of polyamide plastic and one central piece of polytetrafluoroethylene (PLASTIGEN, Chile) with 48 through-holes (w\u0026thinsp;=\u0026thinsp;3 cm, l\u0026thinsp;=\u0026thinsp;1.2 cm, d\u0026thinsp;=\u0026thinsp;0.3 cm) in each piece were used. In addition, four rectangular pieces of polycarbonate membrane (Midland Scientific Inc., CO, USA) with a 0.03-\u0026micro;m pore size, silicone glue, and screws were used to assemble and seal the MWCs for sub-cultivation assays.\u003c/p\u003e \u003cp\u003eTo prepare soil samples inoculum, 1 g of fresh soil collected from P, M, and R were serially diluted (10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) in SDW mixed with 1.5% gellan gum at 40\u0026deg;C (Sigma-Aldrich, Saint Louis, MO, USA) and then inoculated into an MWC by dipping the central plates into each soil sample cell suspension (P, M, and R). The assembly of MWC was as follows: two membranes were adhered to both sides of the central plate using hot silicon glue; the top and bottom plates were aligned and adjusted, and the screws were tightened to provide pressure (Berdy et al. 2017). Finally, the screws and all edges of the MWC were sealed using hot silicone glue to prevent external contamination. Two MWC devices were inoculated and assembled using the same method for each P, M, and R soil subsample. Subsequently, the MWCs were incubated in a sterilized plastic box containing each soil sample (P, M, and R) and maintained for 21 days inside refrigerators at 4\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). After incubation, the MWCs were washed vigorously in particle-free DNA-grade water (Fisher Scientific, Hampton, NH) and disassembled under laminar flow. The first MWC was disassembled, and their samples (P, M, and R soils suspension in gellan gum) contained inside of the device were transferred entirely to Petri dishes containing R2A in order to obtain culturable bacteria lawn for further metabarcoding analysis from pre-incubated assays by ISC. On the second MWC, R2A plates were inoculated with five wells and incubated at 15\u0026deg;C until colonies were observed. Experimental procedures are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. Colonies with different phenotypes (color, size, shape, texture, brightness, and elevation) were randomly chosen, transferred, and grown in fresh R2A plates. Three hundred ninety (390) isolates were purified by streaking on agar plates. Isolates were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in R2A:glycerol (7:3) and used for further assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Culturable bacteria lawns and isolation bacterial strains by plating-on agar method\u003c/h2\u003e \u003cp\u003eTraditional direct plating-on agar (PM) methods were carried out to obtain bacteria lawns and isolate pure culture bacteria colonies, then compared with those obtained using the ISC approach (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). For the PM bacteria lawns, the same soil inoculum used for ISC (1:1000 dilution) was plated directly (in triplicates) onto the R2A agar medium (1.5%) and incubated at 15\u0026deg;C until complete bacteria lawns were observed on the plate. In parallel, another set of was also plated following the above-mentioned steps and incubated until single colonies were visible. Those were streaked, incubated, and purified onto R2A medium. Colonies grown on agar plates were counted after 4 days of incubation at 15\u0026deg;C. Colonies with different phenotypes (color, size, shape, texture, brightness, and elevation) were randomly chosen, transferred, and grown in fresh plates. Three hundred twenty-eight (328) isolates were purified by streaking on agar plates. Isolates were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in R2A:glycerol (7:3) and used for further assays.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.6 Metabarcoding 16S rRNA of culturable bacteria lawns community from in situ cultivation and plating-on agar methods\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe genomic DNA (gDNA) of both bacteria lawns (ISC and PM) was extracted via DNeasy PowerSoil Kit (Qiagen, Inc.). Two ml of SDW was added to the bacteria lawn, and bacteria were scratched out from the lawn and suspended in the SDW with a spreader. An aliquot of 200 \u0026micro;L of the obtained suspension was used for DNA extraction of \"total culturable bacteria\" samples. DNA sample integrity and concentration were fluorometrically determined and adjusted to ~\u0026thinsp;20 ng uL-1. Bacteria communities from each extraction were explored by Illumina MiSeq, following the instructions of Yarimizu et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, the v4-v5 region of the 16S rRNA gene was amplified with the 341F (5'-CCT ACG GGN GGC WGC AG-'3) and 805R (5'-GAC TAC HVG GGT ATC TAA TCC-'3) primer set coupled in the 5'-end to Illumina overhang sequences. Libraries were indexed using Nextera XT v2 indexes, and paired-end sequenced (2x300 bp) in Illumina MiSeq (Illumina Inc.). The resulting reads were processed with SHI7 to keep only QC\u0026thinsp;\u0026gt;\u0026thinsp;35 reads() [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]., Trimmed sequences were rarefied to 25,000 reads in QIIME2, denoised and analyzed as amplicon sequence variants (ASVs) via DADA2. In parallel, biodiversity analysis for richness (observed ASVs, abundance-based coverage estimates, Chao1), diversity (coverage, Shannon, and Simpson indexes), and taxonomic assignation of the bacteria lawn communities were also performed with the same tool [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The community taxonomic compositions were plotted in R (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.r-project.org/\u003c/span\u003e\u003cspan address=\"https://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Principal component (PCA) and Redundancy (RDA) analyses were used to ordinate the samples, with and without soil physicochemical parameters as constraints, respectively. In parallel, differences in beta diversity among the community were evaluated by analysis of similarity (PERMANOVA) using Bray-Curtis dissimilarity matrices [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Genotyping and molecular identification of cultured bacterial strains of in situ and plating-on agar methods\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from the 390 and 328 bacterial strains isolated using ISC and PM methods, respectively. The DNA purification was done using the Wizard\u0026reg; Genomic DNA Purification Kit (Promega, Madison, WI, USA). The Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction (ERIC\u0026ndash;PCR) was performed to avoid isolates genotypic redundancy as described by Cid et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNon-redundant isolates from ISC and PM culture collections were characterized by partial sequencing of their 16S rRNA genes. Partial amplification of the 16S rRNA gene was performed by endpoint PCR using the 27f (5'‒AGA GTT TGA TCC TGG CTC AG‒3') and 1492r (5'‒TAC GGY TAC CTT GTT ACG ACT T‒3') primer set [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and sanger-sequenced. The sequences were compared with the available NCBI GenBank database using the BLASTn tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Antibiotic susceptibility testing\u003c/h2\u003e \u003cp\u003eBacterial culture collection obtained from ISC and PM antibiotic susceptibility was evaluated by the Kirby\u0026ndash;Bauer disc diffusion method in triplicates on Mueller\u0026ndash;Hinton agar (Merck, Darmstadt, Germany). The bacteria culture collections obtained from ISC and PM were tested. Susceptibility (or resistance) was evaluated for 24 antibiotic classes and tested in the bacterial culture collections using commercial disks for susceptibility testing (Bio-Rad, Hercules, CA, USA). The tested antibiotic were grouped per family as follow: aminoglycosides (amikacin 30 \u0026micro;g; gentamicin 10 \u0026micro;g; kanamycin 30 \u0026micro;g), fluoroquinolones (levofloxacin 5 \u0026micro;g; ciprofloxacin 5 \u0026micro;g), glycopeptides (vancomycin 30 \u0026micro;g), glycylcyclines (tigecycline 30 \u0026micro;g; fosfomycin 200 \u0026micro;g), amphenicols derivatives (nitrofurantoin 300 \u0026micro;g); oxazolidinones (linezolid 30 \u0026micro;g), lincosamides (lincomycin 2 \u0026micro;g), lipopeptides (daptomycin 30 \u0026micro;g), rifamycins (rifampicin 5 \u0026micro;g), sulfonamide-trimethoprim-combinations (sulfamethoxazole/trimethoprim 25 \u0026micro;g), fifth-generation cephalosporins (cefadroxil 30 \u0026micro;g; cefazolin 30 \u0026micro;g), second-generation-cephalosporins (cefoxitin 30 \u0026micro;g; cefaclor 30 \u0026micro;g), third-generation-cephalosporins (ceftazidime 50 \u0026micro;g; ceftazidime/avibactam 30 \u0026micro;g), fourth-generation-cephalosporins (cefepime 30 \u0026micro;g), fifth-generation cephalosporins (ceftolozane/tazobactam 40 \u0026micro;g), betalactams (amoxicillin/clavulanic-acid 30 \u0026micro;g; cefoxitin 30 \u0026micro;g; ampicillin/sulbactam 20 \u0026micro;g; oxacillin 1 \u0026micro;g), macrolide (erythromycin 15 \u0026micro;g), polymyxins (colistin 4 \u0026micro;g), bacitracin 10 \u0026micro;g, carbapenems (ertapenem 10 \u0026micro;g), monobactams (aztreonam 30 \u0026micro;g), and penicillins (piperacillin 100 \u0026micro;g). All plates were incubated at 15 \u0026ordm;C for 2 days. Then, the inhibition halo diameter was measured considering \"resistant\" the determined antibiotic when the diameter was \u0026le;\u0026thinsp;10 mm and \"sensible\" when the halo measured\u0026thinsp;\u0026ge;\u0026thinsp;11 mm, as described by Marcoleta et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Potential Virulence Activity\u003c/h2\u003e \u003cp\u003eIsolates obtained from both ISC and PM were phenotypically assessed for the presence of VFs, meaning, such as deoxyribonuclease (DNase), hemolytic activity lecithinase and pyocyanin production using agar plate methods with some modifications described by Marcoleta et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The DNAse activity was detected in DNAse agar (1% NaCl) containing 0.01% (w/v) of toluidine blue, and the incubation was performed at 15\u0026deg;C for 48h. For hemolytic activity, the bacterial strains were cultured on tryptone soy agar (TSA) plates containing 1% NaCl and supplemented with 5% sheep red cells (bioM\u0026eacute;riux SA, Marcy-l'\u0026Eacute;toile, France) incubated at 35\u0026deg;C for 48h. For lecithinase and pyocyanin production tests were assays used TSA supplemented with 5% (v/v) egg yolk emulsion and \u003cem\u003ePseudomonas\u003c/em\u003e agar medium, respectively (Winkler Ltda. Santiago, Chile), with incubation at 15\u0026deg;C for 48h. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain ATCC 27853 and \u003cem\u003eEscherichia coli\u003c/em\u003e strain ATCC 25922 were used as positive and negative controls, respectively. All clear halo-producing bacteria in the agar media were considered positive.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Chemical properties of frozen and ice-free soils\u003c/h2\u003e \u003cp\u003eSoil chemical properties were analyzed (Tukey's post-hoc test, P\u0026thinsp;\u0026le;\u0026thinsp;0.05) from P, M, and R soil samples (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The low pH values were observed in R samples (pH 5.3) related to P and M, which had neutral pH values (7.8 and 7.6, respectively). A higher EC was also observed in the R (51.2 \u0026micro;s cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), compared with M and P with values 31.6 51.2 \u0026micro;s cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 29.951.2 \u0026micro;s cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Similarly, TP and available P (Olsen) were ~\u0026thinsp;3-fold higher in R samples (366 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 119 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively) compared to M and P samples. Moreover, TC and OM content were low in P and M (~\u0026thinsp;3.5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.4, respectively) samples, while R obtained 79.7 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10.6 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Similarly, TN was higher in R samples (8.5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than in M and P with values\u0026thinsp;~\u0026thinsp;1 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e samples. Similarly, exchangeable cations content differed among frozen and ice-free soils, with the R containing the highest amounts of Mg\u003csup\u003e+\u003c/sup\u003e Na\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e+\u003c/sup\u003e, and Fe\u003csup\u003e2+\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of permafrost (P), moraine (M), and rhizosphere (R) soils from Ecology Glacier.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003cp\u003e(\u0026micro;s cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOM\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTP\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003csub\u003eolsen\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTC\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTN\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMg\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eFe\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e109\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2357\u0026thinsp;\u0026plusmn;\u0026thinsp;158 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e402.6\u0026thinsp;\u0026plusmn;\u0026thinsp;42.c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e645.3\u0026thinsp;\u0026plusmn;\u0026thinsp;64 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e645.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e108.7\u0026thinsp;\u0026plusmn;\u0026thinsp;14.4 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3303.3\u0026thinsp;\u0026plusmn;\u0026thinsp;365 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e678.3\u0026thinsp;\u0026plusmn;\u0026thinsp;102 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e460\u0026thinsp;\u0026plusmn;\u0026thinsp;26 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e552.7\u0026thinsp;\u0026plusmn;\u0026thinsp;43 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.2\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e366\u0026thinsp;\u0026plusmn;\u0026thinsp;38.2 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e119\u0026thinsp;\u0026plusmn;\u0026thinsp;36.1 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e79.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.8 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1402.5\u0026thinsp;\u0026plusmn;\u0026thinsp;232 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1013.3\u0026thinsp;\u0026plusmn;\u0026thinsp;175 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e557.8\u0026thinsp;\u0026plusmn;\u0026thinsp;88 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e554.4\u0026thinsp;\u0026plusmn;\u0026thinsp;51 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e152.1\u0026thinsp;\u0026plusmn;\u0026thinsp;32.7 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003eP\u0026thinsp;=\u0026thinsp;Permafrost; M\u0026thinsp;=\u0026thinsp;Moraine; R\u0026thinsp;=\u0026thinsp;Rhizosphere; EC\u0026thinsp;=\u0026thinsp;electro conductivity; TP\u0026thinsp;=\u0026thinsp;total phosphorus; P\u003csub\u003eOlsen\u003c/sub\u003e = available phosphorus; TC\u0026thinsp;=\u0026thinsp;total carbon; OM\u0026thinsp;=\u0026thinsp;organic matter; TN\u0026thinsp;=\u0026thinsp;total nitrogen; Ca\u003csup\u003e2+\u003c/sup\u003e = exchangeable calcium ion; Mg\u003csup\u003e+\u003c/sup\u003e = exchangeable magnesium ion; Na\u003csup\u003e+\u003c/sup\u003e = exchangeable sodium ion; K\u003csup\u003e+\u003c/sup\u003e = exchangeable potassium ion; Fe\u003csup\u003e2+\u003c/sup\u003e = exchangeable iron ion.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003e All deviation values represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error from n\u0026thinsp;=\u0026thinsp;6.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003e\u0026Dagger;\u003c/sup\u003eDifferent lower letters represent significant differences (one-way ANOVA, Tukey HSD test, P\u0026thinsp;\u0026le;\u0026thinsp;0.05) among samples.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Plate count of total culturable bacteria from frozen and ice-free soils\u003c/h2\u003e \u003cp\u003eTotal bacterial counts on agar plates revealed a significant difference (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) in the abundance of culturable heterotrophic bacteria among frozen and ice-free soil samples from Ecology Glacier. The abundance of culturable bacteria was greater in rhizosphere samples (from 2.94\u0026times;10\u003csup\u003e5\u003c/sup\u003e to 8.21\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw) than in the moraine and permafrost samples (from 1.39\u0026times;10\u003csup\u003e3\u003c/sup\u003e to 6.35\u0026times;10\u003csup\u003e3\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Higher diversity in bacterial phenotypes was observed across samples using R2A media (2.86\u0026times;10\u003csup\u003e3\u003c/sup\u003e to 8.21\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw) than LB 1:10 (1.95\u0026times;10\u003csup\u003e3\u003c/sup\u003e to 4.97\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw), and NM1 (1.39\u0026times;10\u003csup\u003e3\u003c/sup\u003e to 2.96\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dw) during the 30 d of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Pearson correlation showed significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) positive correlations among bacterial abundance with most nutrients but negatively correlated with pH and Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e (Supplementary, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Metabarcoding of culturable bacteria lawns\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Alpha and beta diversity\u003c/h2\u003e \u003cp\u003eThe 16S rRNA gene metabarcoding of culturable bacteria lawns from frozen and frozen-free soil recovery by using PM and ISC revealed differences in ASVs between R (164) and M (125) samples but not with P (146) in PM methods (P\u0026thinsp;\u0026le;\u0026thinsp;0.05). Alpha diversity showed that the Chao1 index was influenced by the culturing method and significantly higher in the R (PM: 165.3 and ISC: 134.8) compared to M (PM: 125.3 and ISC: 125.5) and P (P: 146.8 and ISC: 119.5). Concerning culture methods, the highest ASV and richness values were found using PM compared to ISC methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, Shannon and Inverse Simpson indexes were contrasting, with higher values in the R using PM (3.3 and 14.7, respectively). In contrast, by using ISC, the diversity index was higher in M samples (2.9 and 11.6, respectively). In addition, the P communities were identified as the less diverse (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) in this study, independently of isolation methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe beta diversity showed noticeable differences in the bacteria community lawn structure between both soil sample type and isolation methods, as revealed by PCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In this analysis, both PC1 and PC2 axes explained 54% of the variability of the bacterial communities (P\u0026thinsp;\u0026le;\u0026thinsp;0.05), with the culturing method appearing more relevant than the soil sample type. The PERMANOVA analysis confirmed the differences in the bacterial community structure in lawns associated with the method (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.30; P\u0026thinsp;\u0026le;\u0026thinsp;0.001) and as well sample type (frozen and ice-free soils; R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.34; P\u0026thinsp;\u0026le;\u0026thinsp;0.001). In this sense, a clear differentiation in bacterial community structure was observed according to the recovery isolation methods used. Regarding soil type, the bacteria communities showed more similarity between the M and P samples than those in the R samples, independently of cultivation methods. Once physicochemical variables were introduced into the structure analysis, the redundancy analysis (RDA) showed that pH (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.12, P\u0026thinsp;\u0026le;\u0026thinsp;0.003) and electroconductivity (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.13, P\u0026thinsp;\u0026le;\u0026thinsp;0.001) were the main soil chemical factors with a significant effect on bacterial community lawns. However, their inclusion reduced the variance explanation to 44.1%, revealing that underlying variables not measured in this study are significant for each bacteria community structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Taxonomic assignation of bacterial community lawns\u003c/h2\u003e \u003cp\u003eThe taxonomic assignment of 16S rRNA sequences of bacterial composition from lawns revealed phyla Pseudomonadota (55\u0026ndash;75%), Actinomycetota (20\u0026ndash;35%), and Bacteroidota (5\u0026ndash;10%) as the most abundant bacterial taxa in culturable lawn bacteria lawn communities. In addition, members of Gammaproteobacteria were the most abundant class in frozen and ice-free soils, with an average relative abundance varied from 50.1\u0026ndash;51.6%, from 40.6\u0026ndash;74.4%, and from 51.4\u0026ndash;37.8% for P, M, and R using PM and ISC, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). On average, the Actinobacteria class was also the most dominant phyla in the R using ISC with 33.2% relative abundance, while M and P samples ranged from 18.7\u0026ndash;14.8%, respectively. In contrast, when using PM, a higher abundance of Actinobacteria was observed in M (29.9%) and P (31.8%) than those obtained in R samples (13.8%). Similarly, Bacteroidia was the second dominant class in P (30.6%) under the ISC method, but their abundances were less observed in M and R samples under PM varied from 14.0\u0026ndash;11.6%. Remarkably, this class was less than 2% abundant in P samples using PM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Noteworthy, the Bacilli class was only observed in samples subjected to the PM method, with values of 12.2%, 7.9%, and 7.3% in M, P, and R samples, respectively (Supplementary, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the dominance of the same classes for both methods and all soil samples, differences were observed across samples at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Pseudomonas was the most abundant genus across soil samples, with values ranging from 32.1\u0026ndash;16.3% for PM and 35.8\u0026ndash;23% for ISC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Moreover, the \u003cem\u003eJanthinobacterium\u003c/em\u003e genus was only observed in M and P using both PM (3.5\u0026ndash;5.1%) and ISC (7.4% and 16.4%) culturing methods. Notably, members of the Stenotrophomonas genus were abundant across all samples, varying from 7.6\u0026ndash;15.9% using PM. In addition, \u003cem\u003eSphingobacterium\u003c/em\u003e (11.8\u0026ndash;13.4%) genera were only observed in bacteria lawns from moraine samples obtained exclusively using PM. It is important to mention that R samples showed a significantly different pattern in bacterial taxonomic assignations compared to M and P, independently of the culture method used (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). \u003cem\u003ePedobacter\u003c/em\u003e genus was only observed in rhizosphere samples, ranging from 12.9\u0026ndash;13.7% by PM and from 10.9\u0026ndash;11.3% via ISC methods. Additionally, rhizosphere bacteria lawns composition under PM was presented members of \u003cem\u003eBacillus\u003c/em\u003e (4.3\u0026ndash;12.9%), \u003cem\u003eRhodanobacter\u003c/em\u003e (3.6\u0026ndash;4.0%) and \u003cem\u003eLysobacter\u003c/em\u003e (1.6\u0026ndash;2.2%) genera, while \u003cem\u003eFlavobacterium\u003c/em\u003e (~\u0026thinsp;4.9%) \u003cem\u003eMicrobacterium\u003c/em\u003e (~\u0026thinsp;30.9%), \u003cem\u003eSphingomonadaceae\u003c/em\u003e family and \u003cem\u003eArthrobacter\u003c/em\u003e (2.3\u0026ndash;6.4%) genera were dominant in bacteria lawns only recovery by ISC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Bacterial culture collections and isolate strain identification\u003c/h2\u003e \u003cp\u003eIn total, 390 and 328 bacterial strains were isolated from iced and iced-free soil samples in Ecology Glacier using PM and ISC, respectively. Based on ERIC-PCR assays, 21% (82/390) of isolates recovered with PM methods showed distinct genetic variability (24, 33, and 25 from M, P, and R-associated isolates, respectively). In addition, using ISC methods, 76/328 (23%) genotypes were classified as non-redundant isolates, which included 19, 27, and 30 for M, P, and R, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Despite the isolation method used, M samples showed the highest percentage of genetic variability than those observed in R and P samples, respectively (26,4% ISC and 28.2% PM). Alpha diversity analysis showed higher richness values in R samples using both isolation methods (PM:9 and ISC: 8). Moreover, ISC methods also revealed a higher richness in M (8) and P (7) samples compared to those obtained via PM in the M and P samples (6 and 4, respectively). Similarly, the Shannon index reveals the higher diversity in M (1.590) and P (1.120) samples using ISC methods compared to values obtained via PM (1.516 and 0.495, respectively). Based on the Simpson index, the culturable community by PM was more diverse in M and R samples (0.722 and 0.725, respectively) than the measured by ISC (0.687 and 0.609, respectively). Despite this, the ISC method (0.488) was able to recover more diverse bacterial isolates compared to PM (0.222) in P samples, as shown in the Simpson index (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe taxonomic characterization of isolated strains showed that the most abundant phyla were Pseudomonadota (76%), followed by Actinomicetota (17,7%), Bacteroidota (3,15%) and Bacillota (3,15%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A total of 24 different bacterial genera were isolated from all soil samples using both ISC (13 genera) and PM (15 genera) approaches (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In detail, Pseudomonas was the most isolated genus across soil samples using PM (51 isolates) and ISC (47 isolates) methods (48, 29, and 21 isolates from P, R, and M, respectively). Other isolates within the Pseudomonadota phylum were assigned to the \u003cem\u003eStenotrophomonas\u003c/em\u003e (5 isolates) obtained by both methods. Moreover, the \u003cem\u003eBrucella\u003c/em\u003e genus (3 isolates) was isolated by PM and the \u003cem\u003eJanthinobacterium\u003c/em\u003e genus (3 isolates) by ISC. Finally, using both methods, \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eSphingomonas\u003c/em\u003e genera (2 isolates) were isolated from the R soil samples. The next dominant phylum was Actinomycetota, represented by the \u003cem\u003eArthrobacter\u003c/em\u003e (10 isolated by PM and 6 by ISC), distributed as 6 isolates from M, 5 isolates from R, and 4 isolates from P soil samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Moreover, members of the \u003cem\u003eMicrobacterium\u003c/em\u003e (6 isolates) and \u003cem\u003eCurtobacterium\u003c/em\u003e (2 isolates) genera were only isolated by the ISC method. In contrast, PM exclusively obtained isolates belonging to the \u003cem\u003ePseudarthrobacter\u003c/em\u003e genus (3 isolates). Isolates belonging to Bacteroidota phylum were distributed among the \u003cem\u003eFlavobacterium\u003c/em\u003e (3 isolates) and \u003cem\u003ePedobacter\u003c/em\u003e (2 isolates) genera obtained by PM and ISC methods, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn particular, members of the Bacillota phyla were obtained exclusively using the PM method, coincidentally with the high-throughput sequencing (HTS) approach results. Members of rare taxa (unique colonies observed in culture collection) \u003cem\u003eCurtobacterium\u003c/em\u003e, \u003cem\u003eAchromobacter\u003c/em\u003e, \u003cem\u003eBosea\u003c/em\u003e, \u003cem\u003eMassilia\u003c/em\u003e, \u003cem\u003eRhodococcus\u003c/em\u003e, \u003cem\u003eSporosarcina\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003ePhyllobacterium\u003c/em\u003e, and \u003cem\u003ePolaromonas\u003c/em\u003e genera were also isolated by the ISC method. Surprisingly, poorly studied bacterial taxa from Antarctic soils, such as \u003cem\u003eRathayibacter\u003c/em\u003e and \u003cem\u003eRugamonas\u003c/em\u003e, were isolated using ISC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Antibiotic resistance and virulence patterns of plate and in situ cultured bacterial strains\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Qualitative antibiotic resistance\u003c/h2\u003e \u003cp\u003eThe resistance (or sensibility) to 35 types (24 classes) of antimicrobial agents was tested in the bacterial collection obtained by the disk diffusion susceptibility test (Kirby-Bauer technique). Antibiotic pattern distribution was higher in isolated strains from rhizosphere samples than those obtained from isolates of permafrost and moraine samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Based on the method, a higher count of resistances was observed in strains isolated by the ISC approach compared to PM, mainly in bacterial culture collections obtained from M and P soil samples. A higher number of bacteria (86%) were resistant to beta-lactams, followed by Cephalosporins (77%), Oxazolidines (71%), and Liconsamids (70%), among others, across all soil samples. Bacterial strains belonging to the \u003cem\u003eFlavobacterium\u003c/em\u003e genus resisted more than 22 different antibiotics, while \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eCurtobacterium\u003c/em\u003e genera showed multi-resistance over 18 different antibiotics independently of isolated methods used. Remarkably, we found 42 isolates resistant to Carbapenems, the majority members of \u003cem\u003ePseudomonas\u003c/em\u003e (35 isolates), \u003cem\u003ePedobacter\u003c/em\u003e, and \u003cem\u003eCurtobacterium\u003c/em\u003e (2 isolates, respectively). Additionally, we found resistance to some antibiotics defined as \"last-line,\" such as Colistin, in members of \u003cem\u003ePseudomonas\u003c/em\u003e (30 isolates), \u003cem\u003eArthrobacter\u003c/em\u003e (4 isolates), \u003cem\u003ePseudoarthrobacter\u003c/em\u003e, and \u003cem\u003eFlavobacterium\u003c/em\u003e (2 isolates).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 Potential virulence activity\u003c/h2\u003e \u003cp\u003eThe cultured bacteria collections (76 isolates by ISC and 82 by PM) were subjected to virulence potential assessment, including deoxyribonuclease (DNase), hemolytic activity lecithinase, and pyocyanin production. A total of 12 of 23 genera (52.2%) carried out at least one virulence factor evaluated, representing 41 isolates and 58 isolates (99 isolates strains) from ISC and PM, respectively. A higher number of virulence factors was observed in isolates from P compared to R and M samples, independently of the culture method (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Based on those, higher virulence was observed among bacteria isolated by PM compared to ISC. Overall, virulence factor assays revealed a prevalence of \u0026gt;\u0026thinsp;35% for both lecithinase activity (75 isolates) and hemolytic activity (68 isolates) among isolates from all the soil samples, while pyocyanin production was positive to 39 isolates (20%) from 158 strains evaluated Additionally, DNAse activity was observed with low frequency (~\u0026thinsp;7%; 13 isolates) among the isolates tested. In detail, members of the Pseudomonas genus were positive for all virulence factors evaluated independently of isolated methods used. In contrast, isolated strains belonging to \u003cem\u003eMicrobacterium\u003c/em\u003e, \u003cem\u003eBrucella\u003c/em\u003e, and \u003cem\u003eSporosarcina\u003c/em\u003e genera carried out only the DNAse activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), while pyocyanin production was the main activity found in isolated \u003cem\u003eSphingomonas\u003c/em\u003e and Staphylococcus genera.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study represents the first effort to investigate the culturable bacterial communities in the soils surrounding the Ecology Glacier, Antarctica, using traditional and \u0026lsquo;\u003cem\u003ein situ\u003c/em\u003e\u0026rsquo; cultivation methods, focusing on characterizing the antibiotic resistance profiles and virulence factors exhibited by the isolated bacterial strains. A higher abundance of bacteria counts and diversity was found in R compared to M and P samples. The rhizosphere is the primary hotspot for microbial colonization and activity in soils, highlighting the influence of plant-microbe interactions on shaping soil microbial communities [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Root exudates and decomposing plant material provide a rich source of nutrients and create micro-niches that support a greater abundance and diversity of bacteria [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These results concord with those published by Purcell et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], in which they observed higher bacterial counts in early terrestrial zones with apparent vegetation on King George Island. The differences in bacterial counts could be attributed to the freezing soil, such as moraine and permafrost, which are considered a restricted niche where microbial colonization depends on limited water and nutrient availability [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe use of both traditional plating-on agar method (PM) and \"\u003cem\u003ein situ'\u003c/em\u003e' cultivation (ISC) proved crucial in capturing a broader spectrum of bacterial diversity. The ISC approach, utilizing MWC devices, allowed the recovery of similar ASVs but a significantly more diverse bacterial community lawn than traditional culturing across all soil samples. This observation contrasts with several studies where similar \"in situ\" techniques have demonstrated increased ASV recovery from various environments, such as marine sponges [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], corals [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and rhizosphere [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This discrepancy could be attributed to inherent characteristics of the soil and water availability required for the ISC methods, which might have limited the growth of some bacterial taxa. Despite this, differences in the diversity of lawn bacteria communities were found in the R compared with the M and P soil samples independently of isolation methods. Previous studies on culturable bacterial communities of M samples from Ecology Glacier pointed out that the chemical changes in soils modulated the taxonomic diversity of culturable bacterial communities [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Based on the RDA analysis, significant differences were observed in the structure of bacteria lawn obtained by using ISC and PM, modulated by the electronic conductance and pH. Several studies have shown that soil salinity and pH can modify the composition of the bacterial community in Antarctic soils [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The observed differentiation in diversity and composition of the bacterial community could be attributed to the type of samples, where the nutrient content of R was higher than M and P, given the plant influence, as evidenced by our analytical findings (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIndependent of the isolation method, the Illumina-based sequencing analyses revealed the dominance of members of the phyla Gammaproteobacteria in all studied bacteria lawn communities. Members of Proteobacteria are the most abundant phyla in Antarctic soils [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, using both methods, Actinobacteria and Bacteroidetes phyla were prevalent in bacteria lawns recovery from soil samples. The Actinobacteria and Bacteroidetes are the dominant bacterial groups in soils in general, as well as in the Antarctic soil microbiome [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. At the genus level, differences were observed between bacteria lawns from all soil samples, as members of \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eMicrococcaceae\u003c/em\u003e family, and \u003cem\u003eFlavobacterium\u003c/em\u003e were most abundant in moraine and permafrost by ISC methods. These genera are common glacier-ice lineages present in Tibetan Plateau ice cores [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], China glaciers [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and Antarctica [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Similarly, bacteria lawn communities from R were dominated by \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eMicrobacterium\u003c/em\u003e, \u003cem\u003eStenotrophomas\u003c/em\u003e, and \u003cem\u003ePedobacter\u003c/em\u003e genera. This differentiation concerning alpha and beta diversity analysis in bacteria lawns from frozen and iced-free Antarctic soils is known. The highest diversity found in R samples suggests that heterotrophic bacteria are abundant in Antarctic vascular plants rhizosphere. These differences may result from the significant influence of birds and animals around vegetation, which could exert selective pressure on the rhizosphere microbiome [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Based on the methods, the R bacteria lawn composition under PM was dominated by members of the Bacillus genus. In contrast, the \u003cem\u003eArthrobacter\u003c/em\u003e genus was dominant in bacteria lawns, only recovered by ISC. Members of the bacterial \u003cem\u003eArthrobacter\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e genera are both readily cultured and commonly identified in Antarctic soil communities [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding isolated bacterial strains, our study showed affiliations to 24 different bacterial genera, where \u003cem\u003ePseudomonas\u003c/em\u003e members were the most dominant taxa for both PM and ISC methods. After \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e, \u003cem\u003eArthrobacter\u003c/em\u003e, and \u003cem\u003eMicrobacterium\u003c/em\u003e (Actinomicetota) were the most dominant taxa. The isolated strains belonging to Bacillota phyla were mainly represented by the Bacillus genus, only recovered by PM. These results are coincident, as observed from the bacteria lawns community analysis. Using ISC, isolating poorly studied genera (such as \u003cem\u003eBosea\u003c/em\u003e, \u003cem\u003eRathayibacter\u003c/em\u003e, and \u003cem\u003eRugamonas\u003c/em\u003e) was possible. Studies have shown that up to 80% of bacteria from Antarctic soils are \"yet-to-be cultured\", including phylotypes from well-studied phyla such as the Actinobacteria [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, only a few taxa were unique in culture collections between ISC and PM culture methods, as revealed by the diversity index, suggesting there are still considerable culturomic challenges associated with bacteria domestication [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Despite this, P and M showed higher diversity when pre-culture incubation by ISC was applied compared to R samples. This finding underscores the limitations of relying solely on traditional cultivation techniques, which often favor fast-growing and readily culturable microorganisms. Employing diverse cultivation strategies is essential for a more comprehensive understanding of soil bacterial diversity and ecological function in Antarctic soils.\u003c/p\u003e \u003cp\u003eThe widespread antibiotic resistance patterns observed among the isolates recovered by ISC and PM revealed resistance to several antibiotics, including beta-lactams, tetracyclines, and aminoglycosides, indicating a broad resistance profile in culturable bacteria communities. Numerous studies have demonstrated the occurrence and abundance of a natural resistome in iced soils as a response to microbial competition and environmental pressures in the Antarctic ecosystem [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Despite this, animal and human influence are often described as an important source of bacteria-resistant dissemination [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Our results revealed differences in the distribution of antibiotic patterns among isolate strains, where the ones from the R samples were resistant against a broad spectrum of antimicrobial drugs compared to those recovering from M and P samples. Calisto et al. [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] evidenced that 63% of isolates (47 strains) from R of \u003cem\u003eD. antartica\u003c/em\u003e were multidrug-resistant to 14 over 21 antibiotics tested. However, \u003cem\u003ePseudomonas\u003c/em\u003e resistance phenotypes observed in P samples significantly differ between the R and M isolates, emphasizing the AMR role as a competition mechanism in driving species survival. Based on the method, ISC yielded more resistant bacterial strains than PM, particularly in bacterial cultures obtained from M and P samples. Although most carbapenem-resistant isolates belonged to Pseudomonas (35 isolates), the ISC also facilitated the isolation of carbapenem-resistant Pedobacter and \u003cem\u003eCurtobacterium\u003c/em\u003e. These results conflict with those published by Wong et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], in which they reported that \u003cem\u003ePedobacter\u003c/em\u003e sp. strains BG5 isolated from soils of King George Island, Antarctica, were sensitive to imipenem, indicating its lack of resistance to this carbapenem antibiotic. Moreover, \u003cem\u003eCurtobacterium\u003c/em\u003e species are cosmopolitan plant pathogens exhibiting distinct diverse antibiotic resistance patterns [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], but studies based on AMR in Antarctic isolates are still scarce. Notably, 86% and 76% of the isolated strains resisted beta-lactams and cephalosporins, respectively. Previous studies have detected multidrug-resistant bacteria collected from soils, wildlife, freshwater, and glacier environments, among other habitats in Antarctica [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. These studies reported bacterial isolates with high levels of antibiotic resistance, including aminoglycosides, β-lactams, and trimethoprim, consistent with our findings.\u003c/p\u003e \u003cp\u003eAdditionally, isolates express multidrug-resistant phenotypes with susceptibility to 'last-line' antibiotics, such as Colistin, mainly in \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eArthrobacter\u003c/em\u003e, \u003cem\u003ePseudoarthrobacter\u003c/em\u003e, and \u003cem\u003eFlavobacterium\u003c/em\u003e. \u003cem\u003ePseudomonas\u003c/em\u003e are common resistant-bacteria taxa whose resistome compromised more than 170 genes (ancestral genes) that could confer resistance to natural or synthetic antibiotics [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Remarkably, Flavobacterium strains in this study resisted more than 22 clinical antibiotics. These results are in agreement with a previous report of \u003cem\u003eFlavobacterium\u003c/em\u003e identified from iced and iced-free soils in the Arctic and Antarctica exhibiting distinct resistance mechanisms [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Although the genera \u003cem\u003eArthrobacter\u003c/em\u003e and \u003cem\u003ePseudoarthrobacter\u003c/em\u003e comprise numerous species isolated from the Antarctic region [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], data on antibiotic susceptibility of environmental isolates from iced soils is scarce [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, detecting VFs in many isolates suggests potential pathogenicity within these culturable bacterial communities. It is known that VFs and antibiotic resistance are recognized as crucial mechanisms for adaptation, competition, and colonization processes under harsh conditions [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The results also revealed that the isolates had positive percentages of \u0026gt;\u0026thinsp;40% lecithinase activity, 35% hemolytic activity, 21% pyocyanin production, and 5% DNAse activity. Several VFs have been reported from the Arctic and Antarctic bacterial strains, mainly proteinase and DNase enzymes [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In addition, previous studies reported that 11 virulence factors amongst the Antarctic isolates were characteristic of E. coli strains more commonly associated with humans [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The ability to produce enzymes like lecithinase and DNase, exhibit hemolytic activity, and produce toxins like pyocyanin indicates the potential for these bacteria to cause harm to plants or animals. In addition, metagenomic analyses revealed the occurrence of VFs genes (VFGs) in soil from the Fildes peninsula in Antarctica [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], indicating that resistance mechanisms would result from thousands of years of evolution [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. While the data on antibiotic resistance and VFs in Antarctic Glaciar are scarce, we have previously published a study that reported the resistome and virulome in Union Glaciar using metagenomic and culture-dependent approaches [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], in which the bacterial isolate was resistant to up to 24 clinical antibiotics. In addition, some isolates produced putative VFs, including siderophores, pyocyanins, and exoenzymes with hemolytic activity, lecithinase, protease, and DNase. It is relevant to mention that these resistant bacterial isolates mainly belonged to the \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eArthrobacter\u003c/em\u003e, \u003cem\u003ePlantibacter\u003c/em\u003e, and \u003cem\u003eFlavobacterium\u003c/em\u003e genera, similar to our findings. Despite the importance of horizontal transference of antibiotic resistance genes and VFs to microbial communities, no previous reports have explored potential VFGs or the production of VFs among bacteria from Antarctica. While additional research is needed to assess the actual risk posed by these microorganisms, their presence warrants careful monitoring. These results highlight the importance of the One Health approach, emphasizing the need for multidisciplinary efforts to understand the pathways of resistant bacteria and virulence factors, especially in the current climate emergency scenario.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study revealed a remarkably diverse culturable bacterial community inhabiting the soils surrounding the Ecology Glacier, with Pseudomonadota, Actinomicetota, and Bacteroidota as the dominant phyla. Our selected \"\u003cem\u003ein situ\u003c/em\u003e\" cultivation method successfully recovered a wider range of bacteria genera than the traditional plating-on agar method, including less studied genera such as \u003cem\u003eBosea\u003c/em\u003e, \u003cem\u003eRathayibacter\u003c/em\u003e, and \u003cem\u003eRugamonas\u003c/em\u003e. This finding emphasizes the importance of comprehensively employing diverse culture techniques to represent soil bacterial diversity. Many isolated bacterial strains exhibited multidrug resistance, particularly against beta-lactams, cephalosporins, oxazolidinones, and lincosamides. In many isolates, virulence factors, including lecithinase, hemolytic activity, pyocyanin production, and DNase activity, suggest potential pathogenicity within these bacteria communities. These approaches to Antarctic bacteria must be considered in the near future to properly understand the effects of ice melting in Antarctica and possibly in other environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, A.E.M, F.P.C., V.C., M.A.J. and J.J.A.; formal analysis, C.V., N.H., M.A.C. and J.J.A.; data curation, N.H. and J.R.; investigation, C.V., N.H., A.E.M., F.P.C., L.A.B., V.C., M.A.J. and J.J.A.; writing\u0026mdash;original draft preparation, C.V. and J.J.A.; writing\u0026mdash;review and editing, C.V., M.A.C., J.R., A.E.M., F.P.C., L.A.B., V.C., H.S., M.A.J and J.J.A.; funding acquisition, A.E.M., F.P.C., V.C., M.A.J. and J.J.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was funded by The Regular Research Team Projects in Science and Technology \u0026amp; Thematic Research Team from The Chilean National Agency for Research and Development (ANID), code ACT210044. Partial support was also provided by The National Fund for Scientific and Technological Development (FONDECYT), Project no.1240602 and 1221228 (to M.A.J and J.J.A.), and by the Millennium Science Initiative Program, code ICN2021_044 (to V.C. and J.J.A.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e All sequences obtained in this study were deposited in the NCBI GenBank Nucleotide database (https://www.ncbi.nlm.nih.gov/genbank/) under bioproject PRJNA1095903. Additional raw data from this study is available under request to the corresponding author (J.J.A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors thank the Chilean Antarctic Institute (INACH) staff for their support during the Scientific Antarctic Expedition ECA58 developed from December 2021 to March 2022, and for the permit to collect soil samples at an Antarctic specially protected area (ASPA N\u0026deg;128; Permit N\u0026deg;200; INACH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e Authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThomas, C. D. et al. 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Total Environ.\u003c/em\u003e \u003cb\u003e957\u003c/b\u003e, 177594 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacterial diversity, Antibiotic resistance, virulence factors, Antarctica, iChip","lastPublishedDoi":"10.21203/rs.3.rs-6667169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6667169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlacier forelands in the Antarctic Peninsula are increasingly affected by climate change. However, the impact on the composition of culturable soil bacteria communities remains unclear. Here, we explored the culturable bacterial communities from permafrost (P), moraine (M), and \u003cem\u003eDeschampsia antartica\u003c/em\u003e rhizosphere (R) soil samples collected near the Ecology Glacier, Antarctica. Using traditional plating-on agar (PM) and '\u003cem\u003ein situ\u003c/em\u003e' cultivation (ISC) methods, bacterial counts were significantly higher in R (8.2\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e soil) than in M and P (~\u0026thinsp;3.9 \u0026times;10\u003csup\u003e3\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e soil). Culturable lawn bacteria communities and 158 genotypically different isolated strains (76 by ISC and 82 by PM) were identified, purified. And their antibiotics multiresistance (AMR) and virulence factors (VFs) were also screened. Our results revealed phyla Pseudomonadota (55\u0026ndash;75%), Actinomicetota (20\u0026ndash;35%), and Bacteroidota (5\u0026ndash;10%) as the most abundant bacterial taxa in culturable bacteria lawn communities. The isolated strains belonged to 24 different bacteria genera, where Pseudomonadota (76%), Actinomicetota (18%), Bacteroidota (4.6%), and Bacillota (3.2%) were the most dominant phyla. Using ISC, a wider genera diversity (e.g., \u003cem\u003eBosea\u003c/em\u003e, \u003cem\u003eRathayibacter\u003c/em\u003e, and \u003cem\u003eRugamonas\u003c/em\u003e) was isolated. On the other hand, \u003cem\u003eBacillus\u003c/em\u003e exclusively grew on PM. Among these isolates, 86% were resistant to beta-lactams, 77% to cephalosporins, and 71% to oxazolidines. Interestingly, some \u003cem\u003eFlavobacterium\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eCurtobacterium\u003c/em\u003e strains showed AMR to \u0026gt;\u0026thinsp;18 different antibiotics. For VFs assays, we also observed\u0026thinsp;\u0026gt;\u0026thinsp;35% lecithinase and hemolytic activity, 20% pyocyanin production, and 7% DNAse activity among all isolates. A high diversity of AMR and VFs was observed in culturable bacteria inhabiting the surrounding soils of the Ecology Glacier.\u003c/p\u003e","manuscriptTitle":"Characterization of Culturable Bacterial Communities in Permafrost, Moraine, and Rhizosphere Soils near the Ecology Glacier (King George Island, Maritime Antarctica): Patterns of Antibiotic Resistance and Virulence Factors in Isolated Bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 18:45:01","doi":"10.21203/rs.3.rs-6667169/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12019357-0733-47b2-b878-1af584937fa4","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50291356,"name":"Biological sciences/Microbiology"},{"id":50291357,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2026-01-21T11:54:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-20 18:45:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6667169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6667169","identity":"rs-6667169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}