Molecular Characterization of Antibiotic Resistant Genes In Psychrophilic Bacteria Isolated From Glaciers

Abstract

The cryosphere was once thought uninhabitable due to life-detrimental conditions but is found teeming with extremophiles of all life forms including viruses, bacteria, fungi, and Eukaryotes, and a decade ago scientists started to explore the polar cold regions for the occurrence of antibiotic resistance bacteria harbored with antibiotic-resistant determinants. Antibiotic production and resistance are prehistoric and currently, its spread is the preeminent global threat. The bacteria inhabiting the natural pristine cold environments are thought to be the potent reservoir of antibiotic-resistant genes. Besides the polar cold regions, the occurrence of the most congested non-polar glaciers in the region of Hindukush, Karakorum, and Himalayas, turned the attention to exploring the antibiotic-resistant bacteria in these areas since they are uncharted regarding the abundance of antibiotic-resistant genes. Therefore, the current study aims to explore the Passu glacier (Karakorum mountains range) for the presence of antibiotic resistant culturable bacteria, the curing effect of acridine orange on glacier bacteria susceptibility to antibiotics, and the carriage of plasmid-mediated antibiotic resistance, the abundance of antibiotic-resistant genes, and mobile genetic elements like integron integrases, and their cloning efficacy in antibiotic-sensitive mesophilic bacteria. In this regard, we collected the glacial sediment, ice, and meltwater samples from the Passu glacier. Bacterial isolation was performed by evaluating the colony morphology and identified through 16S rRNA amplicon sequencing. Kirby Bauer agar disc diffusion method was utilized to determine the bacterial in vivo antibiotic susceptibility profile. The molecular detection of antibiotic-resistant genes and the abundance of integron integrase genes were evaluated through conventional and real-time PCR amplification and quantification, respectively. To characterize the antibiotic resistant genes, Sanger sequencing was performed and amino acids-based alignment with the counter reference antibiotic-resistant genes was performed to call the amino acids coverage and consensus variation, followed by tracing their evolutionary history through the Neighbor joining method on Mega X software. The acridine orange in different concentrations, as a curing agent, was used to check the MICs of tested bacteria plus its plasmid elimination effect and to make a correlation between the plasmid-containing and plasmid-deprived bacteria, while evaluating their pre and post-curing antibiotic susceptibility profiling. The glacier's bacteria (psychrophiles) harbored antibiotic-resistant genes and were cloned in a pCR 2.1 vector utilizing TOP TA cloning kit and an antibiotic-sensitive competent E. coli (mesophile) was used to uptake the vector embedded with the desired antibiotic-resistant genes of interest, followed by the blue-white screening of transformed colonies. The Plasmid-PCR and Sanger ix sequencing of each vector was performed, utilizing the M13 Forward, and reverse primers plus each ARG primer, to analyze the transformants, and SnapGene Software was utilized to map and annotate each plasmid vector construct. From the Passu glacier, a total of 65 culturable bacterial isolates were retrieved. Predominantly identified bacteria were gram-negative 43 (66.15%) whereas, gram-positive isolates were 22 (33.84%). Among gram-negative bacteria, Gammaproteobacteria were dominant (62.79%), followed by Betaproteobacteria (18.60%) and Alphaproteobacteria (9.30%), and among gram positive bacteria, Actinobacteria (50%) and Firmicutes (40.90%) were predominant. The glacial bacteria showed significant antibiotic resistance against a panel of 29 antibiotic groups and PCR amplification showed phylum Proteobacteria predominantly detected with 21 antibiotic-resistant genes including blaAmpC 6 (100%), blaVIM-1, blaSHV, and blaDHA 5 (100%) each, blaOXA-1 1 (100%), blaCMY-4 4 (100%), followed by Actinobacteria 14, Firmicutes 13 and Bacteroidetes 11 antibiotic-resistant genes, whereas, all isolates were negative for blaKPC, qnrA, vanA, ermA, ermB, intl2, and intl3. The multiple antibiotic resistance indexes were higher for gram-negative, compared to gram-positive. Alignment of protein homology sequences of antibiotic-resistant genes with counter reference genes revealed amino acids coverage and consensus variations for blaNDM-1, blaOXA-1, blaSHV, mecA, aac(6)-Ib3, tetA, tetB, sul2, qnrB, gyrA, and intI1. When different concentrations (0 to 200 µg/mL) of acridine orange were subjected to bacterial growth, 17/43, (39.53%) gram-negative and 15/22 (68.18%) gram positive bacteria retained their slight growth up to 75 µg/mL acridine orange concentration, whereas, compared to gram-negative bacteria, gram-positive yielded higher MICs values. While comparing the pre and post-curing verificatory antibiotic susceptibility assays, 21/43 (48.83%) gram-negative and 7/22 (31.81%) gram-positive bacteria revealed no change in susceptibility pattern. The plasmid-cured bacterial strains showed 100% susceptibility to levofloxacin, ciprofloxacin, piperacillin, and imipenem whereas a major MAR index decline was observed for Staphylococcus equorum (HP19), Leucobacter aridicollis (HP22) and Arthrobacter psychrochitiniphilus (LP2) (0.7 to 0.2 each), Serratia marcescens (HP50) (0.6 to 0.1), Flavobacterium antarcticum (HP20) (0.6 to 0.2), Flavobacterium saliperosum (HP8) (from 0.5 to 0.1) and Brevundimonas diminuta (HP21) (0.7 to 0.4). The real-time PCR assay for the screening of integron integrase class 1, 2, and 3, among 28 multi-drug resistant bacteria carrying disparate antibiotic-resistant genes, revealed 20 strains (71.4%) were positive for integron integrase class 1, 12 (42.8%) for integron integrase class 2 while all negative for integron integrase class 3. Overall, gram-negative bacteria were more ubiquitous with integron x integrase class 1 (14, 70%) and integron integrase class 2 (10, 83.3%) with the lowest Ct (cycle threshold) values. A total of 12 (60%) IntI1 positive strains were detected with strong gene copy/µL (Ct value ≤ 29), while all intI2 positive bacteria had Ct > 29. Among the total 12 (42.8%) bacteria, gram-negative 10 (35.7%) while gram-positive 2 (7.1%) harbored both intI1 and intI2 genes. Strains from the glacier’s sediment sample were more copious in intI1 (16, 80%) and intI2 (9, 75%), followed by water and ice samples. The ARGs (blaCTXM-15, blaNDM 1, aac(6)-lb3, and gyrA), detected among the MDR psychrophilic bacteria Brevundimonas diminuta, Rahnella inusitata, Staphylococcus equorum, and Alcaligenes faecalis were PCR amplified and poly-adenylated amplicons were purified and quantified for vector insertion. The desired ARGs of interest were successfully cloned in a pCR2.1-Topo vector and transformed using competent E. coli (One-Shot Mach1-T1) cells with ampicillin drug selection. Through the alpha complementation system (blue-white screening), transformed E. coli cells were screened out. The extracted plasmid vectors containing the desired gene of inserts were PCR amplified using appropriate primers and exact amplicons were visualized on the gel electrophoresis. The nucleotide sequences of blaCTXM-15, blaNDM-1, aac(6)-lb3, and gyrA cloned plasmid vectors were processed through SnapGene Software, and mapping and annotation were performed. The complete sequence of each plasmid vector construct with blaCTXM-15, blaNDM-1, aac(6’)-lb3, and gyrA was obtained with disruption of the lac operon lacZ gene. The present study manifests the first comprehensive assessment of the presence of antibiotic resistant bacteria harbored with antibiotic-resistant genes and class 1 and 2 integron integrase genes, inhabiting a non-polar Passu glacier. The attained results highlight the mandatory focus to evaluate other non-polar glaciers regarding the bacterial diversity carrying antibiotic resistant genes and mobile genetic elements. The glaciers located in the HKKH region provide agriculture and domestic water to billions of people in various countries, moreover, due to global warming effects, the glacier meltwater intermixes with the rivers and rainwater which poses a serious threat to the community’s health and requires quintessential approach.