Genetic structure and biological properties of the first ancient multiresistance plasmid pKLH80 isolated from a permafrost bacterium

Abstract

A novel multidrug-resistance plasmid, pKLH80, previously isolated from Psychrobacter maritimus MR29-12 found in ancient permafrost, was completely sequenced and analysed. In our previous studies, we focused on the pKLH80 plasmid region containing streptomycin and tetracycline resistance genes, and their mobilization with an upstream-located ISPpy1 insertion sequence (IS) element. Here, we present the complete sequence of pKLH80 and analysis of its backbone genetic structure, including previously unknown features of the plasmid's accessory region, notably a novel variant of the b-lactamase gene bla . Plasmid pKLH80 was found to be a circular 14 835 bp molecule that has an overall G+C content of 40.3 mol% and encodes 20 putative ORFs. There are two distinctive functional modules within the plasmid backbone sequence: (i) the replication module consisting of repB and the oriV region; and (ii) the mobilization module consisting of mobA, mobC and oriT. All of the aforementioned genes share sequence identities with corresponding genes of different species of Psychrobacter. The plasmid accessory region contains antibiotic resistance genes and IS elements (ISPsma1 of the IS982 family, and ISPpy1 and ISAba14 of the IS3 family) found in environmental and clinical bacterial strains of different taxa. We revealed that the sequences flanking bla RTG-6 and closely related genes from clinical bacteria are nearly identical. This fact suggests that bla RTG-6 from the environmental strain of Psychrobacter is a progenitor of bla RTG genes of clinical bacteria. We also showed that pKLH80 can replicate in different strains of Acinetobacter and Psychrobacter genera. The roles of IS elements in the horizontal transfer of antibiotic resistance genes are examined and discussed.

Back to the future in a petri dish: Origin and impact of resurrected microbes in natural populations

“…Many of these resistance genes were highly similar to resistance genes found in pathogenic bacteria today, confirming the hypothesis that the antibiotic resistance genes of clinical bacteria originated from environmental bacteria. Taken together, these results support the hypothesis that a reservoir of resistance genes existed in a range of bacteria species prior to the anthropogenic use of antibiotics and contribute to a growing body of evidence demonstrating that antibiotic resistance evolved alongside antibiotic production in the natural environment (Perron et al., ; Petrova et al., ). These results also support the growing body of evidence that nonpathogenic environmental organisms, including those present in the permafrost, are a reservoir of resistance genes that have the potential to be transferred into pathogens, and thus greatly affect the evolution of multidrug‐resistant bacteria in clinical settings (Bhullar et al., ).…”

Plasmids of psychrophilic and psychrotolerant bacteria and their role in adaptation to cold environments

“…The ability to confer resistance to various heavy metals or metalloids was demonstrated for pGIAK1, originating from the obligate alkaliphilic and halotolerant Bacillaceae strain JMAK1, pSinA of arsenite oxidizing Sinorhizobium sp. M14 and pSCD of sulfur oxidizer S. denitrificans skB26 (Drewniak et al, 2013; Guo and Mahillon, 2013; Watanabe et al, 2014); while pKLH80 of Psychrobacter maritimus MR29-12, a strain isolated from ancient permafrost, is a novel multidrug-resistant plasmid, carrying genes determining streptomycin, tetracycline and β-lactam resistance (Petrova et al, 2014) (Table 2). …”

“…Moreover, it was previously shown that insertion sequence IS Ppy1 of pKLH80 can mobilize for transposition streptomycin ( strAB ), tetracycline ( tetH ) and β-lactam ( bla RTG −6 ) resistance modules (Petrova et al, 2009, 2014). The presence of the resistance modules within transposable elements is of a great importance, since these elements contribute highly in dissemination of the resistance determinants among diverse plasmids co-residing in a single bacterial cell.…”

“…Many plasmid-harboring bacterial strains have been isolated from permanently cold environments including the Arctic (e.g., Dziewit et al, 2013a; Moller et al, 2014), Antarctica (e.g., Kobori et al, 1984; Dziewit et al, 2013b) and Siberia (e.g., Petrova et al, 2014). Several plasmids were also identified during genomic projects of psychrophilic bacteria, e.g., five plasmids were recognized in the genome of Carnobacterium gilichinskyi WN1359T, a strain originating from Siberian permafrost (Leonard et al, 2013).…”

The Prehistory of Antibiotic Resistance

“…The DNA from permafrost can be isolated and queried experimentally for antibiotic resistance genes (among other things). The seminal work of D 'Costa et al (2011) on Beringian permafrost is complemented by studies showing that functional resistance genes can be retrieved from 5000-year-old DNA (Perron et al 2015), and that resistance had mobilized to plasmids and transposons in ancient times (Mindlin et al 2005;Petrova et al 2011Petrova et al , 2014. Modern day microorganisms found in a cave that has been isolated from the surface for four million years have also been shown to harbor functional antibiotic resistance genes (Bhullar et al 2012).…”