Conjugative transfer of multi-drug resistance IncN plasmids from environmental waterborne bacteria to Escherichia coli.

Watersheds contaminated with municipal, hospital, and agricultural residues are recognized as reservoirs for bacteria carrying antibiotic resistance genes (ARGs). The objective of this study was to determine the potential of environmental bacterial communities from the highly contaminated La Paz River basin in Bolivia to transfer ARGs to an Escherichia coli lab strain used as the recipient. Additionally, we tested ZnSO4 and CuSO4 at sub-inhibitory concentrations as stressors and analyzed transfer frequencies (TFs), diversity, richness, and acquired resistance profiles. The bacterial communities were collected from surface water in an urban site close to a hospital and near an agricultural area. High transfer potentials of a large set of resistance factors to E. coli were observed at both sites. Whole-genome sequencing revealed that putative plasmids belonging to the incompatibility group N (IncN, IncN2, and IncN3) were predominant among the transconjugants. All IncN variants were verified to be mobile by a second conjugation step. The plasmid backbones were similar to other IncN plasmids isolated worldwide and carried a wide range of ARGs extensively corroborated by phenotypic resistance patterns. Interestingly, all transconjugants also acquired the class 1 integron intl1, which is commonly known as a proxy for anthropogenic pollution. The addition of ZnSO4 and CuSO4 at sub-inhibitory concentrations did not affect the transfer rate. Metal resistance genes were absent from most transconjugants, suggesting a minor role, if any, of metals in the spread of multidrug-resistant plasmids at the investigated sites.

An assay for determining minimal concentrations of antibiotics that drive horizontal transfer of resistance.

Ability to understand the factors driving horizontal transfer of antibiotic resistance from unknown, harmless bacteria to pathogens is crucial in order to tackle the growing resistance problem. However, current methods to measure effects of stressors on horizontal gene transfer have limitations and often fall short, as the estimated endpoints can be a mix of both the number of transfer events and clonal growth of transconjugants. Our aim was therefore to achieve a proper strategy for assessing the minimal concentration of a stressor (exemplified by tetracycline) that drives horizontal transfer of antibiotic resistance from a complex community to a model pathogen. Conditions were optimized to improve a culture-based approach using the bacterial community of treated sewage effluent as donor, and fluorescent, traceable Escherichia coli as recipient. Reduced level of background resistance, differentiation of isolates as well as decreased risk for measuring effects of selection were achieved through the use of chromogenic medium, optimization of conjugation time as well as applying a different antibiotic for isolation of transconjugants than the one tested for its ability to drive transfer. Using this assay, we showed that a very low concentration of tetracycline, 10µg/L i.e. 150 times below the minimal inhibitory concentration of the recipient, promoted horizontal transfer of multiple antibiotic-resistance determinants. Higher concentrations favoured selection of a tetracycline-resistance phenotype along with a decline in the number of detectable transfer events. The described method can be used to evaluate different environmental conditions and factors that trigger horizontal dissemination of mobile resistance elements, eventually resulting in the formation of drug-resistant pathogens.

Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids.

In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+C content of 53.75%. A total of 135 open reading frames (ORFs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. ORFs encoding proteins with putative functions in stress response, transposition and site-specific recombination were also predicted. Analysis of the organization and nucleotide sequence of pD2RT backbone region revealed high degree of similarity to the draft genome sequence data of the plant-pathogenic pseudomonad Pseudomonas syringae pv. glycinea strain B076, exhibiting relatedness to pPT23A plasmid family. The pD2RT backbone is also closely related to that of pGRT1 of Pseudomonas putida strain DOT-T1E and pBVIE04 of Burkholderia vietnamiensis strain G4, both plasmids are associated with resistance to toluene. The ability of pD2RT to self-transfer by conjugation to P. putida recipient strain PaW340 was experimentally determined. Genetic organization of toluene-degrading (xyl) genes and flanking DNA segments resembles the structure of Tn1721-related class II transposon Tn4656 of TOL plasmid pWW53 of P. putida strain MT53. The complete sequence of the plasmid pD2RT extends the known range of xyl genes carriers, being the first completely sequenced TOL plasmid, which is not related to well-studied IncP plasmid groups. We also verified the functionality of the catabolic route encoded by pD2RT by monitoring the expression of the xylE gene in pD2RT bearing hosts along with bacterial strains containing TOL plasmid of IncP-9 group. The growth kinetics of plasmid-bearing strains was found to be affected by particular TOL plasmid.

Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes.

A set of phenol-degrading strains of a collection of bacteria isolated from Baltic Sea surface water was screened for the presence of two key catabolic genes coding for phenol hydroxylases and catechol 2,3-dioxygenases. The multicomponent phenol hydroxylase (LmPH) gene was detected in 70 out of 92 strains studied, and 41 strains among these LmPH(+) phenol-degraders were found to exhibit catechol 2,3-dioxygenase (C23O) activity. Comparative phylogenetic analyses of LmPH and C23O sequences from 56 representative strains were performed. The studied strains were mostly affiliated to the genera Pseudomonas and Acinetobacter. However, the study also widened the range of phenol-degraders by including the genus Limnobacter. Furthermore, using a next generation sequencing approach, the LmPH genes of Limnobacter strains were found to be the most prevalent ones in the microbial community of the Baltic Sea surface water. Four different Limnobacter strains having almost identical 16S rRNA gene sequences (99%) and similar physiological properties formed separate phylogenetic clusters of LmPH and C23O genes in the respective phylogenetic trees.

Occurrence of plasmids in the aromatic degrading bacterioplankton of the baltic sea.

Plasmids are mobile genetic elements that provide their hosts with many beneficial traits including in some cases the ability to degrade different aromatic compounds. To fulfill the knowledge gap regarding catabolic plasmids of the Baltic Sea water, a total of 209 biodegrading bacterial strains were isolated and screened for the presence of these mobile genetic elements. We found that both large and small plasmids are common in the cultivable Baltic Sea bacterioplankton and are particularly prevalent among bacterial genera Pseudomonas and Acinetobacter. Out of 61 plasmid-containing strains (29% of all isolates), 34 strains were found to carry large plasmids, which could be associated with the biodegradative capabilities of the host bacterial strains. Focusing on the diversity of IncP-9 plasmids, self-transmissible m-toluate (TOL) and salicylate (SAL) plasmids were detected. Sequencing the repA gene of IncP-9 carrying isolates revealed a high diversity within IncP-9 plasmid family, as well as extended the assumed bacterial host species range of the IncP-9 representatives. This study is the first insight into the genetic pool of the IncP-9 catabolic plasmids in the Baltic Sea bacterioplankton.

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20.

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 ISPre1-like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 ISPre2-like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer - we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of ISPre2-like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.