Understanding the genetic basis of the incompatibility of IncK1 and IncK2 plasmids

Antimicrobial resistance is a persistent challenge in human and veterinary medicine, which is often encoded on plasmids which are transmissible between bacterial cells. Incompatibility is the inability of two plasmids to be stably maintained in one cell which is caused by the presence of identical or closely related shared determinants between two plasmids originating from partition or replication mechanisms. For I-complex plasmids in Enterobacteriacae, replication- based incompatibility is caused by the small antisense RNA stem-loop structure called RNAI. The I-complex plasmid group IncK consists of two compatible subgroups, IncK1 and IncK2, for which the RNAI differs only by five nucleotides. In this study we focussed on the interaction of the IncK1 and IncK2 RNAI structures by constructing minireplicons containing the replication region of IncK1 or IncK2 plasmids coupled with a kanamycin resistance marker. Using minireplicons excludes involvement of incompatibility mechanisms other than RNAI. Additionally, we performed single nucleotide mutagenesis targeting the five nucleotides that differ between the IncK1 and IncK2 RNAI sequences of these minireplicons. The obtained results show that a single nucleotide change in the RNAI structure is responsible for the compatible phenotype of IncK1 with IncK2 plasmids. Only nucleotides in the RNAI top loop and interior loop have an effect on minireplicon incompatibility with wild type plasmids, while mutations in the stem of the RNAI structure had no significant effect on incompatibility. Understanding the molecular basis of incompatibility is relevant for future in silico predictions of plasmid incompatibility

I-complex plasmids – a story about incompatibility and host adaptation.

Antimicrobial resistance (AMR) is a growing problem worldwide. This is mainly the result of the presence of resistance genes in bacteria. When these genes are located in small pieces of DNA, so called plasmids, they can easily spread between bacteria. This thesis gives an overview the different types of plasmids, all with their different resistance genes, and their presence around the globe. Bacteria may contain a number of different plasmids in a single cell. whether any of these plasmids can be stably maintained depends on several factors. One of these factors is called incompatibility, which is the inability of plasmids to be maintained in one cell. Based on this incompatibility plasmids can be categorized in 40 different “Inc” groups, a biological phenomenon that was long used as a typing system to study epidemiology. This thesis focusses on the members of the “I-complex” incompatibility group, found in Enterobacteriaceae and frequently associated with important resistance gene classes such as the so-called Extended Spectrum Beta-lactamases (ESBLs). The I-complex includes IncI1α, IncI1γ, IncI2, IncK, IncB/O and IncZ plasmids. In this thesis the basis of their incompatibility is analysed and the results show that a single mutation (change in DNA) can determine if two plasmids are compatible or not. It concludes that IncB/O and IncZ plasmids, which were previously considered compatible and thus different plasmids, were actually incompatible and should be considered as highly similar. It also shows that the IncK plasmid group, which is an important carrier of resistance genes in human and poultry bacteria, consists of two compatible plasmid lineages, designated IncK1 and IncK2. Studies into the molecular effects of these plasmid types on the physiology of their host cell showed that IncK2 plasmids provide a fitness advantage at a higher environmental temperature which may have resulted in their predominance in poultry over IncK1, which is mostly found in mammals. Overall, the results presented in this thesis highlight the importance of plasmids in the spread of AMR, and it underlines the importance of understanding the plasmid compatibility and its adaptation to the bacterial or animal host

I-complex plasmids – a story about incompatibility and host adaptation.

Antimicrobial resistance (AMR) is a growing problem worldwide. This is mainly the result of the presence of resistance genes in bacteria. When these genes are located in small pieces of DNA, so called plasmids, they can easily spread between bacteria. This thesis gives an overview the different types of plasmids, all with their different resistance genes, and their presence around the globe. Bacteria may contain a number of different plasmids in a single cell. whether any of these plasmids can be stably maintained depends on several factors. One of these factors is called incompatibility, which is the inability of plasmids to be maintained in one cell. Based on this incompatibility plasmids can be categorized in 40 different “Inc” groups, a biological phenomenon that was long used as a typing system to study epidemiology. This thesis focusses on the members of the “I-complex” incompatibility group, found in Enterobacteriaceae and frequently associated with important resistance gene classes such as the so-called Extended Spectrum Beta-lactamases (ESBLs). The I-complex includes IncI1α, IncI1γ, IncI2, IncK, IncB/O and IncZ plasmids. In this thesis the basis of their incompatibility is analysed and the results show that a single mutation (change in DNA) can determine if two plasmids are compatible or not. It concludes that IncB/O and IncZ plasmids, which were previously considered compatible and thus different plasmids, were actually incompatible and should be considered as highly similar. It also shows that the IncK plasmid group, which is an important carrier of resistance genes in human and poultry bacteria, consists of two compatible plasmid lineages, designated IncK1 and IncK2. Studies into the molecular effects of these plasmid types on the physiology of their host cell showed that IncK2 plasmids provide a fitness advantage at a higher environmental temperature which may have resulted in their predominance in poultry over IncK1, which is mostly found in mammals. Overall, the results presented in this thesis highlight the importance of plasmids in the spread of AMR, and it underlines the importance of understanding the plasmid compatibility and its adaptation to the bacterial or animal host

Understanding the genetic basis of the incompatibility of IncK1 and IncK2 plasmids.

Antimicrobial resistance is a persistent challenge in human and veterinary medicine, which is often encoded on plasmids which are transmissible between bacterial cells. Incompatibility is the inability of two plasmids to be stably maintained in one cell which is caused by the presence of identical or closely related shared determinants between two plasmids originating from partition or replication mechanisms. For I-complex plasmids in Enterobacteriacae, replication- based incompatibility is caused by the small antisense RNA stem-loop structure called RNAI. The I-complex plasmid group IncK consists of two compatible subgroups, IncK1 and IncK2, for which the RNAI differs only by five nucleotides. In this study we focussed on the interaction of the IncK1 and IncK2 RNAI structures by constructing minireplicons containing the replication region of IncK1 or IncK2 plasmids coupled with a kanamycin resistance marker. Using minireplicons excludes involvement of incompatibility mechanisms other than RNAI. Additionally, we performed single nucleotide mutagenesis targeting the five nucleotides that differ between the IncK1 and IncK2 RNAI sequences of these minireplicons. The obtained results show that a single nucleotide change in the RNAI structure is responsible for the compatible phenotype of IncK1 with IncK2 plasmids. Only nucleotides in the RNAI top loop and interior loop have an effect on minireplicon incompatibility with wild type plasmids, while mutations in the stem of the RNAI structure had no significant effect on incompatibility. Understanding the molecular basis of incompatibility is relevant for future in silico predictions of plasmid incompatibility

Incompatibility and phylogenetic relationship of I-complex plasmids

Plasmid incompatibility is the inability of two plasmids to be stably maintained in one cell, resulting in loss of one of the plasmids in daughter cells. Dislodgement is a phenotypically distinct form of incompatibility, described as an imperfect reproduction, manifesting in rapid exclusion of a resident plasmid after superinfection. The relationship between plasmids of the phenotypic incompatibility groups IncB/O and IncZ is unclear. Their inability to co-exist was initially referred to as dislodgement while other research reached the conclusion that IncB/O and IncZ plasmids are incompatible. In this manuscript we re-evaluated the relationship between IncB/O and IncZ plasmids to settle these conflicting conclusions. We performed dislodgement testing of R16Δ (IncB/O) and pSFE-059 (IncZ) plasmids by electroporation in a bacterial cell and checked their stability. Stability tests of the obtained plasmid pair showed that the IncB/O plasmid was exclusively and almost completely lost from the heteroplasmid Escherichia coli population. Other IncB/O - IncZ pairs could not form a heteroplasmid population, using conjugation or electroporation. Our data supports the previous suggestion that IncB/O and IncZ plasmids may be considered phenotypically incompatible

Successful host adaptation of IncK2 plasmids

The IncK plasmid group can be divided into two separate lineages named IncK1 and IncK2. IncK2 is found predominantly in poultry while IncK1 was reported in various mammals, including animals and humans. The physiological basis of this distinction is not known. In this manuscript we examined fitness cost of IncK1 and IncK2 plasmids at 37 and 42°C, which resembles mammalian and chicken body temperatures, respectively. We analyzed conjugation frequency, plasmid copy number and plasmid fitness cost in direct competition. Additionally, we measured levels of σ-32 in Escherichia coli carrying either wild type or conjugation-deficient IncK plasmids. The results show that IncK2 plasmids have a higher conjugation frequency and lower copy number at 42°C compared to IncK1. While the overall fitness cost to the host bacterium of IncK2 plasmids was higher than that of IncK1, it was not affected by the temperature while the fitness cost of IncK1 was shown to increase at 42°C compared to 37°C. These differences correlate with an increased expression of σ-32, a regulator of heat-shock protein expression, in E. coli with IncK2 compared to cells containing IncK1. This effect was not seen in cells containing conjugation deficient plasmids. Therefore, it is hypothesized that the assembly of the functional T4S may lead to these increased levels of σ-32. Increased activation of CpxR at 42°C may explain why IncK2 plasmids, and not IncK1, are predominantly found in chicken isolates

Successful Host Adaptation of IncK2 Plasmids.

The IncK plasmid group can be divided into two separate lineages named IncK1 and IncK2. IncK2 is found predominantly in poultry while IncK1 was reported in various mammals, including animals and humans. The physiological basis of this distinction is not known. In this manuscript we examined fitness cost of IncK1 and IncK2 plasmids at 37 and 42°C, which resembles mammalian and chicken body temperatures, respectively. We analyzed conjugation frequency, plasmid copy number and plasmid fitness cost in direct competition. Additionally, we measured levels of σ-32 in Escherichia coli carrying either wild type or conjugation-deficient IncK plasmids. The results show that IncK2 plasmids have a higher conjugation frequency and lower copy number at 42°C compared to IncK1. While the overall fitness cost to the host bacterium of IncK2 plasmids was higher than that of IncK1, it was not affected by the temperature while the fitness cost of IncK1 was shown to increase at 42°C compared to 37°C. These differences correlate with an increased expression of σ-32, a regulator of heat-shock protein expression, in E. coli with IncK2 compared to cells containing IncK1. This effect was not seen in cells containing conjugation deficient plasmids. Therefore, it is hypothesized that the assembly of the functional T4S may lead to these increased levels of σ-32. Increased activation of CpxR at 42°C may explain why IncK2 plasmids, and not IncK1, are predominantly found in chicken isolates

Successful host adaptation of IncK2 plasmids

The IncK plasmid group can be divided into two separate lineages named IncK1 and IncK2. IncK2 is found predominantly in poultry while IncK1 was reported in various mammals, including animals and humans. The physiological basis of this distinction is not known. In this manuscript we examined fitness cost of IncK1 and IncK2 plasmids at 37 and 42°C, which resembles mammalian and chicken body temperatures, respectively. We analyzed conjugation frequency, plasmid copy number and plasmid fitness cost in direct competition. Additionally, we measured levels of σ-32 in Escherichia coli carrying either wild type or conjugation-deficient IncK plasmids. The results show that IncK2 plasmids have a higher conjugation frequency and lower copy number at 42°C compared to IncK1. While the overall fitness cost to the host bacterium of IncK2 plasmids was higher than that of IncK1, it was not affected by the temperature while the fitness cost of IncK1 was shown to increase at 42°C compared to 37°C. These differences correlate with an increased expression of σ-32, a regulator of heat-shock protein expression, in E. coli with IncK2 compared to cells containing IncK1. This effect was not seen in cells containing conjugation deficient plasmids. Therefore, it is hypothesized that the assembly of the functional T4S may lead to these increased levels of σ-32. Increased activation of CpxR at 42°C may explain why IncK2 plasmids, and not IncK1, are predominantly found in chicken isolates

Incompatibility and phylogenetic relationship of I-complex plasmids

Plasmid incompatibility is the inability of two plasmids to be stably maintained in one cell, resulting in loss of one of the plasmids in daughter cells. Dislodgement is a phenotypically distinct form of incompatibility, described as an imperfect reproduction, manifesting in rapid exclusion of a resident plasmid after superinfection. The relationship between plasmids of the phenotypic incompatibility groups IncB/O and IncZ is unclear. Their inability to co-exist was initially referred to as dislodgement while other research reached the conclusion that IncB/O and IncZ plasmids are incompatible. In this manuscript we re-evaluated the relationship between IncB/O and IncZ plasmids to settle these conflicting conclusions. We performed dislodgement testing of R16Δ (IncB/O) and pSFE-059 (IncZ) plasmids by electroporation in a bacterial cell and checked their stability. Stability tests of the obtained plasmid pair showed that the IncB/O plasmid was exclusively and almost completely lost from the heteroplasmid Escherichia coli population. Other IncB/O – IncZ pairs could not form a heteroplasmid population, using conjugation or electroporation. Our data supports the previous suggestion that IncB/O and IncZ plasmids may be considered phenotypically incompatible.</p

Plasmids of distinct IncK lineages show compatible phenotypes

IncK plasmids are one of the main carriers of bla CTX-M-14 and bla CMY-2 genes and show high similarity to other plasmids belonging to the I complex, including IncB/O plasmids. Here, we studied the phylogenetic relationship of 37 newly sequenced IncK and IncB/O plasmids. We show that IncK plasmids can be divided into two compatible lineages named IncK1 and IncK2