Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae

Bacterial antimicrobial resistance (AMR) is constantly evolving and horizontal gene transfer through plasmids plays a major role. The identification of plasmid characteristics and their association with different bacterial hosts provides crucial knowledge that is essential to understand the contribution of plasmids to the transmission of AMR determinants. Molecular identification of plasmid and strain genotypes elicits a distinction between spread of AMR genes by plasmids and dissemination of these genes by spread of bacterial clones. For this reason several methods are used to type the plasmids, e.g. PCR-based replicon typing (PBRT) or relaxase typing. Currently, there are 28 known plasmid types in Enterobacteriaceae distinguished by PBRT. Frequently reported plasmids [IncF, IncI, IncA/C, IncL (previously designated IncL/M), IncN and IncH] are the ones that bear the greatest variety of resistance genes. The purpose of this review is to provide an overview of all known AMR-related plasmid families in Enterobacteriaceae, the resistance genes they carry and their geographical distribution

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

Occurrence and diversity of IncZ plasmids in Europe

IncZ plasmids belong to the I-complex together with IncI1, IncI2, IncIγ, IncK1, IncK2 and IncB/O. The first IncZ plasmid was described in 1983. Later, seven additional distinct IncZ plasmid groups have been described. The most commonly used in silico plasmid typing scheme does not discriminate between the different plasmids within the IncK/B/O/Z group. The aim of this study was to investigate occurrence and diversity of IncZ plasmids in Europe. In this study 410 E. coli and Shigella sonnei isolates carrying IncK/B/O/Z replicons were collected in 10 European countries and Lebanon and subjected to short-read whole genome sequencing. The isolates originated from humans (n=124), poultry (n=233), pigs (n=28), dairy cattle (n=4), veal cattle (n=19) and environment (n=2). IncK/B/O/Z plasmids were further typed based on their RNAI sequence and categorized into IncK1, IncK2, IncB/O and IncZ plasmid incompatibility groups. Among the 410 isolates, IncZ was the most observed type within the IncK/B/O/Z plasmid group with 213 positive isolates (52%). IncZ plasmids carried a wide variety of genes conferring resistance to β-lactams, sulfonamides, aminoglycosides and colistin. The results indicate that human clinical samples have a significantly higher (p<0,001) occurrence of IncZ plasmids compared to carriage samples from humans. Additionally, a phylogenetic analysis based on the RNAI sequence suggests the existence of new clusters in addition to the previously described IncZ plasmid groups.This study found a high occurrence of IncZ plasmids within the IncK/B/O/Z-harboring isolates from human and animal sources in Europe, carrying a wide variety of antimicrobial resistance genes. Additionally it revealed existence of potentially new IncZ plasmid types. In order to get more insight to this plasmid type, there is a need for a better typing and surveillance

Colistin-resistant Enterobacterales among veterinary healthcare workers and in the Dutch population.

Plasmid-mediated colistin resistance can be transferred from animals to humans. We investigated the prevalence of carriage of mcr-mediated colistin-resistant Escherichia coli and Klebsiella pneumoniae (ColR-E/K) in veterinary healthcare workers and in the general population in the Netherlands

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