Temperature dependency suggests that these plasmids are potential vectors for the dissemination of genes among bacterial species in aqueous and soil environments. Many of them are reported to be associated with multidrug resistance because, besides ESBL genes, they often carry genes encoding for resistance to sulphonamides, aminoglycosides, tetracyclines and streptomycin Figures 2 and 3.
IncHI1 plasmids are considered as a main carrier of a multiple resistant phenotype in Salmonella Typhi. IncHII plasmids were described to carry resistance to trimethoprim, streptomycin and spectinomycin, which were part of Tn 7 , and ampicillin, amikacin, chloramphenicol, gentamicin, kanamycin and tetracycline. The copy number is controlled by iterons which are also targeted in the PBRT scheme. Later, six subgroups of IncP plasmids were defined. IncP plasmids are often isolated from Salmonella Infantis from broilers in Japan.
Recently, an IncP plasmid was reported to be associated with the colistin resistance gene mcr-1 and its variant mcr Originally the plasmids IncL and IncM were separate groups. Richards and Datta proposed placing IncL plasmids in the IncM group because repeated incompatibility experiments showed that IncL plasmids were incompatible with IncM plasmids.
In contrast, Carattoli et al. Incompatibility tests did not confirm compatibility of IncM1 and IncM2 plasmids. In hospitals, K. IncN, MOB F according to relaxase typing, is a group of broad-host-range plasmids, for which the copy number is controlled by iterons. It was observed that plasmids belonging to the IncN group are often colocalized with IncF plasmids. Recently, a new plasmid type, named IncN2, carrying a novel replicase gene encoding Rep was described.
Garcia-Fernandez et al. It involves three target genes: repN , traJ and korA. It is disseminated throughout Europe and isolated mainly from E. It was mainly isolated from human K. Colicins, which belong to the family of bacteriocins, are proteins produced by some strains of E. Colicins are encoded by genes predominantly located on plasmids. There are two groups of colicinogenic plasmids.
These plasmids have been frequently used for genetic engineering and biotechnology such as construction of vector pBR A single mutation in this region can give rise to two different ColE1 plasmids, with independent copy numbers, replication and resistance level. Although ColE plasmids have been found to carry different AMR genes, they are predominantly associated with spread of qnrS1 and qnrB19 genes.
ColE plasmids were reported to carry novel colistin resistance genes mcr-4 and mcr Johnson et al. Recently, based on differences in the taxC gene, novel subgroups X5 and IncX6 were identified. IncX plasmids were present in Salmonella strains which were isolated before antibiotics were commonly used.
In addition, tetracycline and trimethoprim resistance determinants can be carried by IncX plasmids Table S1. A number of plasmids are not often reported in literature but, as most of them have a broad host range and can carry multiple AMR genes, these are involved in the continuous spread of resistance genes.
IncW plasmids were found in many bacterial sources in the s. The reference IncW plasmid pSa was shown to carry genes conferring resistance to chloramphenicol, tetracyclines, sulphonamides, gentamicin and trimethoprim. These groups are not detected by PBRT. These plasmids have a broad host range including Alpha-, Beta-, Delta- and Gammaproteobacteria and Cyanobacteria.
It was proposed that its broad host range is a result of the presence of genes required for plasmid replication. IncQ plasmid incompatibility is expressed through direct repeats at oriV , which was confirmed in both RSF and R This suggests that the lack of a partitioning or stability system leads to a high copy number, which prevents plasmid loss.
Additionally, it was proven that members of subclasses of the IncQ family are compatible with each other due to evolution of their iteron sequences. Rawlings and Tietze suggested dividing the IncQ family into two groups based on their Rep protein similarities. Early isolated IncT plasmids were carrying kanamycin- Rst1 or sulphonamide resistance genes R Plasmid Rms has been classified as a novel IncG plasmid; however, later studies showed that this plasmid is a member of the IncU group.
IncU plasmids were reported to carry resistance to: trimethoprim, chloramphenicol, ampicillin, tetracyclines, sulphonamides, kanamycin and streptomycin. The first IncD plasmid was mentioned by Datta. However, the group is not included in the PBRT scheme. IncD plasmids belong to the IncF-like group of plasmids based on classification by genetic relatedness and pilus structure.
Transfer between other families has not been determined. They can carry resistance determinants to ampicillin and kanamycin. Unfortunately, no plasmid of this incompatibility group was fully sequenced and there are no reports revealing the functional biology or prevalence of these plasmids. In Coetzee et al. Later it was discovered that R rather belongs to the group of integrative and conjugative elements ICEs.
These elements are integrated in the chromosome, but after excision they circularize, replicate autonomously and are self-transmissible via conjugation. Recent work of Carraro et al. Additionally, these plasmids encode the toxin—antitoxin system hipAB , although this is not highly conserved. These results suggest that ICEs are more similar to plasmids than was previously thought but, as these are not actually plasmids, they are not detected by the PBRT scheme. IncY is a group of prophages which replicate as autonomous plasmids.
IncY were confirmed to confer resistance to ampicillin and carry the bla SHV-2 gene. Although the PBRT scheme is widely used, it is recognized that it cannot detect all known plasmid types.
Another consideration is the continuous rearrangement and mutations that plasmids undergo, which may also occur in the regions that are used for plasmid typing. This may result in novel untypeable plasmids evolving from currently well-studied plasmid types. Since the first incompatibility experiments performed by Couturier et al. Different typing methodologies are used in literature, which hampers a comparison of results from these studies.
Nowadays, the PBRT scheme is the most commonly used technique for plasmid typing of Enterobacteriaceae, as it facilitates rapid identification of the dominant replicon types. Its use has led to a more unified way of plasmid identification, which in turn has resulted in a large expansion of our knowledge of plasmid epidemiology.
The commercially available PBRT kit is kept up to date by periodic inclusion of newly described targets for plasmid identification. The main disadvantage, however, is that it can only detect plasmids included in the scheme, and that some plasmids harbour more than one replication machinery. Typing plasmids according to the relaxase gene has a higher discriminatory power, but it misses plasmids which do not contain a relaxase gene.
Carattoli 6 has provided an extensive overview of plasmids and their associated resistance genes. The work presented here provides an update about all known resistance plasmids in Enterobacteriaceae.
A great variety of plasmids can be found in human, animal and environmental isolates. There are differences in prevalence of certain plasmids from different sources and on different continents.
Animals in Europe are mainly colonized by E. ESBLs are the most frequently described enzymes conferring resistance to antimicrobials encoded on plasmids. Enzymes hydrolysing aminoglycosides and genes encoding for resistance to quinolones and sulphonamides are often co-transferred through transposons located on a plasmid.
We also show that various plasmids seem to be associated to a different range of antibiotic resistance gene classes, e. However, the exact nature of these specific relationships is still not fully understood. Given the fact that the number of studies performed on all continents varies and certain resistance determinants are studied more intensively, the data presented in this article will inevitably be slightly biased.
Therefore, the observed differences should be interpreted with care. Most papers describe data from Europe. Furthermore, most plasmids are typed using the PBRT scheme, which means that the prevalence of the plasmids not included in that scheme can be underestimated.
The positions and opinions presented in this article are those of the authors alone and are not intended to represent the views or scientific works of EFSA. Abraham EP , Chain E. An enzyme from bacteria able to destroy penicillin. Rev Infect Dis ; 10 : — 8. Google Scholar. Sex compatibility in Escherichia coli. Genetics ; 37 : — Hayes W. The kinetics of the mating process in Escherichia coli. J Gen Microbiol ; 16 : 97 — Episome-mediated transfer of drug resistance in Enterobacteriaceae.
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Most research on resistance plasmids has focused either on comparisons of resistance genes, or on one type of plasmid, or on mobile genetic elements 38 , as opposed to overall plasmid composition in multiple strains. In this study, we attempt to find common elements and unifying principles in sets of plasmids found in E.
While in no way repudiating the value of other approaches, we feel that this point of view focusing on architecture and shared components adds novel insights to the understanding of plasmids. Two separate types of regions can be distinguished in plasmids; conserved and variable regions The conserved regions mostly contain genes involved in replication, stability and mobility, while the variable regions contain accessory genes.
In the plasmids described in this study, the consensus sequence, defined as the conserved regions per incompatibility group, is composed of genes for plasmid transfer and maintenance.
The variable regions that contain the resistance genes are located in the gaps seen in the consensus sequence or in mobile genetic element sites Figs. This implies that the plasmid can preserve its integrity by inserting new genes solely into designated regions and between essential genes. Within these E. A variation in conserved regions within the IncF group was earlier also observed by Fernandez-Lopez et al.
These classes, however, did not always correspond to the range found in this study. The consensus of IncI, IncX1 and IncX4 outlines the minimum requirement for a plasmid to be classified as this type of plasmid according to this dataset.
This set of similar genes suggests that crucial genes for classification are those that are involved in plasmid maintenance and plasmid transfer 18 , Possibly these represent the minimum number of genes necessary for transfer and maintenance for the corresponding plasmid type. This study supports the theory of incompatibility 16 , as no two plasmids belonging to the same incompatibility group were located in the same strain.
Incompatibility is caused by the competition between two plasmids using the same replication and partitioning system 40 , Hence, in theory, plasmids of two similar but not identical incompatibility groups could co-exist within one strain, if these plasmids also harbor other genes enhancing their survival such as partitioning genes or addiction systems.
The less similar their incompatibility region is, the more likely plasmids are to be able to coexist within one cell In contrast, it is common to find multiple replicons of the same incompatibility group within one plasmid, especially for IncF and IncI type plasmids Strains to be included in the study were selected for either ES BL or tetracycline resistance, not for multiple resistance, though further characterization often showed this.
Resistance genes were frequently assembled in clusters flanked by MGEs suggesting that multiple types of resistance can be easily transferred from plasmid to plasmid or from plasmid to chromosome Since clusters of this kind are highly mobile, they stimulate the spread of antimicrobial resistance The presence of these clusters sometimes multiple times within a single strain is in line with the concept of mobile genetic elements being able to move around separately from the plasmid 38 , Two types of multiple resistance clusters have been identified: 1 Sulphonamide, macrolide and aminoglycoside resistance and 2 lincosamide and aminoglycoside resistance Fig.
The extended spectrum beta-lactamases and tetracycline resistance genes are more often located each in a separate cluster, suggesting that these MGEs only transfer one type of resistance. In a dataset comprising bacterial genomes antimicrobial resistance clusters were positioned close to heavy metal or chemical resistance genes This observation supports the idea that these categories of genes only insert within specific places within a plasmid rather than randomly Tetracycline clusters flanked by Tn10 are wide-spread in gram-negative and gram-positive bacteria 48 , The various clusters of CTX-M genes are highly prevalent in plasmids 32 , This in turn suggests that this class of genes might be transferred more often from cell to cell or from plasmid to plasmid than other resistance genes.
Indeed, other studies have shown that ESBL plasmids can be transferred at a higher rate than tetracycline resistance plasmids 54 , The presence of TEM clusters in multiple plasmids within one cell suggests that the transposition of these genes had already taken place.
When beta-lactam resistance is induced, a section of the genome of E. Addiction systems that allow plasmids to maintain themselves within a cell by producing a stable toxin and unstable anti-toxin 25 are widespread 35 , Addiction systems can explain why multiple large plasmids remain in the cell over generations even in the absence of selective pressure Almost all plasmids in this study contained a toxin-antitoxin system.
Whether these addiction systems are associated with certain plasmid types is a matter of debate. Addiction systems were judged to be randomly distributed over plasmids rather than to be associated with specific types In conclusion, resistance plasmids recovered from foodborne E.
The shared features include essential genes for plasmid transfer and maintenance. Most plasmids maintain their presence in the cell with the assistance of addiction systems Specific resistance clusters can be positioned in any of the plasmids investigated.
As a result, resistance genes move easily and rapidly between plasmids and from plasmid to chromosomes 5 , 6.
Strains originated from chicken, turkey or bovine meat and were selected for having either a beta-lactam or tetracycline resistance.
Plasmid presence was initially confirmed by the by detection of the incompatibility group 62 and later confirmed by successful plasmid transfer experiments 54 and gel electrophoresis.
All E. One colony was picked and grown first in 5 ml LB with antibiotics before scaling up to ml LB with antibiotics. From the final volume, cell pellets were collected.
The plasmids were isolated using a Qiagen Plasmid Maxi Kit, checked for purity with Nanodrop and if samples had low concentration of DNA or were too contaminated with salts they were purified with ethanol precipitation.
Sequencing was performed by BaseClear B. Hybrid de novo assembly was performed using both the generated Illumina short read and PacBio long read data. In most cases several plasmids originated from a single strain and as a result the correct assembly of the plasmids turned out to be problematic and to pose severe methodological problems.
Short read data only were insufficient for plasmid assembly. In the end hybrid assembly combining long and short read data has provided high quality scaffolds that are reliable and translated back into single plasmids. Hybrid assembly was performed by first improving the quality of the Illumina reads by trimming of low-quality bases using BBDuk, part of BBMap suite version High-quality reads were assembled into contigs using ABySS version 2.
Based on these alignments, the contigs were linked together and placed into scaffolds. Using Illumina reads, gapped regions within scaffolds were partially closed using GapFiller version 1.
Finally, assembly errors and the nucleotide disagreements between the Illumina reads and scaffold sequences were corrected using Pilon version 1. The assembly of strains that afterwards still showed too low quality were not included in this paper. Low quality refers to too many unassembled small scaffolds, too many gaps and too low coverage of reads mapping to a scaffold.
Hybrid assembly can sometimes result in the artificial fusion of plasmids within one scaffold, as also seen by Margos et al. This artifact was mostly caused by identical regions present in two plasmids within the same cell. These were separated afterwards, based on gel photos showing multiple bands, Illumina only data showing these specific plasmids as separated and transfer experiments resulting in the transfer of single separated plasmids.
Fusion plasmids or hybrid plasmids do also occur in nature and this needs to be taken into account before splitting fused plasmids The scaffolds thus obtained were tested for circularity to determine if one scaffold corresponds to one full plasmid.
Circularity was also based on the annotation similarities between the start and end of the sequence, such as genes commonly found next to each other. Scaffolds not corresponding to a full sequence are distinguished from the other plasmids by name. Establishing circularity was also attempted using other softwares and then seems highly likely, but could not be proven beyond any doubt. The scaffold sequences were screened for resistance genes and their incompatibility group using CGEs ResFinder 4.
Full annotation was performed afterwards using RAST 2. The annotated sequences were further analyzed using Snapgene viewer 5. Imperfectly assembled plasmids were included in the alignment, but are distinguished by adding scaffold to the name. CLC workbench alignments provided a general view of matching parts in the sequences and were used to create a consensus sequence for each incompatibility group.
BRIG was used to visualize the annotated consensus and the matching plasmids sequences. Carattoli, A. Plasmids and the spread of resistance. Levy, S. Antibacterial resistance worldwide: Causes, challenges and responses. Lopatkin, A. Persistence and reversal of plasmid-mediated antibiotic resistance. Sommer, M. Prediction of antibiotic resistance: Time for a new preclinical paradigm?. Dionisio, F. Plasmids spread very fast in heterogeneous bacterial communities.
Genetics , — Threlfall, E. The emergence and spread of antibiotic resistance in food-borne bacteria. Food Microbiol. Tacconelli, E. Discovery, research, and development of new antibiotics: The WHO priority list of antibiotic-resistant bacteria and tuberculosis.
Dis 18 , — PubMed Article Google Scholar. Verraes, C. Antimicrobial resistance in the food chain: A review. Public Health 10 , — Stine, O. Widespread distribution of tetracycline resistance genes in a confined animal feeding facility. Agents 29 , — Kaesbohrer, A. The resistant phenotypes of 35 transconjugants for 15 kinds of antibiotics compared to the donor strains were shown in Table 2.
The results showed that all transconjugants and donor strains were capable of multiple antibiotic resistance for three or more antibiotics compared to recipient strain E.
So transconjugants which had a narrowed antibiotic resistance spectrum, lost one or several antibiotic resistances which were present in the donor bacteria, or had a broadened antibiotic resistance spectrum and gained one or several antibiotic resistances which were not present in the donor bacteria.
In a word, the antibiotic resistant spectrum of transconjugants narrowed after exposure to the donor bacteria at the rate of The resistant gene phenotypes of 35 transconjugants compared to its donor strains by PCR were shown in Table 3. The bla SHV gene was transferred successfully at the rate However, only one strain of the qnrS gene was transferred at the rate of 4.
Table 3. The multiple antibiotic resistant genotypes of 35 strains of donors and transconjugants. A plasmid harbored in E. The sequence analyzing results of the plasmid showed that E. Escherichia coli are important opportunistic pathogens that cause urinary tract infections and sepsis in animals and humans Lewis et al. The prevalence of multiple antibiotic resistant Enterobacteriaceae in the world has been increasing in recent decades. The selective pressure created by the abuse of these agents has led to the development of multiple antibiotic resistant bacteria.
Bacterial genes encoding ESBLs are often located on the same plasmid with other antibiotic resistance genes, leading to multiple bacterial resistances, causing great difficulties in clinical treatment of infectious diseases Ben-Shahar et al.
The genes encoding ESBLs are located on the plasmids. Due to the different geographical and antibiotic habits, the prevalence of genotypes in different countries, regions, and environments varies Fabre et al. One of the plasmids in transconjugants was sequenced to detect the transfer of the plasmids in the bacteria. Although WTPs can significantly reduce the microbial load in water, it cannot completely eliminate antibiotic resistance bacteria. On the contrary, these selective pressures increase the resistance of certain bacteria.
It will enter the local environment, resulting in the spread of resistant bacteria. On the other hand, untreated wastewater overflow into the surface during rainstorms may be one of the sources of ESBLs-producing E. No OXA genotype was detected in this study and a small amount of the fluoroquinolone resistance gene was detected.
The mechanism of bacterial resistance is quite complex. However, great progress has been made in the research of this topic. In particular, research of the R plasmid confirms that the genetic material contains the natural resistance gene in bacteria.
Acquired antibacterial resistance is gained via selective stress. Conjugation is the most common way genetic information is transferred and plays a very important role in the spread of multiple antibiotic resistance genes.
The results show that under certain selective pressures, the plasmid is very easily transferred between E. The antibiotic resistant spectrum of transconjugants narrowed compared to the donor bacteria at the rate of This could mean that the antibiotic resistance gene may be located in the movable elements such as plasmids rather than the genomes Park et al. However, the antibiotic resistance spectrum of transconjugants broadened compared to donor bacteria at the rate of 5. In addition, transconjugants which lost one or more antibiotic resistances also added one or more antibiotic resistances at the rate of These are why antibiotics should be used with caution so as not to cause an increase in antibiotic resistance.
At the same time, there was a significant increase in the resistance to STR, which may be caused by the enhanced expression of aadA1 and aadA2 gene cassettes located on the transferred plasmid, showing resistances that are not in donor bacteria Zhao et al.
This proved that the plasmids in E. The resistance genes are located on plasmids which have the ability to transfer in vitro , and the plasmids in E. QL performed the experiments and analyzed the data. WC drafted the manuscript. HZ and DH collected wastewater samples and some data.
Looking around the lab, you'll likely find many of the antibiotics listed in the table below. Note, in this post we'll focus primarily on antibiotics against Gram negative bacteria. In f uture posts, we'll detail selection in non-bacterial cells such as yeast or mammalian cells. The above table lists some antibiotics commonly found in the lab, their mechanism for killing bacteria, and general working concentrations. For instructions on how to prepare antibiotic stocks, see Addgene's Reference Page.
Historically, antibiotics have also been used to disrupt genes at the chromosomal level. Scientists introduce an antibiotic resistance cassette within the coding region of the gene they are trying to disrupt or delete, which both inactivates the gene and acts as a marker for the mutation. When designing these types of experiments it is best practice not to use the same resistance cassette for the mutation and for plasmid selection.
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