Genomic sequencing of E. faecalis V583 and the recent E. faecium Aus0004 showed presence of multiple nomadic familial elements in the genomes. Mobile familial elements nowadays in the strains are prophages, interpolation sequence elements, jumping genes and plasmids. For this chapter, plasmids of the Incompatibility group 18 and pheromone-responsive plasmids are described alongside with jumping genes of the Tn3-family, composite and conjugative categories. Some of the plasmids and jumping genes described contain virulency and antibiotic opposition factors, which were really likely to be acquired from elsewhere. Plasmids and jumping genes besides contribute to the airing of this factors and has been noted to lend to the transportation of vancomycin opposition to methicillin-resistant S. aureus ( MRSA ) .
4.1 Genomicss of the Enterococcus
Sequencing of bacteriums genomes has created a hoarded wealth trove of informations and started off many research countries. Analyzing of virulency factors are now made easier with cognition of this.
It is of no surprise that a bacterium with vancomycin opposition has already been sequenced. The strain, E. faecalis V583, a clinical isolate from the United States has already been to the full sequenced at The Institute for Genome Research ( TIGR ) .
4.1.1 V583
Based on informations that is widely available to the populace, V583 contains a 3218030 base brace chromosome and 3 round plasmids. The GC content of the chromosome is 37.53 % and has 3257 unfastened reading frames. The 3 plasmids, identified as pTEF1, pTEF2 and pTEF3 contains 66320, 17963 and 57660 bp severally. Table 1 shows the distribution and the map of the cistrons in the genome.
Function
No of Genes
Amino acid biogenesis
37
Biosynthesis of cofactors, prosthetic groups
49
Cell envelope
133
Cellular procedures
96
Cardinal intermediary metamorphosis
38
Disrupted reading frame
2
Deoxyribonucleic acid metamorphosis
107
Energy metamorphosis
208
Fatty acid and phospholipid metamorphosis
34
Conjectural proteins
1561
Other classs ( plasmid, prophage or transposon maps )
77
Protein destiny
77
Protein synthesis
131
Purines, pyrimidines, nucleosides and bases
69
Regulative maps
221
Signal transduction
2
Transcription
35
Conveyance and binding proteins
368
Unknown map
238
Viral maps
19
Table 4.1. Distribution of cistrons identified on E. faecalis V583 genome ( table adapted from Gilmore et al. )
pTEF1 and pTEF2 are similar to pAD1 and PCF10, doing them pheromone conjugative plasmid, while pTEF3 is similar to pAMI?1, which is an Inc18 plasmid.
Vancomycin opposition in V583 is encoded within a nomadic component, at EF2282 to EF2334. EF1955 to EF1963 besides encodes a Vancocin opposition which has the same sequence to the vanB cistrons found on a conjugative jumping gene, Tn1549. A pathogen island in the cistron, from EF0479 to EF0628 has been found in V583. It contains cistrons for collection, cell lysis and perchance many others and grounds on the islands show that it has been integrated into the genome from elsewhere by looking at the different per centums in GC content in different parts of the island. There are besides presence of other leftovers of incorporate plasmids in the chromosome and adding on to the fact that they have 3 plasmids shows that V583 depend a batch on plasmids for genome development.
4.1.1 Aus0004
Aus0004 is a vanB-positive, vancomycin resistant E. faecium that was isolated from a patient in Austin Hospital, Melbourne, Australia. The strain contains a round chromosome that is 2,955,294 bp in length and has three round plasmids. The plasmids are named Aus0004_p1, Aus0004_p2 and Aus0004_p3 and are 56,520 bp, 4,119 bp and 3,847 bp severally.
In the genome itself, Tn1549-like conjugative jumping gene incorporating the vanB phenotype is found, along with several infective islands. The infective island contains the clairvoyance cistron which is needed for colonisation in urinary piece of land infection and may play a function in biofilm formation.
Three prophages were present in the genome of Aus0004 and are similar to many other phages found in other species such as Clostridium and Staphylococcus.
21 different interpolation sequence elements that are in 76 distinguishable transcripts were found in Aus0004 and distributed among the chromosome and plasmids. A certain interpolation component, ISEf1 is non merely normally found in Aus0004 but is besides one of the dominant ISEs found in V583.
Present in Aus0004 are cistrons that code for haemolysin, collagen-binding adhesin but unlike other strains of E. faecium and E. faecalis, gelatinase, collection substance, lipase and hemagglutinin were non found in this genome.
Merely like V583, a big part of Aus0004 is comprised of nomadic DNA. This is shown by the presence of nomadic familial elements such as prophages, interpolation sequence elements, infective islands and plasmids.
4.2 Mobile Familial Elementss
The nomadic familial elements in the enterococcus are chiefly the plasmids and jumping genes. This subdivision is of peculiar significance to the clinical community due to the fact that it normally contains antibiotic opposition cistrons and straight take part in the airing of it.
Not merely that, it besides contains hemolysins, bacteriocins and even opposition to ultraviolet visible radiation.
As shown in the 2 illustration strain of E. faecalis and E. faecium, nomadic familial elements are to a great extent evolved in the development of the bacterium. The phenomenon is known as genomic malleability, where the bugs evolve to counter the force per unit area exerted from different environments, such as traveling from the colon so to the urinary vesica to do infection, or the presence of antibiotics.
In this subdivision, some of the nomadic familial elements involved in the spread of virulency factors and opposition in the Enterococci would be explored.
4.2.1 Plasmids
There are three types of plasmids in the enterococcus bacteriums: the turn overing circle replicating ( RCR ) plasmids, the Inc18 ( mutual exclusiveness group 18 plasmids and the pheromone-responsive plasmids.
The RCR plasmid is capable of reproduction in Gram-positive and Gram-negative species while Inc18 plasmids can merely retroflex in Gram-positive bacteriums.
The pheromone-responsive plasmids are merely capable of reproduction in the enterococcus spp. merely.
In this subdivision, the Inc18 plasmids and the pheromone-responsive plasmids would be discussed but non the RCR. This is because RCR is non native to the enterococci but it is used a vector that is constructed for the usage in this species.
4.2.1.1 Inc18 plasmids
There are two well-characterized Inc18 plasmids that are isolated. First, the pAMI?1 was foremost isolated from E. faecalis strain DS5 and the other one is pIP50 which was found in Streptococcus agalactiae. However, this peculiar type of plasmid is normally found in E. faecium. There are other plasmids that autumn under this class, such as pIP816 and pRUM, and each of it incorporate its ain cistrons cistrons coding for antibiotic opposition. Some of the belongingss of this plasmid are that the sizes range from 25 to 30kb in size, transcript Numberss range from 10 to 15 per chromosome. Many are self-conjugative and can be transferred to and replicated in several species of the Streptococci. The pAMI?1 plasmid contains erythromycin opposition due to the presence of ermB, which besides contains other opposition to antibiotics like macrolides and streptogramin B.
Numerous surveies have shown that these plasmids can reassign antibiotic opposition to other Gram-positive bacteriums, such as Listeria. spp and Lactococcus. spp. . pIP501, in peculiar, is able to confabulate antibiotic opposition to Gram-negative bacteriums, such as E. coli.
The reproduction of the Inc18 plasmids have received considerable attending and it has been found that it replicates via a alone type of mechanism Initiation of reproduction of the pAMI?1 plasmid requires the RepE instigator protein, a short 44bp beginning located downstream of the repE cistron which is the originating site for leading-strand synthesis and the host ‘s DNA polymerase I. Synthesis begins at the booster of RepE with the devising of a 10bp primer, so DNA polymerase I and reproduction would continue for about 150bp until a site for dawdling strand priming is exposed. At this point, DNA polymerase I is replaced by DNA polymerase III which will direct reproduction for both lagging and prima strands.
RepE is rate restricting for reproduction, so hence, synthesis should be tightly controlled. There are two mechanisms to its ordinance, one being the CopF protein and the other being an antisense RNA which is produced from the 5 ‘ terminal of the instigator cistron. Antisense RNA binds to the 5 ‘ terminal that is complementary to the non-coding part that is upstream of the repE messenger RNA and it consequences in expiration of written text. Cop proteins stops written text of their several rep cistrons by adhering to a rep booster sequence.
4.2.1.2 Pheromone-responsive plasmids
Plasmid transportation by pheromone is an of import mechanism for distribution antibiotic-resistance and virulency in the enterococcus bacteriums.
Fig. 4.1 Map of pAD1 shows notation of ORFs in the sequence. Red contains cistrons related to reproduction and care. Green regulates pheromone response. Dark bluish contains structural cistrons related to junction and purple contains opposition to ultraviolet visible radiation. Picture taken from Clewell et all, 2002 ) The pheromone-responsive plasmids are a household of 20 different plasmids which has been isolated from 14 different enterococcal isolates. These plasmids range in size from 37 to 91kb maintained in low transcript figure of up to 2-4 per chromosome equivalent and are by and large found in E. faecalis merely except for one which was isolated in E. faecium. They are characterized by its ability to organize sums in stock when assorted with plasmid-free cells which is formed due to copulating.
They are so named because conjugative transportation starts off with at least 5 different “ sex pheromone ” secreted by plasmid-free cells to other cells that contain plasmids. The giver cell, incorporating the plasmids that are specific to the pheromone, will react by synthesising plasmid-encoded collection substance ( AS ) which would adhere to the surfaces of both cells, incorporating enterococcal binding substance ( EBS ) . This binding allows for the formation of a channel which plasmid Deoxyribonucleic acid can reassign from giver to recipient. As a consequence, it is non surprising to happen that a strain would be transporting multiple pheromone-responsive plasmids.
Plasmids that have been described for it are pAD1, pCF10, pPD1 and pAM373. pAD1 encodes hemolysin or bacteriocin, pCF10 codifications for Achromycin opposition and pPD1 encodes bacteriocin.
pAM373 plasmid ‘s map is still non known yet. Terminology of the plasmids is dependent on the pheromone it responds to. For illustration, pAD1 responses to the pheromone cAD1. A map of pAD1 can be seen in figure 4.1. Pheromones are little peptide from 7 to 8 amino acid residues that are by and large hydrophobic in nature. They are synthesized as a precursor, normally up to 275 amino acids depending on the plasmid strain, that requires treating to organize the pheromone ( see figure 4.2 ) .
Further surveies has shown that pheromone signaling is non every bit simple as it was thought to be, where a coupling response is conferred by giver cell after having of a pheromone. After some clip, it became evident that it does non explicate plenty, as pheromones are chromosomally encoded while response is plasmid determined. This would logically intend that the bacteriums itself could react to its ain sex pheromone. However, this has ne’er seen to be go oning. This was explained by giver cells showing the ability to encode little peptides that act as competitory inhibitors to forestall its ain plasmids from acknowledging its endogenously secreted pheromone. The inhibitor peptide is secreted as a precursor up to 23 amino acids, where the last 7-8 amino acids in the C-terminal constitute mature inhibitors ( see figure 4.2 ) . To explicate how this inhibitor peptide does non suppress exogenic pheromones, Mori et Al showed that E. faecalis cells release a mixture of iCF10 and cCF10 in a molar ratio of 50-100/1, which allows the giver cells to stay sensitive to ‘legit ‘ pheromones while neutralizing endogenous pheromones.
As mentioned earlier in this chapter, pheromone-responsive plasmids have non been identified in other species of bacteriums. The cAM373 peptide, which induces a response from pAM373, has a similar activity produced by Staphylococcus aureus. However, the construction is different, with a difference of one amino acid at the C-terminus and there is presently no grounds that this is more than merely a happenstance. Recent surveies utilizing a pAM373: :PAD2 conintegrate plasmid showed that the plasmid can be stably transferred from E. faecalis to S. aureus and was able to retroflex. Add on to the fact that cAM373 is able to bring on a response from a late identified pAM368 plasmid that encodes a vancomycin-resistance cistron shows that S. aureus achieving this virulency factor is coming nearer.
Fig. 4.2 Shows the virulency, plasmids, pheromone, inhibitors and their corresponding precursors. Taken from Clewell et al. , 2000.
4.2.2 Transposons
Transposon in the Enterococcus spp. by and large falls into 3 categories: the Tn3- household, composite and
Transposon in the Enterococcus spp. by and large falls into 3 categories: the Tn3-
conjugative jumping genes. All 3 jumping genes are widespread in many bacteriums and have been well-characterized in other Gram-negative bacteriums for the first 2 categories and Gram-positive bacteriums for the last category.
Transposons have a function to play in the airing of antibiotic-resistance cistrons, as it has a wide host scope and some categories are able to undergo conjugative heterotaxy even in the absence of plasmid DNA.
In this subdivision, the function of jumping genes in the Enterococcus would be discussed.
4.2.2.1 Tn3 jumping genes
The Tn3 household of jumping genes normally encode ampicillin opposition in Gram-negative bacteriums.The household are classed together by their similarity in their upside-down repetition and heterotaxy proteins and in their mechanisms of heterotaxy. This group of jumping genes do non encode any conjugative maps, which means that transportation between bacteriums cells require aid by incorporating into a plasmid. At least even without a conjugative map, they have one advantage over the conjugative and composing jumping genes, is that they are replicative and both giver and mark molecules possess a transcript.
Tn917 and Tn1546 are two transposon under this household that are good studied. Tn917 was the first jumping gene identified in enterococcus and contains a 5.4kb Erythrocin opposition cistron on the plasmid pAD2. It besides encodes opposition to lincosamides and streptogramin B due to the ermAM cistron, which protects the ribosome from being binded by macrolides through methylation. Expression of it is can be induced by macrolides and Erythrocin, but non clindamycin, even if ermAM is immune against the latter drug. Tn551, a jumping gene found in S. aureus is basically the same as Tn917 despite a few differences except that it can constitutively show the MLS opposition.
Tn1546, a 10.9kb Tn3 -family transposon that encodes vancomycin opposition whose phenotype is the VanA-type glycopeptode, was isolated from E. faecalis. The phenotype can be seen expressed in one-fourth of the patients transporting the enterococcus bacteriums in intensive attention unit. Like Tn917, it does non hold any conjugative maps and its spread is done by incorporating itself into conjugative plasmids.
A reappraisal by Palmer et Al has mentioned that Tn1546 resides in plasmids of the Inc18 category, such as pIP816 which is similar to the pAMI? category. It besides mentioned that Inc18-type plasmid is associated with Tn1546 for most of the transportation between the enterococci and MRSA.
The reproduction of the Tn3 jumping gene is mediated by a transposase and resolvase. First, the plasmid incorporating Tn3 forms a cointegrate by blending with the mark plasmid with the aid of the transposase enzyme. The mark plasmid would have the transcript of the jumping gene through DNA reproduction into dual stranded Deoxyribonucleic acid. After that, resolvase will divide the mark and donor DNA and the terminal consequence is that each plasmid would hold its ain transcript of the jumping gene.
4.2.2.2 Composite jumping genes
Fig. 4.3 Structure of a composite jumping gene. Note the antibiotic opposition cistrons sandwiched in between two IS. ( Taken from hypertext transfer protocol: //www.zo.utexas.edu/faculty/sjasper/images/18.18.gif )
A composite jumping genes, in this context, consist of an antibiotic cistron flanked by presence of terminal interpolation sequences, or IS elements. Both IS elements, normally of the same type, provide mobility for the cistron.
Tn5281, a 4.7kb plasmid incorporating a opposition to aminoglycosides and flanked by upside-down transcripts of IS256 and IS257 was first described in a gentamicin-resistant strain of enterococcus on pBEM10. The plasmid is similar to Tn4001 and Tn4031 found in the staphylococci. Like Tn5281, it is besides flanked with IS256. This group of jumping genes carry the aacA-aphD bifunctional aminoglycoside modifying enzyme cistron that besides shows opposition against Garamycin.
IS256 is common among enterococci, even those without any antibiotic opposition and this state of affairs could lend to the formation of plasmids with multiple antibiotic opposition. IS1216 is besides another IS of importance in this jumping gene household, as it has been implicated in the spread of antibiotic opposition cistrons. A survey by Heaton et Al has shown that the jumping gene, with the aid of IS1216, was able to reassign itself from a nonconjugative plasmid to a pheromone-responsive-plasmid in E. faecium. They concluded that similar events such as this could go on elsewhere in nature, lending to the spread of antibiotic opposition.
Tn5384 is a jumping gene of involvement. It contains antibiotic opposition elements against Erythrocin and Garamycin. The formation of this jumping gene is through the cointegration of a plasmid from enterococcus and staphylococci, each holding belongingss of being a wide host scope and anti beta-lactam antibiotics severally. This provide support for the function of staphyloccocus in the development of the enterococcus.
4.2.2.3 Conjugative jumping genes
Conjugative jumping genes are nomadic familial elements that could reassign itself from one genome of a bacterium to another genome without the usage of a plasmid Deoxyribonucleic acid, but merely through intercellular contact. Not merely that, its name was besides given because of its ability to besides permute intracellularly.
Designation of this jumping gene was discovered through DS16, a E. faecalis strain in 1981. In the survey where it was discovered, E. faecalis was used which contained 2 plasmids, pAD1 and pAD2 contain multiple antibiotic opposition cistrons. When DS16 was mated with plasmid-free receivers, it was reported that the Achromycin determiner is capable of junction even with the absence of plasmid DNA. It was found that this determiner is present on a jumping gene, called Tn916.
Tn916, a 18kb jumping gene, contain cistrons required for junction, antibiotic opposition and DNA cleavage. There is a similar jumping gene, besides well-characterized and has many visual aspects aboard Tn916 in literature, which is called Tn1545. A difference between the two plasmid is that Tn1545 carries a Erythrocin and kanamycin opposition on top of tetracyclin. Tn916 is promiscuous, able to conjugate into an highly wide host scope, up to 50 species into legion sites in the chromosome of the receiver bacteria, which has been shown in vitro. It can besides reassign itself onto low-copy figure plasmids and pAD1.
Junction of Tn916 involves an deletion event and a non-replicative round intermediate and it besides must bear an oriT site, which is the beginning of transportation. They are transposon like because they can strike from and so besides incorporate itself into Deoxyribonucleic acid but their mechanism is different than other jumping genes, such as Tn5 and Tn10. The heterotaxy is similar to plasmids, in that they have a round intermediate, but the chief difference is that it does non retroflex. Figure 3 shows intercellular heterotaxy.
Fig. 4.4 A transportation intermediate is formed by first deletion of the incorporate jumping gene. A individual strand is transferred into the receiver, where it the other strand gets synthesized. After that, integrating back or into the chromosome occurs.
Conjugative jumping genes are common in enterococci and by and large shack on the chromosome. This jumping genes plays an of import function in the spread of antibiotic opposition. Being tetracycline opposition, the spread of this would ensue in more bacteriums being antibiotic resistant.
4.2.3 Deductions of the Mobile Genetic Elementss
Mobile familial elements are normally associated with acquisition of antibiotic opposition in clinical scenes and it is good known that this can do a broad assortment of jobs in handling patients, particularly MRSA.
Horizontal cistron transportation has really annihilating effect, particularly when taking into consideration the antibiotic-producing Gram-positive Actinomycetes spp. and the infective Enterococcus spp. and Staphylococcus aureus. This allows bacteriums that commonly causes disease in worlds easy get antibiotic opposition. There is besides another job of potentially fatal MRSA geting vancomycin opposition with the usage of conjugative jumping genes and research workers have investigated the possibility of acquisition through vancomycin-resistant Enterococci, which by nature besides employs the usage of nomadic familial elements for endurance.
Back so, conjugative transportation of vancomycin-resistance between S. aureus and enterococci have merely been achieved in the lab. Nobel et Als have noted that conjugative transportation of Vancocin was achieved in the research lab puting between enterococci and staphylococcus though there was no transportation between staphylococci itself.
However, 4 old ages subsequently after Baronial et Al ‘s paper, the first MRSA to get Vancocin opposition has been isolated in a Nipponese patient and incidence of now what is called VRSA ( vancomycin-resistant S. aureus ) infections has been increasing after that.
There are possible accounts for this phenomenon. Showsh et Al has noted that conjugative plasmid pAM368 incorporating vancomycin-resistance found in E. faecalis responses to the pheromone peptide cAM373 produced by S. aureus. Another survey done by Weigel et Al has found that Tn1546 encoding vancomycin-resistance was able to reassign itself from E. faecalis to S. aureus onto its pLW1043 plasmid. This plasmid is so able to distribute to other staphylococci, which has the possible to do the growing of opposition against the lone presently utile drug more widespread.
4.3 Decision
Mobile familial elements are indispensable for the continued development of the enterococci. What was used to antagonize environmental alterations has been used against antibiotics.
In this chapter, each to the full sequenced strain of E. faecalis and E. faecium has been analyzed from the current information available. Unlike V583, Aus0004 have merely merely been out for a few months when this chapter was written and therefore it is non really good characterized yet. However, current analysis of it showed that it is really similar to V583, in the fact that it contains many interpolation sequences and other marks of nomadic familial elements being involved in its development.
Furthermore in this chapter, it has been shown that each of the plasmids and jumping genes found in the enterococcus has been noted to hold at least one antibiotic opposition cistron. The deductions of this, particularly composite jumping genes, is that it can be transferred to a wide host scope, including the already multi-antibiotic immune S. aureus. In fact, it has already been reported that strains of Vancocin immune S. aureus has already appeared.
However, more research remains to be done, particularly on the genomic differences between E. faecalis and E. faecium. Besides, an attack to proteomic analysis could state us more about the different proteins produced depending on the conditions.
