Horizontal Gene Transfer (hgt)
The enormous increase of drug resistance and emergence of MDR (Multi Drug resistant) bacterial strains of clinical and Nosocomial source are found to be a heavy task for clinicians. There is an array of factors may be responsible for drug resistance. The factors include, the bacterial own nature and characteristics, the non- systematic use of anti bacterials and more over the social economic conditions. But the spread and acquisition of drug resistance among the human pathogenic bacteria strains are brought about by shuttle of drug resistance genes (R-genes) through different vectors. The vectors are otherwise known as “Mobile elements” are able to insert themselves from donor bacterial strains to a compatible recipient strain. The “Mobile elements” having specific characteristic features can express the “Gene cassette” for drug resistance in the recipient cell. Further this R gene is integrated into another recipient cell converting it into a resistant strain. And this process goes on and finally the whole population of bacterial strains are invaded with drug resistant genes. Therefore the bacterial genomes are always in a state of dynamism. As the spread of R genes is in horizontal manner that is within a generation, this type of drug resistance is known as “Horizontal gene transfer” (hgt).
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In prokaryotic genomes, assessing GC content change is useful in identifying horizontally-transferred genes, which usually have distinct GC content from the host genome. Pathogenic islands comprise relatively large genomic regions, e.g., 10-200 Kb, which are acquired by core genomes via horizontal gene transfer.
In the present scenario, it is observed that horizontal gene transfer is an enduring problem although several novel approaches like “Gene editing”, combinatorial therapy have some certainty, but it is obligatory to study the biology of horizontal gene transfer (hgt) those have skewed towards the diagnostic bacteriology.
Origin of hgt
The gene exchange network among bacteria probably also relies on transduction and transformation and is assumed to have an important impact on the dynamics of bacterial communities. Agreed that human pathogens were susceptible to antibiotics before the use of these drugs for the treatment of infections, the origin of antibiotic resistance determinants.
(a)The role of anti metabolite producing human commensals
The human commensals can provide antibiotic resistance to pathogens. However, in most cases, the antibiotic resistance genes have originated in the environmental microbiota. Several antibiotics are produced by environmental bacteria. A plausible explanation may attached here that antibiotic-producing organisms could be the origin of HGT-acquired antibiotic resistance genes, because these micro-organisms must have inherent systems to escape from the activity of the anti metabolites which they produce.
(b) Opportunistic human commensals
The human commensal bacterial strains are considered to be “Opportunistics” if they are able to produce infections only in people who are immunosuppressed (for instance, those with AIDS, under chemotherapy or after transplantation), debilitated or with a basal disease. Generally, these bacterial strains do not infect persons having active immunity. These opportunistic bacterial strains are observed to be acquisition of Resistant genes from their community. Hence there is origin of hgt among bacterial strains. The infection caused by the ubiquitous bacterial strain namely Pseudomonas aeruginosa is reported to be opportunistic and originated from its community members.
(C ) Use of Sub Inhibitory Concentration (SIC) of Antibiotics
This is reported that some antibiotics may serve for signalling purpose at their sub inhibitory concentrations. The minimum concentration of antibiotic required for inhibition of growth of bacterial population is known as “Minimum inhibitory concentration” of the prescribed antibiotic. However, there is a requirement to study the efficacy study of the antibiotics. Therefore there is need to conduct experiments to observe the cidal or static effect of respective drugs against the specific bacterial strains under study. The cidal effect is the killing of bacteria completely. The static effect is the temporary inhibition of metabolic activity of bacterial cells. Given opportunity, they used to resume their viability. In this context the static effect is the consequence of use of Sub Inhibitory Concentration (SIC) of Antibiotics. This low concentrations of antibiotics trigger specific transcriptional changes, which are independent of general stress response bacterial network.
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(d) Human use of Antibiotics
Acquired antibiotic resistance is an event in the evolution of human pathogens in which the main selective force has been the human use of antibiotics. This is observed that plasmids of invaded bacterial species have acquired resistance genes after antibiotics were introduced for therapy. As mentioned above, the event of hgt may be initiated with contact between a commensal bacterial strain and an invading bacterial strain.
(e ) Existence of same R-genetic elements in related pathogenic strains
The existence of same R gene podium (F plasmids, Transposons, Integrons etc.) between related pathogenic bacterial strains may prompt the event of transfer of R-genes horizontally. This is true for the existence of hgt among the bacterial strains belonging to Enterobacteriaceae group.
(f) Co -existence of Metal resistance and hgt
Heavy metal resistance and biocide resistance genes can be associated with antibiotic resistance resistant gene shuffling. The bacteria strains with heavy metal resistance are reported to be take advantage of horizontal gene transfer programmes. The heavy metal contamination and use of biocides are the selecting factors for budding Resistant genes and their movements among the bacterial strains.
(g) Biofims are ideally suited for hgt
Microbes often construct and live within surface-associated multicellular communities known as biofilms. In the biofilms, the bacteria are embedded in an extracellular polymeric matrix, and are protected against environmental stresses, antimicrobial treatment, and the host immune system. The human pathogenic bacterial strains are reported with biofilm production in a variety of human infections, such as endocarditis, osteomyelitis, chronic otitis media, foreign-body-associated infections, gastrointestinal ulcers, urinary tract infections, chronic lung infections in cystic fibrosis patients, caries, and periodontitis. The causative agents for these infections belong to both Gram positives (Staphylococcus spp., Streptococcus spp., Bacillus spp., etc.) and Gram negatives (Escherichia coli, Pseudomonas aeruginosa, Enterococcus spp. Moraxella spp., etc). Additionally the acid fast intracellular bacterium Mycobacterium tuberculosis is also reported to be associated with biofilm production and drug resistance. Baceiro et al., (2013) reported that Biofilms play an important role in hgt. The architecture of biofilms promotes hgt, especially by conjugation, and because of the high density and close proximity of the cells, the conjugation itself can even stimulate biofilm production. Indeed, transformation appears to be necessary for biofilm formation and stabilization. A type IV secretion system is involved in biofilm formation and contributes to cell-to-cell contact, thus mediating DNA transfer. There is therefore a positive feedback between the horizontal exchange of genes and biofilm formation, which favors movement of resistance genes and virulence factors, especially in the presence of antibiotic selective pressure. Biofilms are ideally suited to the exchange of genetic material of various origins, and it has been shown that bacterial conjugation occurs within biofilms. The direct contribution of conjugative plasmids themselves to the capacity of the bacterial host to form a biofilm. Natural conjugative plasmids expressed factors that induced planktonic bacteria to form or enter biofilm communities, which favour the infectious transfer of the plasmid. This general connection between conjugation and biofilms suggests that medically relevant plasmid-bearing strains are more likely to form a biofilm (Ghiego, 2001).
(h) Presence of extracellular DNA
The biofilm is network of hetero genous group of molecules which are tightly knitted. The molecules like extracellular polysaccharides, surface proteins and more over extracellular DNA are involved in Biofilm composition. Extracellular DNA can contribute to <1–2% of the biofilm matrix.. It is required for attachment and aggregation of microcolonies during biofilm development and also functions as a mere structural support to maintain biofilm architecture. The extracellular DNA within the biofilm matrix binds to and sequesters the cations. The extracellular DNA might have persisted from other bacterial strains carrying the drug resistant genes. While inside the web of biofilm these small fragment of DNA molecules may be potential candidates for transfer of drug resistance.
Figure # The biofilm formation in Escherichia coli cells isolated from a clinical sample (Source: Laboratory of Medical Microbiology, School of Life Sciences, Sambalpur University, Odisha)
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Figure # The process of biofilm formation by a Gram negative bacteria
Figure # The process of hgt among biofilm producing bacteria
Mechanism of Horizontal Gene Transfer (hgt)
There are three major underlying mechanisms behind the horizontal gene transfer (hgt) among bacterial strains. They are
Plasmid mediated Conjugation
Conjugation requires cell-to-cell contact, usually via a pilus or pore that forms a channel that allows for the passage of plasmids. The plasmids carrying the drug resistance, metal resistance genes are called R plasmids. The examples of R plasmids are R4, R1, R6 etc, are didtrubuted among an array of bacterial pathogenic strains like Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter spp., Mycobacterium tuberculosis spp, Salmonella spp., Shigella spp, Vibrio cholerae, Streptococcus spp. Staphylococcus spp., Micrococcus spp., Listeria monocytogenes, Moraxella spp., Acenetobacter spp., Haemophilus influenzae, Helicobacter pylori and many more. The genes are responsible to express the proteins for inactivation of drugs. The bactaria carrying the R plasmids are able to escape or tolerate an array of narrow spectrum and broad spectrum antibiotics including synthetic, semisynthetic and natural antibiotics.
Conjugative plasmids present in 1-2 copy number/cell carrying a gene cassette for transfer of genes through the process of conjugation is called Fertility (F-factor). The molecular weight of plasmids varies beween 80-100kb. The plasmids are much prevalent in Escherichia, Citrobacter and Salmonella spp.
A variant of Quinolone-resistance gene,qnrA1 are carried on plasmids. The qnrgenes encoding DNA gyrase, topoisomerase IV, outer membrane proteins and drug-efflux pumps in Enterobacteriaceae are involved in quinolone-resistance. Together withqnrgenes and the plasmid-mediated integration mechanism,CTX-MESBLs is responsible for the rapid horizontal and vertical dissemination of antibiotic-resistance genes among bacterial species.
A block of transfer genes called tra genes are consisting of about 28 genes. This is an operon comprising a set of genes which are expressed constitutively through a poly cistronic mRNA. The TraT protein, which is an external outer membrane lipoprotein associated with plasmid conjugation and also with several virulence mechanisms (e.g., serum resistance, phagocytosis, and biofilm formation), also plays an important role. Thus, plasmids have a doubly important role, as they spread resistance genes and the traT genes are directly involved in bacterial virulence.
The F plasmids can also become integrated into the recipients core genome and may co-replicate with the chromosomal DNA. There are reports regarding sporadic transfer of a copy of plasmid to another plasmid lacking bacterial cell. Conjugation requires two sets of genes, the mobility (MOB) genes and the mating pair formation (MPF) genes. The MOB genes code for a relaxase and DNA processing proteins, responsible for the relaxosome, and for the coupling protein that links the relaxosome to the mating channel. MPF genes encode for the membrane–associated mating pair formation complex, a form of type 4 secretion system (T4SS) that provides the mating channel. The relaxosome docks to the coupling protein which helps mediate transport through the T4SS into the recipient cell, followed by establishment and replication of the plasmid in the recipient.
There is no phenotypic distinction between the donor and recipient cells. But the cells should be compatible with each other. The bacteria cell carrying the F plasmid is named as F+ and the cells lacking the F plasmid called F- cell. Prior to conjugation, the F+ cell expresses a protein, Pillin for a tubular extension of the bacterial cell surface called as F pilus. The F pilus interacts with specific receptor molecules present on cell surface of F- bacterial cell. Finally, a conjugation bridge is put down between the two compatible cells. Following this event, a nick is formed by using an endonuclease in a strand of super coiled F plasmid DNA at a specific sequence called oriT. The accessory SSB (Single Stand binding) proteins are expressed because of tra genes activity. The SSB proteins bind the exposed 5’terminus and lead the strand in the direction of lumen of the conjugation tube. In the meanwhile, it undergoes polymerisation reaction by adding mono nucleotides at its 3’terminus. This type of DNA replication is called “Rolling circle replication mechanism” The polymerisation process continues until the 5’ terminus enters and circularise in the form of a circular DNA in the cytoplasm. Prior to incorporation of the F+ strand, the F- plasmid replaces some its nucleotides. So the transferred strand carrying R genes in Toto or in partial form is incorporated. The gap is sealed and a semi conservative forms of plasmids are ```found both in F+ and F- cell. In the end the R gene is transferred from one bacterial cell to other bacterioal cell.
Available evidence suggests that pathogens with multiple mutations and combinations of r R genes evolve and survive successfully in vivo.
The major incompatibility (Inc) group involved in transfer of resistance and virulence genes is the IncF group; transmission of IncF plasmids. Tthe IncFII group plasmids (e.g., pEK499) often carry the CTX-M-15 -lactamase and may be involved in spread of the clone. These plasmids carry resistance genes in multiple families and also carry virulence genes (e.g., the pEK499 plasmid carries two copies of the vagC-vagD system, which is involved in cellular division and is necessary to maintain the virulence of K. pneumonia.
A virulence plasmid of a porcine enterotoxigenic E. coli (ETEC) strain carries a Tn10 transposon that carries the tetracycline resistance genes tetA and tetC (encoding efflux systems). The toxin-specific locus caused the enterotoxigenicity of the strain, which contains two heat-stable enterotoxin genes, sta and stb. In this example, the authors observed coselection of virulence and tetracycline resistance, but they did not analyze the biological cost of carrying this virulence plasmid. Coselection of tetracycline was also observed in C. perfringens in a study that analyzed the nonreplicating transposon Tn916, which is involved
in the conjugation of a replicating plasmid carrying putative virulence genes and two tetracycline resistance mechanisms, tetA(P) (efflux system) and tetB(P), which provide ribosomal protection.
Integrative and Conjugative Elements
Integrative and conjugative elements (ICEs) are self-transmissible mobile genetic elements that help in the horizontal dispersal of genes located in another higher DNA molecule with replicative capacity, such as plasmids and chromosomes.
ICEs may have more significant impact than plasmids in HGT
Analysis of some isolates from cholera outbreaks has revealed the involvement of SXT/R391-like genes in the transmission of virulence and resistance. The in vitro plasticity and capacity of the SXT-related ICE from Vibrio cholerae to transfer virulence genes to other species such as E. coli has also been demonstrated.
Phage mediated transduction
In transduction, fragments of bacterial DNA are included as part of viral DNA, and when this viral particle infects other bacterial cells, DNA is integrated and replicated in the host bacterial cell. There are numerous examples of different virulence genes carried by prophages in E. coli: e.g., Shiga toxin (STX); genes of effector proteins such as bacteriophages, nleA-H, and Cif; and cytolethal distending toxin (CDT).
Shiga toxin-producing E. coli (STEC) strain belonging to serotype O104:H4 with virulence features common to the enteroaggregative E. coli pathotype, which previously carried the plasmid-encoded TEM-1 and CTX-M-15-lactamases. The Shiga toxin was most likely transduced from other enterohemorrhagic E. coli strains.
The ability to acquire mobile genetic element (MGE) encoding traits such as antibiotic resistance, has contributed to the emergence of enterococci, particularly E. faecium and E. faecalis, as leading hospital pathogens.
Transformation is the process where a cell takes up naked DNA from the extracellular environment. Bacteria that have the ability to undergo transformation is said to be competent.
Host range of hgt
Enterococci have been shown to transfer resistance genes to clinically important bacteria such as Clostridium difficile, E. coli, S. aureus, streptococci and Listeria spp.
Class I Integron
Class I integron is a mutligene-capturing and disseminating system in bacteria, which has often been linked to multidrug resistance. Class I integron is a platform that assembles a large number of antibiotic resistance genes to integrate large multidrug-resistance cassettes into bacterial chromosome. Class I integron may appear in chromosome or plasmid. Plasmid-bearing class I integron could rapidly disseminate antibiotic-resistance genes throughout the species while antibiotic-resistance genes carried by chromosomal class I integron are generally restricted in particular phylogenetic lineages.Once the antibiotic-resistance gene is anchored on chromosomal integron, it will be maintained for many years under antibiotic selection and will only be spread with the resistant bacteria.
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