Antibiotic Resistance Genes in Organisms

Antibiotic Resistance Genes in Organisms

Antibiotic Resistance Genes in Organisms – Resistance has posed numerous clinical challenges. Antibiotic misuse and overprescription, among numerous other factors, have served as a driving force influencing the selection and dissemination of resistance. As a result, a plethora of diverse resistance mechanisms have been identified, and in many cases, multiple resistance mechanisms to a class of antibiotic have emerged in pathogens, compounding the problem (Melano et al., 2003).

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However, it is becoming increasingly evident, however that environmental forces have greatly impacted the determinants that have emerged clinically. Among the first to be recognized publicly was the impact of the agricultural use of antibiotics as animal growth promoter. Since the 1940s and until the past decade, the extensive use  of antimicrobials at subtherapeutic levels has not only been shown to select for resistance to antibacterial agent but also bacterial  DNA contamination from crude antibiotic preparations often used in such applications has been found to contain resistance determinants (Lu, Asano, Davies, 2004). This use of antibiotics in agriculture has resulted in the spread of strains such as vancomycin-resistance enterococci in both farm animals exposed to antimicrobial and humans in contract with the animals and has been directly linked to the development of drug-resistance infections (Hommerum et al., 2000).

          The use of antibiotic therapeutic treatments or as growth promoters and field cultivation of some genetically modified plants (GMPs) are suspected to increase the risk of antibiotic resistance gene dissemination. Several commercial GMPs contain antibiotic resistance genes that are still under the control of bacterial promoters as remnants of the bacterial genes could be transferred more easily than other plant genes to soil bacteria because of a high degree of homology facilitating recombination in potential bacterial recipients. The possible impact of GMPs in terms of gene transfer to bacterial and modification of the soil microbial community with antibiotic resistance genes cannot be evaluated without considering the initial ecology of these genes in the community (Goldstein et al., 2005).

Types of Resistome

According to Gerard DW (2007), resistome is an expression for the collection of all the antibiotic resistance genes and their precursors in pathogenic and non-pathogenic bacteria. The complete set of antibiotic resistance genes is composed of four different types of genes, which include the following:

A.      Resistance genes found on pathogenic bacteria: these are the fewest but also the most problematic ones at present.

B.      Resistance genes found on antibiotic producers: the microorganisms such as soil-dwelling bacteria fungi that naturally produce antibiotics have their own protection mechanismto avoid adverse effects of the antibiotics on themselves. The genes which code for these resistances are a strong source for the pathogenic bacteria.

C.      Cryptic resistance genes: these genes are embedded in the bacteria chromosome but do not obviously confer resistance, because their level of expression is usually low or they are not expressed.

D.      Precursor genes: these genes do not confer antibiotic resistance. However they encode proteins that confer to some kind of basal level activity against the antibiotic molecule or have affinity to the molecule. In both cases this interaction may evolve to a full resistance gene given the appropriate selection pressure (Gerard DW, 2007).

Origin and Causes of Antibiotic Resistance

The origin of antibiotic resistance is an area of active study. There is evidence that naturally occurring antibiotic resistance is common. The genes that confer this resistance are known as the environmental resistome. These genes may be transferred from non-disease causing bacteria to those that do cause disease leading to clinically significant antibiotic resistance.

It was suggested that penicillinase may have emerged as a defense mechanism for the bacteria in their habitats, as in the case of penicillinase-rich Staphylococcus aureus living with penicillin-producing Trichopyton. Search for a penicillinase ancestor has focused on the class of proteins that are capable of specific combination with penicillin.

The resistance to cefoxitin and clindamycin in turn was speculatively attributed to Braine’s and Hartnell’s contact with microorganisms that naturally produce them or to random mutation in the chromosomes of clostridium strains. Moreover, there is evidence that heavy metals and some pollutants may select for antibiotic-resistant bacteria, generating a constant source of them in small numbers. (Abigail A. Salyers, Dixie D. Whitt, 2005).

Antibiotic Producing Bacteria: A Reservoir and Putative Origin of Resistance Determinants

The diversity of microbial life concealed within the soil has been explored in the search for new clinical and medicinal applications. The most significant application to data has been the implementation of natural product antibiotics, a discovery that has revolutionized our approach to treating infectious diseases. Greater percentages of antibiotics in clinical use originate from soil bacteria, either directly as natural products or as their semi-synthetic derivatives. The Actinomycete class of bacteria is responsible for the synthesis of the vast majority of clinically important compounds (Kieser et al., 2000).

Evolution of mechanism of chemical warfare by soil microorganisms has had several implications:

  • Ø It has required the coevolution of mechanisms of self-protection in antibiotic producers, evidenced by the frequent presence of associated resistance genes in antibiotic biosynthetic genes clusters.
  • Ø Other prokaryotes that inhabit similar niches have evolved or acquired resistance, e.g. natural selection. This extensive coevolution of antibiotic biosynthesis and resistance suggests a possible origin of many clinical resistance determinants, as numerous mechanisms in soil bacteria and human pathogens are identical (Hang et al., 2005).

Prevalence of Antibiotic Resistant Bacteria

The successful treatment of bacterial infections in human is being thwarted by the prevalence of multiply antibiotic-resistant bacteria, thereby increasing diseases incidence, longevity and mortality and the length and cost of hospital stays. To confront the resulting health crisis a large arsenal of antimicrobials are needed (Levy and Marshall, 2004) and insight into antibiotic resistance, which will derive from understanding the ecology of resistance genes, including their origins, reservoirs and movement. Although antibiotic resistance has been studied intensively in clinical settings, little is known about the environmental reservoirs of resistance genes and their contribution to resistance in clinical settings (Levy and Marshall, 2004).

Identifying sources of resistance genes and tracking their movement from unmanaged ecosystems to the human milieu will advance the effort to combat antibiotic resistance in human pathogens. Owing to its complex microbial community, the soil is potentially a large environmental reservoir of resistance. A few studies that have addressed antibiotic resistance in the soil community (the soil resistome) have provided evidence that is consistent with the predicted diversity and abundance of resistance determinants (Riesenfeld et al., 2004a; D‘Costa et al., 2006).

One group of resistance determinants predicted to be abundant in soil is β-lactamases. These enzymes hydrolyze the β-lactam class of antibiotics, such as penicillins and cephalosporins. The high efficacy and low toxicity of β-lactam antibiotics makes them among the most frequently prescribed antibiotics for humans and livestock generating a powerful selection pressure for genes encoding resistance elements in environments proximal to human activity (Henriques et al., 2006).

However, the empirical evidence is scarce regarding the origins of these genes or their movement from unmanaged habitats to clinical settings. It seems likely that resistance genes are abundant in soil, even in the absence of anthropogenic selection pressure, because many soils may contain low concentrations of compounds that select for resistance (Garau et al., 2005). The soil is rich for instance, with microorganisms that produce β-lactam antibiotics, such as penicillins and cephalosporins.

Access to the wealth of uncultured bacteria in soil is provided by the culture-independent method metagenomics, which involves extracting and cloning DNA directly from the environment. Analysis of metagenomic clone is often based on random sequencing (Tringe et al., 2005) or PCR amplification of target genes.

Alternatively, functional metagenomics, which consist of heterologous expression of metagenomic DNA in a surrogate host and activity-based screening, provides the means to discover genes whose function might not be obvious from their sequence (Committee on Metagenomics: Challenge and functional Applications National Research Council, 2007).

 Exploring the Soil Antibiotic Resistome

Molecular mechanism of aminoglycoside resistance in soil-dwelling actinomycetes from the genes streptomyes were determined to be identical to those in clinical pathogens. These strains, producer of the aminoglysides kanamyein and neomycin, were capable of drug modification by acctylation and phosporlation, respectively as a means of self-protection (Benveniste, Davies, 1973).

Numerous parallels have been identified between determinants in soil actinomycetes and those in clinically important strain, with respect to both molecular mechanism and protein homology. The most striking instance is that of the glycopeptides antibiotic vancomycin, still considered an important clinical drug of last resort. Clinical resistance is mediated by the reprogramming of the drug target, the D-Ala-D-D-Ala termini of cell wall peptidoglycan, to one with a significantly lower affinity for vancomycin. This most commonly accomplished by three proteins, encoded by the vanHAX cluster of genes. Six years after the mechanism of pathogenic strain was elucidated, it was discovered that not only was this strategy identical to those in glycopeptides producing soil actinomycetes, but primary amino acid sequence homology was also apparent between the associated VanHAX resistance proteins (Marshall et al., 1997).

Approaches have been implemented to characterize the diversity and prevalence of resistance in soil bacteria-the soil antibiotic resistome- as an important reservoir of resistance (Wright, 2007). Hence, creating a functional metagennomic library in which cloned genomic fragments were expressed from DNA isolated directly from soil and selecting for resistance, traditional challenges associated with studying genes of unknown sequence were circumvented. Finally, analysis specifically revealed novel antibiotic resistance proteins that were previously of unknown function and unrecognizable by sequence alone. Thus, this work not only allowed for the identification of aminoglycoside N-acetyltranferases, the O phosphottansferase, and a putative tertracyline efflux pump but also construct with a novel resistance determinant to the aminoglcoside butirosin. This work shows the power of the functional metagenimic approach when applied to a search of activity with a highly selectable phenotype such as antibiotic resistance (Handelsman, 2004).

The diversity of tetracyeline resistance determinants in soil is characterized based on agriculturally associated resistance. Using PCR- based approaches, three resistance genes were identified in the soil and an additional five were found in manure-supplemented soils. This work speaks to the diversity of tetracycline resistance in agricultural soil.

To explore the soil resistome from an evolutionary perspective, D’Costa et al. (2006) established a systematic approach to characterize resistance in actinomycetes as a means of anticipating new mechanisms of resistance that may emerge clinically in the future. By constructing a morphologically diverse library of hundred of spore-forming actinomycetes and screening for resistance to a collection of 21 natural products, semi-synthetic and synthetic antibiotics, and this work was the first attempt to quantify the phenotypic density of resistance in any subset of soil organism. The phonotypic density of resistance and diversity of the resulting profiles were greater than ever anticipated, with strains resistant to an average of seven to eight antibiotics. In addition, this work identified a wealth of antibiotic inactivating enzymes, including novel mechanisms of resistance to the recently approved antibiotics telithromycin and daptomcin (D’costa et al., 2006).

The study of resistance in soil bacteria is rapidly gaining recognition as an important reservoir form which many clinical parallels can be drawn. Further, studies on a more diverse subset of stains, as well as approaches to study slow-growing strains and those difficult to culture will be important to uncover the true extent of the soil resistome.

Acquisition of Resistance to Antibiotics

Horizontal gene transfer (HGT) is the process that confers new metabolic capabilities to the recipient, allowing its adaptation to new ecological niches. Genetic transfer among bacteria accounts for much of the spread of resistance.     the acquisition of exogenous DNA by bacteria is through transformation, conjugation and transduction. Then, it is possible to either integrate the new genetic material into the recipient’s chromosome or replicate independently. The mobile genetic element mediating HGT consist of plasmids and tranposons and the related gene integrating integrons.

v Plasmids, circular double-stranded DNA molecules harboring genetic determinants are capable of replicating independently of the bacteria chromosome.

v Tranposons are flanked by inverted repeat sequences and encode tranposases, enzymes that introduce nicks at the ends of these elements to allow for integration at insertion sequences, sites which are normal constituents of bacteria chromosomes and plasmids. Traansposons can carry multiple gene cassettes and participate in gene mobilization within a chromosome. The mobility of a tranposon can increase if it integrates into a plasmid which is then transmitted to other cells by conjugation or transformation.

v Integrons are assembly platforms which incorporate genetic material through site-specific recombination and contain within a promoter for expression. An integron-encoded integrase carries out the assembly of tandem gene fragments at the attl primary recombination site. Although intergenic rearrangements are possible. Integrons are essentially immobile in the chromosome unless are essentially immobile in the chromosome unless associated with a tranposon (Frost et al., 2005).

Evidence of these genetic elements, or their remnants, have been identified in all available prokaryotic genomes.

The mobile genetic element-associated transmission of antibiotic resistance determinants is probably responsible for the dispersal of some streptogramin B lyases, which is responsible for antibiotic resistance by means of drug inactivation. The analysis of the genetic environment of genes encoding putative lyases (vgb) in many instances reveals the presence of mobile elements such as transposase upstream or downstream of the gene of interest. Multiple resistance determinants can accumulate on a mobile element and upon transmission contribute to multi-drug resistance. In this case, vgb is often in close proximity to the resistance determinant vat, associated with resistance to streptogramin A. Thus, with respect to antibiotic resistance, the presence of mobile genetic elements can play a powerful role in the transmission of resistance between bacterial strain (Gogarten, Townsend, 2005).

Identification of Active Genes and Subcloning BLA

Transposon mutagenesis carried out in vitro with the Genome priming system (GPS-1) and insertion mutants that failed to grow on the appropriate β-lactam antibiotic were identified and reported as having insertions in the active genes and was sequenced using   primers. Additionally, random insertion mutants chosen for sequencing the entire insert of β-lactamase resistance was sequenced with the aid of primers walking. Sequence reads were assembled using seqMan and GeneQuest was used to identify putative open reading frames (ORFs), which were annotated using BLAST (Basic Local Alignment search Tool) (Altschul et al., 1990).

As per the convention in the β-lactamase field, genes encoding β-lactamase were named bla (even though they do not all share a common ancestor), the protein product were named β-lactam     resistance   from soil (LRS).and the ORFs are designated bla LRS.

In determination of functional genes encoding β-lactamase (bla LRS) domains, the full-length ORF and individual domains were subcloned into pCF 430 downstream of an arabinose-inducible promoter using preexisting Nhel and HindIII sites. Appropriate restriction sites and consensus E. coli shine-Dalgarno site were engineered into PCR primers upstream of the start codon. The full-length ORF subclone was designated pCFHBLO1 (primers 5’- 3’, F: cacggctagcaggaggatattaATGAATTTTCGCCACATAGTCATGG, R: gcggaagcttCTAACGCTGGTCCAGTTGATCCAG; capital letters indicate coding region).

The domain I (class D) subclone (pCFHBL02) comprised amino acids met1-Leu252 (primers 5’- 3’, F: caggctagcagcaggaggatattaATGAATTTTCGCCACATAGTCATGG, R: gcggaagcttCTACAAGCCGGGGAGTTCCCTTGAG), whereas the domain cutoff point was determined by BLAST (Altschul et al 1990; 1997) comparison and alignment with classes D and C β-lactamases, respectively; this comparison yielded a clear partition at the designated position.

Identification and Characterization of the Bifunctional Β-lactamases

The bifunctional β-lactamases was encoded by a clone carrying a 42-kb metagenomic DNA insert and a single ORF, designated bla LRS   was responsible for resistance. And its deduced protein contains 609 amino acids. The C-terminus (356 amino acids) aligns with class C β-lactamases and the N-terminus (253 amino acids) aligns with class D β-lactamases. Therefore, bla LRS    appears to be a natural fusion between two full – length enzymes (Kim et al., 2007).

In determination functional domains, the full-length ORF and individual domains of the bifunctional β-lactamases were subcloned and tested for resistance. The subclone containing the full-length ORF exhibits the levels of resistance similar to the parent strain in assays that measures minimum inhibitory concentration (Kim et al., 2007) with minor differences likely attributable to the difference in genes expression level and experimental variation. Each of the two β-lactamase domains contributes to the resistance profile of the full-length ORF, demonstrating that the hybrid β-lactamases is indeed a bifunctional enzyme. Resistance to amoxicillin, ampicillin and carbenicillin is almost exclusively imparted by the N-terminal (class D) domain of the hybrid enzyme and resistance to cephalexin is because of the C-terminal (class C) domain. Generally, expression of the C-terminal domain confers resistance to cephalosporin-type β-lactams; this includes β-lactams to which the full-length clone confers little to no resistance. Intriguingly, there is an overlap in specificity with respect to piperacillin resistance. Therefore, the fusion expands the substrate specificity beyond what either domain could hydrolyze alone (Kim et al., 2007).                      

Phylogenic Analysis of Soil Β-lactamases

The amino-acid sequences of class A β-lactamases are highly divergent from each other. Β-lactamases resistance soil (LRS) clusters with the chromosomally encoded β-lactamases from Burkholderia pseudomalle, Pseudomonas luteola and Yersinia entercolitica. All of the recovered class B β-lactamases fall into one (B3) of the three subgroup of known metallo- β-lactamases.  The lengths of the branches leading to the soil metallo- β-lactamases from the nearest node indicate that they are more closely related to ancestral enzymes. All but two of the soil metallo-β-lactamases diverge deeply from each other and others in subgroup B3. Interestingly, the relatedness of the soil metallo- β-lactamases does not necessarily correspond to the similarity in functional profiles (Rasmussen and bush, 1997).

The soil class C β-lactamases cluster with BlaE from Mycobacterium smegmatis, which is the only class C β-lactamase from a Gram-positive organism.  These sequences form a distinct clade that is different from the clade of class C β-lactamases of Gram-negative organisms.

Functional Metagenomic Analysis of Soil

Functional metagenomic analysis of soil revealed a gene encoding a bifunctional β-lactamases, blaLRS.  Although bifunctional enzymes are rare in bacteria (Kim et al., 2007), four bifunctional aminoglycoside resistance gene have been reported previously. In each of these as in blaLRS the fusion confers expanded substrate specificity compared with either domain alone, suggesting that pathogenic organism harbouring these genes might have a selective advantage in a clinical environment. This advantage may be because of tighter regulation of expression, greater efficiency and convenience for mobilizability of a gene encoding a bifunctional enzyme than two individual resistance genes. Indeed the four aminoglycoside resistance genes fusion events are shown to be recent and because of an extensive selective pressure (Vakulenko and Mobashery, 2003; Kim et al., 2007).

More metallo- β-lactamases than any other class were isolated from Alaskan soil, and of them belong to one (B3) of three subgroups of metallo- β-lactamases Homologs of enzymes in subgroup B3 are found in both bacteria and archaea and the β-lactamases function of B3 enzymes is thought to have evolves more than 2.2 billion years ago in addition to their primordial existence metallo- β-lactamases belong to the zinc metallo-hydrolase family of which there are more than 6000 members that catalyze a broad range of reactions in all domains of life, these data suggest that the presence of metallo- β-lactamases in an environment  that is not highly selective, such as soil could be a result of divergent evolution from non β-lactamases hydrolyzing member of the zinc metallo-hydrolase family. In this case, diverse substrate in addition to β-lactamases, might select for genes that encode metallo- β-lactamases (Bedrone 2007).

Expanding the Resistance: Exploring Environment Diversity

Resistance to antibacterial has been extensively documented both phenotypically and genetically, in general spanning the entire bacterial domain from diverse ecosystems. With respect to environmental resistance to antibiotics, this ability is not simply restricted to soil-dwelling microorganisms (Schmitt et al., 2006).

The field of metagenomics is rapidly expanding the ability to explore the genetic diversity of novel terrestrial and aquatic environments. Metagenomics entails the sequencing of a clone library derived from the total DNA purified from a complex microbial ecosystem. These partial genome fragments are thought to represent the diversity of the community, including strains which cannot be cultured (Handelsman, 2004).

In order to appreciate the diversity and dispersion of the environmental resistance, it is of great value to examine characterized resistance proteins in the context of not only those annotated as putative resistance determinants but also those from previously under-recognized sources of resistance. Additionally, it is important to compare proteins of alternate cellular functions from which these determinants probably evolved.

Focusing on resistance by means of antibiotic inactivation, three examples illustrating the environmental density are described as follows:

  • β-Lactams is the first class of natural product antibiotics to be implemented clinically. It continues to be among the most extensively prescribed antibacterial in North America. This class includes both natural products and semi-synthetic derivatives, which acts by forming covalent intermediates with active site serine hydroxyl residues of cell wall cross linking enzymes, effectively titrating them out as inactive complexes. The most prevalent mechanism of resistance is enzymatic drug inactivation by β-lactamase enzymes, which hydrolyze the β-lactam ring essential for antibiotic activity. Two mechanisms are known: Ser-dependent hydrolysis and metal-dependent hydrolysis (Fisher et al., 2005).
  • The class A TEM subset of β-Lactamases are present in the genomes of many soil-dwelling strains, as well as Gram-negative that are associated with human infectious diseases e. g. Burkholderia psudomallei, Bordatella bronchisptia, and Delftia acidovorans) commonalities in flanking genes are evident in many of these microbes wit respect to genetic background, suggesting evolution from a common ancestor. However, homologs in some of the strains contain integrases upstream, and in salmonella enteria, there is evidence of genetic mobility in nearby regions both upstream and downs stream. Comparison to sequences from oceanic metagenomic and environmental databases suggests the presence of these β-lactamases in diverse environemtal locations speaking to the ubiquitous dispersion of this important resistance determinant. Confirmation of β-lactamase reactivity in these organisms, however, is necessary to fully understand its true diversity (Chouchani et al., 2006).
  • Type B streptogramins exhibit activity by binding to and subsequently obstructin the bacterial ribososome exit tunnel, thereby inhibiting translation. Clinical resistance, documented in strains of staphylococcus aureus, can occur by cleavage of the cylic lactone through an elimination mechanism by the lyase from strain in diverse phyla that include both environmental strains and clinical pathogens. Analysis of the available genetic environment revealed the presence of mobile genetic elements (e. g. transposases and resolvases) in many of the pathogenic strains, illustration the potential for lateral for gene transfer. In addition, searches of environmental genomes and metagenomic datababases revealed an abundance of uncharacterized putative iyases, grouping in many phylogenetic clusters (Mukhatar et al., 2001).


The evidence using both functional and genomic screening show that environmental organisms, harbour a previously under appreciated density of antibiotic resistance genes.

However, certain soil microorganisms are known to produce β-lactamases and although the antibiotics are not present at therapeutic levels, there may be sufficient concentrations to select for resistance in subpopulations within a community. Moreover, antibiotics themselves are only one of the many environmental factors that may affect the frequency of resistance genes. Antibiotic resistance may play roles outside of the ‘war’ metaphor, which is the traditional paradigm for antibiotics and resistance genes.

Moreover, resistance genes residing in the environment reservoir do pose a threat to human health, if they migrate to clinical settings and transfer to pathogens in which they could expressed.

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