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Antimicrobial protection mechanisms

Antimicrobial protection mechanisms

CAS PubMed Antimicrbial Antimicrobial protection mechanisms Google Scholar. A bacitracin-resistant Bacillus subtilis gene Antimicrohial Antimicrobial protection mechanisms homologue mechnisms the membrane-spanning subunit of the Bacillus licheniformis ABC transporter. An expanded arsenal of immune systems that protect bacteria from phages. Surface-attached capsular polysaccharides, on the other hand, have shown the opposite effect, rendering the bacteria more susceptible to AMP action, in comparison with non-encapsulated isogenic mutants Beiter et al. PLoS One 6:e

Antimicrobial protection mechanisms -

Efflux of these antibiotics in S. peucetius occurs by an ABC ATP Binding Cassette family transporter DrrAB coded by the drr AB genes embedded within the gene cluster responsible for biosynthesis of these antibiotics Guilfoile and Hutchinson, The DrrAB system has been studied in significant molecular and biochemical detail.

The DrrAB pump is assembled from two subunits each of the ABC protein DrrA and the integral membrane protein DrrB. DrrA protein functions as the catalytic nucleotide binding domain NBD.

DrrB protein functions as the carrier protein and forms the transmembrane domain TMD. In an in vitro assay using inverted membrane vesicles, the DrrAB proteins were shown to carry out efflux of Dox in ATP or GTP-dependent manner Li et al. Because of the location of the drr AB genes in the Dox biosynthesis gene cluster, this system is considered to be a dedicated transporter of Dnr and Dox in S.

Interestingly, however, recent studies showed that DrrAB pump is a multidrug transporter with broad substrate specificity, and it can transport many previously known MDR multidrug resistance pump substrates such as ethidium bromide, Hoechst , verapamil, and vinblastine, among others Li et al.

In this regard, the DrrAB system is similar to the mammalian ABC multidrug transporter P -glycoprotein Pgp , which is overexpressed in human cancer cells and is one of the major causes for failure of chemotherapy Chufan et al.

Recent studies showed that critical aromatic residues, contributed by multiple helices in DrrB, form part of a large common drug-binding pocket Li et al.

Mammalian Pgp also uses aromatic residues to provide flexibility in substrate recognition, suggesting a common origin for these proteins and an aromatic residue-based mechanism for polyspecificity that is conserved over large evolutionary distances Chufan et al.

Interestingly, OtrC found in oxytetracycline producer Streptomyces rimosus is another example of a self-resistance efflux system that exhibits multidrug specificity.

Self-resistance in S. rimosus is conferred by two efflux proteins: OtrB previously known as TetB located in the biosynthesis cluster, and OtrC located outside of the cluster Mak et al. OtrB belongs to the major facilitator superfamily MFS of transport proteins, but not much is known about its mechanism of action or substrate specificity Ohnuki et al.

OtrC protein is an ABC family protein, and like DrrAB, it also confers resistance to multiple antibiotics and MDR substrates, including ampicillin, oxytetracycline, doxorubicin, ethidium bromide, ofloxacin and vancomycin Yu et al.

Interestingly, the DrrAB and OtrC systems are quite homologous and show high sequence conservation in the previously identified motifs, including the DEAD and the LDEVLF motifs of DrrA Zhang et al. It might be expected that efflux systems found in producer organisms would be specific for the antibiotic that the system is dedicated for.

Surprisingly, however, the two examples DrrAB and OtrC discussed above suggest polyspecific drug recognition in these systems. This raises interesting questions. Why is a multidrug transporter needed in a producer organism?

What is the origin of DrrAB-like polyspecific antibiotic and drug efflux systems? Are most efflux systems associated with biosynthetic gene clusters polyspecific?

Did these systems evolve from possibly even more ancient broad-spectrum efflux systems that might have served as general defense mechanisms against toxins in environmental bacteria?

That transporters involved in antibiotic resistance could have been repurposed from the general defense efflux systems has been suggested previously Dantas and Sommer, ; Martinez, Such an origin could explain why these systems are multi-specific, and how they could be easily adapted by different producer organisms to transport individual antibiotics synthesized by them.

Analysis of many additional efflux systems found in biosynthesis clusters of producer organisms is needed to begin to formulate clear answers to these questions. Many other examples of ABC as well as MFS transporters used for conferring self-resistance in producer organisms to lantibiotic NAI, polyene macrolide natamycin, tylosin, or actinorhodin are known Rosteck et al.

However, their molecular mechanisms and substrate specificities have not yet been elucidated. Sequestration involves the function of drug-binding proteins, which prevent the antibiotic from reaching its target.

In producers of the bleomycin family of antibiotics, the primary mechanism of resistance involves sequestration of the metal-bound or the metal-free antibiotic Sugiyama and Kumagai, by binding proteins TlmA, BlmA, and ZbmA in S.

hindustanus ATCC Gatignol et al. verticillus Sugiyama et al. Each bleomycin-family producer member has one or more genes related to ABC transporters in their biosynthesis clusters Du et al. For additional examples, see references Sheldon et al. Target modification acts as a self-resistance mechanism against several classes of antibiotics, including β-lactams, glycopeptides, macrolides, lincosamides, and streptogramins MLS , and aminoglycosides.

The β-lactam antibiotic has a similar structure to PBP substrates peptidoglycan precursors , thus allowing the antibiotic to associate and cause acylation of the active site serine resulting in its inhibition Yeats et al. The producer Streptomyces species, despite being Gram-positive, are highly resistant to penicillins, which is due to either overproduction of PBPs or synthesis of low-affinity PBPs Ogawara, Three classes of PBPs A, B, and C are found in bacteria Ogawara, Analysis of the biosynthesis clusters of β-lactam producing bacteria showed that they often contain genes for PBPs, suggesting their role in self-resistance Liras and Martin, ; Ogawara, Interestingly, Streptomyces species contain on average more than 10 PBPs, including both Classes A and B, a number much greater than found in other Actinobacteria.

Glycopeptides, such as vancomycin and teicoplanin, inhibit cell wall transpeptidation and transglycosylation by associating with peptidoglycan precursors D -Ala- D -Ala Binda et al. Antibiotic resistance results from a change in the peptidoglycan precursor from D -Ala- D -Ala to D -Ala- D -Lac or D -Ala- D -Ser, which has a and 6-fold reduction in affinity for the glycopeptides, respectively Bugg et al.

Genes conferring vancomycin resistance were initially identified in clinical strains, with the van A cluster van HAX on the transposon Tn being the most commonly seen. Some systems also use VanY, a D , D -carboxypeptidase to produce tetrapeptides incapable of glycopeptide binding Binda et al.

Related core van HAX clusters have been found in producer organisms, suggesting an evolutionary relatedness of resistance within producers and pathogens Marshall et al.

The examples include similar van H Marshall et al. Variants on the core cluster are also reported Schaberle et al. Target modification is also seen for MLS antibiotics, which bind to the 50S ribosomal subunit.

This mechanism involves methylation of 23S rRNA at residue A by 23S rRNA methyltransferases Douthwaite et al.

Monomethylation MLS type I typically provides moderate level of resistance, while dimethylation MLS type II provides strong resistance Fyfe et al. For further information on MLS resistance mechanisms, see reviews Matsuoka and Sasaki, ; Mast and Wohlleben, ; Spizek and Rezanka, Finally, resistance against aminoglycosides by target modification uses 16S rRNA methyltransferases, which methylate at residue A or G Shakil et al.

This mechanism for self-resistance may work in conjunction with the AMEs, which were described earlier. Other resistance mechanisms bypass the original target by producing additional low affinity targets. Examples include synthesis of additional B subunit of DNA gyrase for novobiocin resistance, alternate resistant RNA polymerase for rifamycin resistance, or an alternate fatty acid synthase for resistance to platensimycin Blanco et al.

Antibiotic removal from the target site provides another protective resistance mechanism. rimosus , the antibiotic oxytetracycline is removed by OtrA from the ribosome Doyle et al.

Most producer organisms contain several mechanisms for self-resistance. For example, S. peucetius relies on DrrAB to efflux doxorubicin Li et al. In addition, there is also a serine protease capable of sequestering daunorubicin to prevent its re-entry into the cell following efflux Dubey et al.

Other examples of producers containing several mechanisms for self-resistance include the following: Microbispora ATCC PTA contains both an efflux pump MlbJYZ and a sequestration protein MlbQ to protect against NAI Pozzi et al.

rimosus has an ABC multi-drug efflux pump OtrC Yu et al. fradiae contains several gene products TlrA, TlrB, and TlrD that modify the ribosome to prevent tylosin binding and uses TlrC for efflux Mak et al.

chattanoogensis L10 contains several different efflux pumps for resistance against natamycin Wang et al. Discovery of antibiotics and their development for treatment of infectious diseases is the biggest success story in the history of chemotherapy.

However, widespread and indiscriminate use of antibiotics in the last 70 years has led to selection of resistant strains to every antibiotic that has been introduced so far. With the very first antimicrobial agents, such as sulfonamides, resistance was observed soon after in the late s Davies and Davies, Even before the widespread use of penicillin in clinical practice, penicillinase was discovered in in Staphylococcus aureus and Streptococcus pneumoniae providing evidence that the resistance mechanisms against penicillin were already present in the natural environment Davies and Davies, ; Ogawara, b.

Similarly, after the introduction of methicillin a semi-synthetic penicillin to treat penicillin-resistant S. aureus infections, resistance was once again observed in strains now referred to as MRSA Methicillin-resistant Staphylococcus aureus Davies and Davies, These observations suggest that the use of each and every antibiotic sooner or later results in appearance of resistant strains.

This is a testament to the extreme malleability and plasticity of bacterial genomes and their vast potential for adaptability.

A high rate of spontaneous mutations and widely prevalent DNA exchange mechanisms in bacteria are critical contributors to the emergence of this phenomenon. According to the Centers for Disease Control and Prevention, antibiotic resistance leads to 23, deaths annually in the US alone.

Recently, the development of MDR and XDR extremely drug resistant strains of Mycobacterium tuberculosis, S. aureus , and Acinetobacter baumannii have become a cause for serious concern, leaving limited options for the treatment of infectious pathogens carrying these resistance mechanisms.

aureus and vancomycin resistant enterococci VRE , or intrinsically resistant environmental bacteria that can become opportunistic pathogens, such as Pseudomonas aeruginosa and A. baumannii Wright, ; Miller et al. Intrinsic antibiotic mechanisms are normally chromosome-encoded and include non-specific efflux pumps which likely evolved as a general response to environmental toxins , antibiotic inactivating enzymes, or mechanisms that serve as permeability barriers Fajardo et al.

These mechanisms are fixed in the core genetic make-up of an organism. Vancomycin resistance in E. coli and other Gram-negative bacteria provides another example of intrinsic resistance, which results from the permeability barrier imposed by the outer membrane Arthur and Courvalin, Although intrinsic mechanisms confer low level antibiotic resistance in the original host, normal commensal flora or environmental bacteria containing intrinsic mechanisms can become opportunistic pathogens in immunocompromised patients Wright, The acquired resistance mechanisms, on the other hand, are generally obtained by horizontal gene transfer HGT, described later and include plasmid-encoded specific efflux pumps such as TetK and TetL of S.

aureus and enzymes that can modify the antibiotic or the target of the antibiotic Bismuth et al. These mechanisms pose a more serious threat to human health because of a change in the context of the resistance determinant from chromosomal to plasmid-mediated, resulting in their enhanced expression and dissemination Dantas and Sommer, ; Martinez, A well-documented example of such a phenomenon is mobilization of the chromosomal β-lactamase gene amp C to a plasmid resulting in its worldwide dissemination Dantas and Sommer, Interestingly, the biochemical mechanisms of resistance in clinical isolates are very similar to those found in producer organisms.

Moreover, the resistance genes belong to the same functional families as seen in the producers Benveniste and Davies, ; Marshall et al.

However, the distribution, expression, and genetic context of resistance determinants in clinical strains are strikingly different.

For example, resistance elements found in producer organisms are embedded in the biosynthesis gene clusters, while in clinical strains they are most often located on plasmids and transposons.

For human health reasons, a lot more attention has been given to understanding the molecular and biochemical basis of antibiotic resistance in clinical isolates, and a large number of excellent reviews have been written on this topic Blair et al.

Therefore, the section below provides only relevant additional information about each resistance mechanism in clinical strains, allowing the reader to compare and contrast our understanding of these determinants in clinical strains vs.

the producer organisms while providing a more complete picture of the field of antibiotic resistance. As seen in producers, antibiotic modification is commonly used as a resistance mechanism for aminoglycosides in pathogenic strains.

While these genes are commonly located on the mobile genetic elements MGEs in clinical bacteria, chromosomal determinants for AMEs have also been found in a large number of environmental bacteria, including Providencia and Acinetobacter species Macinga and Rather, ; Yoon et al.

In spite of the presence of a conserved fold, these enzymes exhibit significant sequence, structural, and functional diversity, again implying convergent evolution of these enzymes from distinct housekeeping cellular proteins Stogios et al. These studies further illustrate the plasticity of antibiotic modification enzymes Fong et al.

In addition to AMEs, multiple CAT enzymes have been identified in both Gram-positive and Gram-negative bacteria, which have been extensively reviewed Schwarz et al. While the role of β-lactamases in producer bacteria is still debatable, they are known to play a critical role in β-lactam resistance in Gram-negative clinical bacteria.

More than β-lactamases have been identified from clinical isolates, and this number continues to grow because of the ever-new mutations in the active site allowing it to adapt to newer β-lactams.

An example is the evolution of TEM-3, which can degrade 3rd generation cephalosporins, placing it into the category of ESBLs Extended Spectrum β-lactamases Paterson and Bonomo, , suggesting rapid evolution of β-lactamase genes in clinical strains.

Most β-lactamase genes are carried on MGEs facilitating their rapid spread through populations; however, some β-lactamase genes are also found in chromosomes of members of the Enterobacteriaceae family where they are poorly expressed and function as silent genes.

Once again, it is speculated that, as in the case of AMEs, β-lactamases may also perform dual functions, including housekeeping and antibiotic resistance Martinez, An interesting set of studies indeed suggest that the biological function of β-lactamases may be peptidoglycan recycling Wiedemann et al.

Decreased permeability is important for Gram-negative bacteria because of the presence of the outer membrane, which forms a permeability barrier and offers an intrinsic mechanism for protection against hydrophilic antibiotics and other antimicrobial agents, such as vancomycin Nikaido, In addition, many types of active efflux pumps have been described in Gram-positive and Gram-negative bacteria, which generally belong to one of the five families: ABC, MFS, RND Resistance-Nodulation-Division , MATE Multidrug and Toxin Extrusion , and SMR Small Multidrug Resistance Sun et al.

Of these, only ABC proteins use ATP as a source of energy, while the other four families couple transport of substrates to ion gradients.

Normally transport proteins carry out import or export of only one specific substrate for example, Tet proteins belonging to the MFS family. Genes encoding antibiotic efflux pumps can be either intrinsic or acquired.

coli , nor A in S. aureus , and lmr A in Lactococcus lactis. Although this system carries out efflux of a very broad spectrum of compounds, its biological function is believed to be export of bile salts in Enterobacteriaceae Thanassi et al.

The RND pumps are unique in that they bridge the inner and outer membranes through a fusion protein AcrA in this case and bring about export of antibiotics from the inside to the outside in a single step. The acquired antibiotic efflux determinants, often found on MGEs in clinical isolates, are exemplified by many different types of tet genes at least 22 have been identified located on plasmids in both Gram-negative and Gram-positive bacteria Roberts, Interestingly, RND pumps can act synergistically with the simple Tet pump proteins MFS family , resulting in a significant increase in the minimum inhibitory concentration for tetracycline Lee et al.

This likely occurs when tetracycline exported to the periplasm by a Tet protein can be captured by the RND pump and exported to the outside Nikaido and Takatsuka, , illustrating how acquired resistance mechanisms can be augmented by the intrinsic mechanisms potentially resulting in major implications in the clinic.

A large number of target replacement and protection mechanisms are also found in clinical isolates. The classical example of target modification is seen in MRSA strains where resistance to β-lactams is conferred by an exogenous PBP, known as PBP2a, whose transpeptidase domain is insensitive to the action of several different β-lactams.

Acquisition of PBP2a facilitates bypass of the original sensitive target, however, since it does not contain the transglycosylase activity it functions together with the transglycosylase domain of the native PBP2 to perform cross-linking reaction in the presence of β-lactams.

PBP2a is coded by the mec A gene, which is located on a large MGE called SCC mec Staphylococcal chromosomal cassette in S. Many different types of SCC mec cassettes have been described, which contain varying numbers of accompanying resistance elements Fishovitz et al.

Another example of target modification is vancomycin resistance, which results from acquisition of the van gene cluster and is commonly a problem in enterococci Miller et al. Of the many known types of van clusters, van A and van B, in particular, are a problem in clinical strains as they occur on MGEs.

The similarities in the sequence and arrangement of van genes in producer and clinical strains suggest that they are evolutionarily linked. Other target modification examples in clinical strains include point mutations or enzymatic alteration of the target Munita and Arias, For examples of point mutations in the target, see Hooper, ; Floss and Yu, Enzymatic alteration of the target is best understood in the case of macrolide resistance conferred by a large group of erythromycin ribosomal methylation erm genes.

These enzymes methylate a specific adenine in the 23S rRNA Weisblum, The erm genes in clinical strains are present on mobile genetic elements and are widespread among both Gram-positive and Gram-negative bacteria Roberts, Significant similarities between the methylation enzymes found in the clinical isolates and the producers have been observed, suggesting a common ancestral origin Uchiyama and Weisblum, ; Doi et al.

Finally, known examples of target protection in clinical strains include the Tet M and Tet O proteins commonly encoded by genes located on MGEs in S.

Interestingly, these proteins are homologous to the elongation factors EF-G and EF-Tu, and their binding to the ribosome facilitates removal of tetracycline in a GTP-ase activity-dependent manner Burdett, ; Trieber et al.

Based on the discussion above, it is evident that our understanding of the distribution and function of resistance determinants in clinical isolates is much more advanced as compared to the producer organisms. Indeed, it is the incorporation of such determinants into MGEs in pathogens that poses a serious threat to human health.

Where do antibiotic resistance genes in the clinic come from? This question continues to puzzle scientists and clinicians.

The idea that resistance genes in pathogens may be acquired from antibiotic producer organisms by horizontal transfer was originally proposed in the s Benveniste and Davies, It was based on the observation that the aminoglycoside-modifying enzymes found in actinomycetes exhibit biochemical activities similar to the enzymes found in pathogenic strains.

Another striking example of a strong connection between antibiotic resistance genes in clinical isolates and those found in antibiotic producing bacteria is provided by the van HAX genes, which show considerable protein sequence similarity as well as a conserved arrangement and organization of genes within the cluster Barna and Williams, ; Marshall et al.

Despite strong indications that transfer from producer organisms to the pathogenic strains might occur Figure 2 , Route 1 a direct link between producers and pathogens has, however, been hard to establish, and very rarely have the resistance genes of pathogens been tracked back to the producers.

Altogether, these observations suggest an evolutionary link between determinants of producers and pathogens but not necessarily a direct recent gene transfer from the producers Forsman et al. Nevertheless, transfer from producers could have occurred a long time ago through a series of closely related carriers; for example, first transfer to closely related non-producing actinomycetes in the soil Figure 2 , Route 2A and then finally to proteobacteria and distant pathogenic strains Marshall et al.

FIGURE 2. Schematic showing reservoirs of antibiotic resistance genes found in nature and various pathways for their movement to the clinic. Transfer of resistance genes to clinical isolates could occur by a variety of routes shown by arrows , each using horizontal gene transfer mechanisms potentially involving plasmids, integrons, or transposons.

While direct transfer of resistance determinants from producers in the soil to clinical strains is possible Route 1 , a more likely route may first involve movement from the producer soil bacteria to non-producer soil bacteria for example Mycobacterium species Pang et al.

Another, possibly more important route, could involve direct transfer from environmental bacteria found in bodies of water, aquaculture, livestock animals, wildlife, and plants to clinical isolates Route 3. Routes 2 and 3 are shown as thick red arrows, implying greater probability of these pathways for dissemination of resistance genes to clinical strains.

An alternative school of thought and a growing body of recent literature, however, now seem to suggest that resistance genes found in non-producer environmental bacteria may have played a more important role in shaping the evolution of antibiotic resistance in pathogens Figure 2 , Route 3 Aminov and Mackie, The genome sequence analyses carried out in recent years have also shown that not only are the intrinsic resistance mechanisms widely prevalent in all microbes Fajardo et al.

Analysis of microbial DNA isolated from the dental plaque of ancient human remains showed the existence of gene sequences homologous to those conferring resistance to β-lactams, aminoglycosides, macrolides, tetracycline, and bacitracin in clinical strains Warinner et al.

Interestingly, the van HAX cluster in permafrost DNA exhibited the same invariant organization as seen in modern vancomycin resistant isolates, confirming that these genes predate the use of antibiotics. Other similar studies showing prevalence of resistance determinants in ancient samples, or isolated caves, are also available Bhullar et al.

Therefore, it is proposed that to get a full understanding of the origin of resistance, one must consider the pan-microbial genome consisting of antibiotic producers, pathogens, cryptic genes, and precursor genes Wright, ; Nesme and Simonet, Albeit limited in number, a few reports of direct genetic exchange from producer to non-producer organisms and from environmental organisms to clinical pathogens are indeed available.

In one report, otr A and otr B gene sequences, found in the oxytetracycline biosynthesis cluster in Streptomyces , were identified in mycobacteria variants Pang et al.

Mycobacterium is closely related to Streptomyces , and both are commonly found in the soil, therefore the transfer of otr A and otr B to mycobacteria suggests their role as potential carrier organisms in the soil. Interestingly, the same study also provided evidence for the presence of S.

aureus tetracycline resistance genes Tet K and Tet L in Streptomyces and mycobacteria variants. The sequences isolated from these variants were almost identical to the S. This study therefore shows that resistance genes can move back and forth between producer and non-producer organisms providing support for Route 2A Figure 2.

In another study, bioinformatics analysis was used to obtain evidence for recent inter-phylum transfer of chloramphenicol and lincomycin efflux genes cmx and lmrA from Actinobacteria to Proteobacteria Jiang et al. The proposed mechanism for such inter-phylum exchange is discussed in Jiang et al.

The most compelling evidence of recent transfers from non-pathogenic environmental bacteria to clinical strains Figure 2 , Route 3 comes from three independent reports Dantas and Sommer, ; Forsberg et al.

First report showed that the CTX-M ESBL gene found on plasmids in pathogenic bacteria worldwide is almost identical to CTX-M gene found in the genome of non-pathogenic environmental Kluyvera species Humeniuk et al.

The second report shows that the quinolone resistance determinant qnr located on a conjugative plasmid in Klebsiella , originated from the genome of non-pathogenic environmental Vibrio and Shewanella species Poirel et al.

baumannii and then to members of Enterobacteriaceae family and to Pseudomonas species Yoon et al. These examples provide definitive evidence of genetic transfer from environmental organisms and also illuminate how an intrinsic resistance gene located in the genome of a non-pathogenic organism can result in a pandemic when mobilized to a conjugative plasmid or a phage and transferred to a clinically relevant strain.

Overall, these examples suggest that both producer and non-producer environmental bacteria play a role in dissemination of resistance genes although recent direct transfers to clinical strains seem to have mainly occurred from non-producer environmental bacteria.

Transfer of antibiotic resistance determinants between bacterial populations occurs by genetic exchange mechanisms involving transformation with free DNA, transduction by bacteriophages, or conjugation involving plasmids Wright, ; Hu et al. All three HGT mechanisms are widely used in nature, although certain species of bacteria tend to employ one mechanism more heavily over the others Barlow, For example, streptococci can become naturally competent and thus participate effectively in transformation, whereas enterobacteria commonly use conjugative plasmids for exchange of genetic information.

Transformation is best characterized in Gram-positive Streptococcus pneumoniae and Bacillus subtilis although many Gram-negative bacteria also become competent Johnston et al. The factors that control competence generally include the nutritional status of the bacterium Claverys et al.

Although the physiological role of transformation is still debated, its main purpose is believed to be DNA repair or genetic diversification to enhance adaptability Johnston et al.

Indeed, transformation seems to have played an important role in evolution of antibiotic resistance strains of Streptococcus and Neisseria. For example, it is thought that the persistence of penicillin resistance in S.

pneumoniae may be related to the high frequency of natural transformation in this organism Hoffman-Roberts et al.

Transformation of Neisseria gonorrhoeae with DNA from resistant commensal Neisseria flavescens is believed to have resulted in generation of a mosaic pen A variant that confers resistance to β-lactams in clinical isolates Spratt, ; Spratt et al. Mosaic variants of antibiotic resistance genes have also been reported in several Streptococcus species, implying the role of transformation in incorporating sections of foreign DNA von Wintersdorff et al.

Transduction is believed to play a major role in evolution of resistance in S. aureus , which exhibits high strain variability and carries a large accessory genome consisting of phages, plasmids, transposons, genomic islands, and SCC mec most of which carry resistance genes , it is generally accepted that HGT in general, and transduction in particular, play a major role in antibiotic resistance gene transfer Haaber et al.

Indeed, moderate rates of transfer about 10 -5 or 10 -6 of genes for penicillinase, metallo β-lactamase, and tetracycline resistance by transducing phages have been reported in S. aureus Varga et al.

However, transduction of even the small SCC mecs 20—25 kb in size from MRSA strains of S. aureus to methicillin-sensitive strains was shown to occur at low frequencies 10 -9 to 10 Scharn et al. Another study, which used qPCR to quantify S.

aureus genes in viral particles, showed the presence of parts of the SCC mec element specifically mec A and ccr A1 in phage particles at relatively high frequency of about 10 -4 Maslanova et al. Quantitative studies, however, do not take into consideration the transmission capability of the particles, therefore they likely reflect an overestimation of the transduction frequency Torres-Barcelo, Interestingly, other resistance and virulence genes of S.

aureus associated with special MGEs referred to as PICIs phage-induced chromosomal islands , which include SaPIs S. aureus pathogenicity islands , are known to be transduced by bacteriophages at remarkably high frequencies approaching 10 -1 Chen and Novick, ; Penadés and Christie, These islands include many antibiotic resistance genes, suggesting that transduction may contribute significantly to variability and evolution of resistance in S.

aureus Novick et al. Interspecies and intergeneric transfer of SaPI elements has also been shown to occur between S. aureus, S. epidermidis , and even Listeria monocytogenes , showing a broader host range of staphylococcal phages Maiques et al.

In general, however, because of the difficulty in detecting recombination events outside of the laboratory, the contribution of either transformation or transduction in transferring resistance genes in the clinic or the environment remains unclear. Nevertheless, certain environments considered to be hot-spots for genetic exchange, such as sewage and wastewater treatment plants, hospital effluents, aquaculture, agricultural and slaughterhouse waste, are prime locations for exchange events because of the high density of bacteria, phages, and plasmids in these settings Kenzaka et al.

In one study, qPCR analysis showed that bla TEM , bla CTX-M , and mec A were indeed present in phage particles isolated from sewage samples Colomer-Lluch et al. Other reports showing the prevalence of phage carrying bla TEM and bla CTX-M genes in soil, water, and sewage are also available Balcazar, ; Larranaga et al.

When combined with high selection pressure in these environments, resulting from the presence of sub-inhibitory concentrations of antibiotics, metals, and toxic materials, which can lead to induction of competence Prudhomme et al.

Other settings suitable for genetic exchange via transduction also include the colonized human or animal host McCarthy et al.

A recent report describing the phenomenon of auto-transduction in S. aureus provides further strong support for the important role of phages in delivering antibiotic resistance genes to the host bacteria Haaber et al. Using in vitro and in vivo virulence model, this study by Haaber et al.

Plasmid-mediated conjugation as a gene transfer mechanism is, however, still considered to be far more prevalent in disseminating resistance genes in nature than either transformation or transduction.

Plasmids are capable of autonomous replication, and they carry genes for resistance against all major classes of antibiotics. In fact, plasmids can carry a collection of resistance genes as part of transposons, thus simultaneously conferring resistance to several classes of antibiotics and metal ions Nikaido, Moreover, they can transfer genes over long genetic distances to different species, genera, and even kingdoms depending on the host range of the plasmid.

Using mathematical modeling analysis, one study recently showed that conjugation may be fold more common than transduction as a resistance gene transfer mechanism Volkova et al.

Since gene transfer by conjugation can be easily tracked by DNA sequencing and PCR-based approaches, there is sufficient evidence for its contribution to worldwide dissemination of antibiotic resistance determinants both in community and hospital environments Carattoli, Some of the most successful known plasmids are the ones that have resulted in the spread of carbapenemase, bla CTX-M ESBL, and quinolone resistance genes among Gram-negative bacteria over very large geographical distances Carattoli, In Gram-positive bacteria, other DNA elements, known as conjugative transposons or integrative conjugative elements ICEs , can also mediate conjugation.

These elements integrate into the chromosome but contain the ability to excise and transfer themselves by conjugation. ICEs often carry resistance genes, for example Tn family members that encode tetracycline resistance Roberts and Mullany, The known conditions for resistance gene transfer by conjugation include high density settings, such as the human or animal gut, biofilms, hospitals, and co-infection conditions Weigel et al.

Although some resistance determinants have been plasmid-associated for a long time Barlow and Hall, , others are mobilized to plasmids from chromosomes, and the rate at which these genes are being mobilized has increased since the widespread use of antibiotics about 70 years ago Barlow et al.

Another worrisome emerging trend is the clustering of antibiotic resistance genes on plasmids, perhaps as a response to selective pressures in the environment.

A well-characterized mechanism of clustering is provided by the S. aureus conjugative plasmid pSK41 that contains an insertion sequence IS , which promotes capture of small resistance plasmids Haaber et al. All three HGT mechanisms are subject to limitations imposed by the host range of the incoming plasmid or the phage, the restriction modification systems of the host, ability to form cell-to-cell contacts, fitness cost of acquiring a new gene, as well as the ability of the incoming DNA to recombine with the host DNA Thomas and Nielsen, ; Domingues et al.

Further, the ability of a mobile genetic element to establish in a population also depends on whether it can replicate autonomously and therefore get vertically transmitted.

The most successful conjugative plasmids, such as the incompatibility group IncP, have a broad host range Davies and Davies, , which facilitates their transfer to and maintenance in distantly related phyla Klumper et al. The ability of MGEs or DNA to persist in the environment also determines success of HGT.

For example, while cell-to-cell contact is essential for conjugation, it provides better protection to DNA. On the other hand, naked DNA is vulnerable to being degraded quickly, which reduces the time period during which it remains intact to successfully encounter a competent cell. DNA packed in a phage particle is more protected than naked DNA, although the narrow host range of a phage may determine if it will be in the gene pool long enough to infect a suitable host von Wintersdorff et al.

In spite of the limitations, bacterial genome sequencing efforts have made it abundantly clear that the HGT mechanisms have had a major impact on evolution of bacterial populations Nakamura et al. Our knowledge of the actual steps and carriers involved in moving resistance genes from environmental and producer organisms to the clinic, or from the chromosome to the MGEs, is, however, still rather limited.

It is not clear, however, why and how resistance genes are captured or transferred from chromosome to the plasmids. In addition to the role of insertion sequences and transposons, mobilization of resistance genes may also be greatly aided by the presence of integrons.

While they are not self-mobile, they can be mobilized to plasmids or phages by transposons, thus gaining the ability to move between cells by HGT. Integrons typically contain three genetic elements, which include a gene for site-specific recombination Int I , a site-specific recombination site att I , and a promoter upstream of the att I site used for expression of the recruited gene cassette often containing resistance determinants Domingues et al.

Class 1 integrons found on MGEs, in particular, are widely distributed in clinical settings and are often associated with carrying and spreading antibiotic resistance genes Naas et al. A rather large pool of circular gene cassettes containing the att C site and the promoter-less resistance determinants for almost all classes of antibiotics used clinically are also known to exist in bacteria Partridge et al.

These genes become functional after the cassettes are incorporated and expressed from the promoter sequence in the integron. In this mechanism, conjugation mediated by a broad-host range conjugative plasmid Klumper et al.

Dead actinobacteria cells would release the actinobacterial DNA flanked by proteobacterial DNA into the environment, and proteobacteria can take up this DNA by transformation and incorporate into their genome using homologous recombination.

Using such a mechanism, cmx and lmr A genes are believed to have been recently transferred from Actinobacteria to Proteobacteria with the help of the broad-host range conjugative plasmids and integrons Jiang et al.

Once these genes are transferred to proteobacteria, it is easy to envision their transfer to pathogenic bacteria which also mostly belong to the phylum Proteobacteria. By now it is well-recognized that the environment itself plays an important role in the acquisition of antibiotic resistance by pathogenic organisms.

This process is envisioned to go through four stages: emergence of novel resistance genes, mobilization, transfer to pathogens, and dissemination. While emergence and mobilization events likely occur all the time, environmental factors, such as selective pressure, fitness cost, and dispersal, determine whether these events actually result in establishing novel genes in populations Bengtsson-Palme et al.

What creates selective pressure strong enough to promote persistence and longevity of resistance genes?

Antibiotic producers present one such scenario where resistance genes can be selected naturally in a competitive environment, thus preserving the pool of resistance genes in that niche Laskaris et al. The most important source of selective pressure, however, is the widespread and indiscriminate usage of antibiotics by humans, which results in dominance of resistant and multiply resistant strains of bacteria not only among human pathogens but also in environments where human activities such as antibiotic manufacturing facilities result in pollution with antibiotics Larsson, Such environments are ideal not only for transfer of resistance genes to pathogens, but they can also result in transfer of resistance from pathogens to environmental bacteria or opportunistic pathogens, resulting in persistence and possible reemergence of resistance genes in the future Ashbolt et al.

Recent studies have shown that antibiotic concentrations significantly below the minimum inhibitory concentration for sensitive bacteria can be selective Gullberg et al. Moreover, other contaminants, such as heavy metals, can also co-select for antibiotic resistance Pal et al.

There is indeed evidence that selective pressure caused by human activities in the last 70 years has resulted in a significant enrichment of resistance genes in bacterial populations. One study compared pre-antibiotic era microbes with modern environmental bacteria in archived soils collected from to in the Netherlands and showed that genes conferring resistance to tetracycline, erythromycin, and β-lactams increased in abundance over time Knapp et al.

Interestingly, an increased rate of mobilization of β-lactamase genes from the chromosome to the plasmids was also reported Barlow et al.

Other reports also suggest that antibiotic selection promotes competence in S. pneumoniae Prudhomme et al.

aureus Goerke et al. Interestingly, a more recent study showed that the ratio of transducing particles to virulent phages varies upon induction by sub-inhibitory concentrations of different antibiotics, suggesting that antibiotics affect packaging of genes into phage particles Stanczak-Mrozek et al.

Antibiotic exposure has also been shown to result in increased rates of mutations and recombination as well as an increase in integrase activity Maiques et al.

In conclusion, mitigation strategies focused on limiting selective pressure, for example by reducing unnecessary usage of antibiotics and avoiding settings which select for and promote persistence, are needed to prevent further recruitment of novel resistance genes into pathogens.

Antibiotic producing bacteria of the genus Streptomyces as well as non-pathogenic environmental bacteria are important reservoirs of antibiotic resistance determinants.

These determinants may be transferred to clinical strains by a variety of HGT mechanisms, including transformation of naturally competent bacteria, phages, and the use of conjugative plasmids, transposons, and integrons.

Despite barriers to the exchange of genetic information between different genera of bacteria, widespread transfer of resistance genes from chromosomes of environmental and soil bacteria to the mobilizable elements in clinical isolates seems to have occurred.

Indeed several examples of recent transfers from environmental bacteria to the clinical strains are available Route 3, Figure 2 ; however, very limited evidence for recent direct transfer from producers to clinical strains has been obtained Route 1, Figure 2.

Nevertheless, transfer from producer bacteria to other actinomycetes in soil is possible Route 2A , which could provide a pathway for further transfer of these determinants to proteobacterial clinical strains Route 2B.

Based on the available evidence, we conclude that Routes 2 and 3 are much more prevalent in nature as compared to Route 1 for transfer of resistance genes to pathogens.

To better understand factors that promote dissemination of resistance genes and to elucidate relationships between antibiotic resistance genes of producer, environmental, and pathogenic bacteria, new and improved strategies for sampling and screening of microbial populations and metagenomic libraries are needed.

Moreover, better algorithms and the use of bioinformatics approaches for determining relationships between resistance determinants of different environmental niches will be highly beneficial. Additional genome sequencing data will also help fill the gaps in our knowledge of intermediate stages and carriers for mobilization.

Indeed two databases, the Antibiotic Resistance Database ARDB and the Comprehensive Antibiotic Resistance Database CARD , assembled in the last decade Liu and Pop, ; McArthur et al. This was demonstrated in a recent bioinformatics study using these databases Jiang et al.

It is expected that these bioinformatics tools will unify information on resistance genes and their products found in thousands of bacterial species isolated from the clinic or the environment as well as their associated mobile genetic elements and allow this information to be quickly mined by researchers in this field.

PK supervised the work, collected and reviewed literature, and co-wrote the review article. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Heinzel, P. Relationships between antibiotic and protein kinases. Hoffman-Roberts, H. Investigational new drugs for the treatment of resistant pneumococcal infections. Expert Opin. Although veterinary laboratories originally based interpretations on standards established using human pathogens, it became apparent by the early s that such an approach did not reliably predict clinical outcomes when applied to veterinary practice.

Subsequently, groups were established to develop veterinary-specific standards. Most often, interpretation is reduced to whether the isolate is classified as susceptible, intermediately susceptible, or resistant to a particular antibiotic. It should, however, be remembered that these in vitro procedures are only approximations of in vivo conditions, which can be very different depending on the nature of the drug, the nature of the host, and the conditions surrounding the interaction between the antibiotic and the target pathogen.

One critical aspect is following standardized, quality-controlled procedures that can generate reproducible results. Because of the required culture time, antimicrobial susceptibility testing by the above methods may take several days, which is not ideal, particularly in critical clinical cases demanding urgency.

Often practitioners may use locally established antibiograms as a guideline for therapy. An antibiogram is a compiled susceptibility report or table of commonly isolated organisms in a particular hospital, farm, or geographic area, which can serve as a useful guideline in therapy before actual culture and susceptibility data becomes available for reference.

In some cases, specific resistance gene detection by PCR or direct enzyme testing can provide earlier susceptibility information Example: mecA detection in methicillin-resistant staphylococci. To learn more, read About Antibiograms.

There are several antimicrobial susceptibility testing methods available today and each one has its respective advantages and disadvantages. They all have the same goal, which is to provide a reliable prediction of whether an infection caused by a bacterial isolate will respond therapeutically to a particular antibiotic treatment.

These data may be used as guidelines for treatment, or as indicators of emergence and spread of resistance on a population level based on passive or active surveillance. Some examples of antibiotic susceptibility testing methods are:.

Selection of the appropriate method will depend on the intended degree of accuracy, convenience, urgency, availability of resources, availability of technical expertise, and cost.

Interpretation should be based on veterinary standards whenever possible rather than on human medical standards due to applicability. Among these available tests, the two most commonly used methods in veterinary laboratories are the agar disk-diffusion method and the broth microdilution method.

The broth dilution method involves placing the isolate into several separate broth solutions containing an antimicrobial agent in a series of varying concentrations. Microdilution testing uses about 0. Macrodilution testing uses broth volumes at about 1.

For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited, as evidenced by the absence of visible bacterial growth, is recorded as the minimal inhibitory concentration MIC.

The test is only valid if the positive control shows growth and the negative control shows no growth. A procedure similar to broth dilution is agar dilution. The agar dilution method follows the same principle of establishing the lowest concentration of a serially diluted antibiotic for which bacterial growth is still inhibited.

Because of convenience, efficiency, and cost, the disk diffusion method is probably the most widely used method for determining antimicrobial resistance in private veterinary clinics.

Commercially prepared disks, each of which is preimpregnated with a standard concentration of a particular antibiotic, are evenly dispensed and lightly pressed onto the agar surface. The antibiotic being tested diffuses outward from the diffusion disk and creates an antibiotic concentration gradient in the agar.

The highest concentration of antibiotic is found closest to the diffusion disk with decreasing amount of antibiotic present, further and further from the disk. The zone around an antibiotic disk that has no growth is referred to as the zone of inhibition.

This approximates the minimum antibiotic concentration sufficient to prevent growth of the test isolate. The zone is measured in mm and compared to a standard interpretation chart used to categorize the isolate as susceptible, intermediately susceptible, or resistant.

The MIC measurement cannot be determined from this qualitative test, which simply classifies the isolate as susceptible, intermediate, or resistant. To help your understanding of testing, watch this video example. The e-test is a commercially available test that uses a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic.

The strip also displays a numerical scale that corresponds to the antibiotic concentration. This method is a convenient quantitative test of antibiotic resistance. However, a separate strip is needed for each antibiotic, and therefore the cost of this method can be high. Let's watch a video on e-test for antibiotic susceptibility.

Several commercial systems provide conveniently prepared and formatted microdilution panels, instrumentation and automated plate readings. These methods are intended to reduce technical errors and lengthy preparation times. Most automated antimicrobial susceptibility testing systems provide automated inoculation, reading, and interpretation.

Although these systems are rapid and convenient, one major limitation for most laboratories is the cost associated with the purchase, operation, and maintenance of the machinery.

Resistance may also be established through tests that directly detect the presence of a particular resistance mechanism. For example, ß-lactamase detection can be accomplished using an assay such as the chromogenic cephalosporinase test.

Since resistance traits are genetically encoded, we can sometimes test for the specific genes that confer antibiotic resistance. Even though nucleic acid-based detection systems are generally rapid and sensitive, it is important to remember that the presence of a resistance gene does not necessarily equate to treatment failure, as resistance is also dependent on the mode and level of expression of these genes.

Some of the most common molecular techniques used for antimicrobial resistance detection are as follows:. Skip to main content. Breadcrumb Home Microbiology. Intricate Science How do minute microorganisms actually resist antimicrobial actions? Learning Outcomes This module aims to introduce the microbiological aspects of antimicrobial resistance.

By the end of the module, you will be able to: 1. Bacterial Resistance Strategies To survive in the presence of an antibiotic , bacteria must disrupt a step in the action of the antimicrobial agent see Pharmacology Module, Mechanisms of Action.

Efflux pumps are variants of membrane pumps possessed by all bacteria, both pathogenic and nonpathogenic, to move lipophilic or amphipathic molecules in and out of their cells.

Some efflux pumps are used by antibiotic-producing bacteria to pump antibiotics out of their cells as fast as the antibiotic is made. This constitutes an immunity protective mechanism for the bacteria to prevent being killed by its own chemical weapon Walsh, Want to learn more?

Watch the efflux video. The first antibiotic resistance mechanism described was penicillinase. It was first reported by Abraham and Chain in Abraham, E.

and E. An enzyme from bacteria able to destroy penicillin. Less than 10 years after the clinical introduction of penicillins, penicillin-resistant Staphylococcus aureus was observed in a majority of Gram-positive infections in people.

The initial response by the pharmaceutical industry was to develop ß-lactam antibiotics that were unaffected by the specific ß-lactamases secreted by S.

However, as a result, bacterial strains producing ß-lactamases with different properties began to emerge, as well as those with other resistance mechanisms.

This cycle of resistance counteracting resistance continues even today Bush, Beta-Lactamase Inhibitors from Laboratory to Clinic. Clinical Microbiology Reviews. Some Examples of Bacterial Resistance Due to Target Site Modification Alteration in PBPs reducing affinity of ß-lactam antibiotics Methicillin-Resistant Staphylococcus aureus , S.

pneumoniae , Neisseria gonorrhoeae , Group A streptococci, Listeria monocytogenes Changes in peptidoglycan layer and cell wall thickness reducing activity of vancomycin: Vancomycin-resistant S.

aureus Changes in vancomycin precursors reducing activity of vancomycin: Enterococcus faecium and E. faecalis Alterations in DNA gyrase subunits reducing activity of fluoroquinolones: Many Gram-negative bacteria Alteration in topoisomerase IV subunits reducing activity of fluoroquinolones: Many Gram-positive bacteria, particularly S.

aureus and Streptococcus pneumoniae Changes in RNA polymerase reducing activity of rifampicin: Mycobacterium tuberculosis. Target DNA gyrase and topoisomerase IV of the bacteria and inhibit the necessary step of supercoiling. Target and bind to the 30s ribosomal subunit to cause misreading of the genetic code which results in inhibition of protein synthesis.

Target and bind to 50s ribsomal subunit to inhibit translocation and transpeptidation process, resulting in inhibition of protein synthesis.

Target and bind to 30s ribosomal subunit to prevent aminoacyl-tRNA to attach to RNA-ribosome complex, inhibiting protein synthesis. Targets dyhydropteroate synthase DHPS and prevents addition of para-aminobenzoic acid PABA , inhibiting folic acid synthesis.

ß-lactams Examples: penicillin, ampicillin, mezlocillin, peperacillin, cefazolin, cefotaxime, ceftazidime, aztreonam, imipenem. Destruction of ß -lactam rings by ß -lactamase enzymes. With the ß-lactam ring destroyed, the antibiotic will no longer have the ability to bind to PBP penicillin-binding protein , and interfere with cell wall syntheses.

Resistance of staphylococi to penicillin; resistance of Enterobacteriaceae to penicillins, cephalosporins, and aztreonam.

Changes in penicillin binding proteins. Mutational changes in original PBPs or acquisition of different PBPs will lead to inability of the antibiotic to bind to the PBP and inhibit cell wall synthesis.

Porcin channel formation is decreased. Since this is where ß-lactams cross the outer membrane to reach the PBP of Gram-negative bacteria, a change in the number or character of these channels can reduce ß-lactam uptake.

Alteration in the molecular structure of cell wall precursor components decreases binding of vancomycin so that cell wall synthesis is able to continue. Aminoglyosides Examples: gentamicin, tobramycin, amikacin, netilmicin, streptomycin, kanamycin.

Modifying enzymes alter various sites on the aminoglycoside molecule so that the ability of this drug to bind the ribosome and halt protein synthesis is greatly diminished or lost entirely.

Change in number or character of porin channels through which aminoglycosides cross the outer membrane to reach the ribosomes of gram-negative bacteria so that aminoglycoside uptake is diminished. Modification of ribosomal proteins or of 16s rRNA. This reduces the ability of aminoglycoside to successfully bind and inhibit protein synthesis.

Quinolones Examples: ciprofloxacin, levofloxacin, norfloxacin, lomefloxacin. Changes in DNA gyrase subunits decrease the ability of quinolones to bind this enzyme and interfere with DNA processes. Lack of appropriate cell wall precursor target to allow vancomycin to bind and inhibit cell wall synthesis.

Lack of uptake resulting from inability of antibiotics to achieve effective intracellular concentrations. Now it's time to Check Your Understanding of your comprehension of this section. Via acquisition of mecA genes which is on a mobile genetic element called "staphylococcal cassette chromosome" SCCmec which codes for penicillin binding proteins PBPs that are not sensitive to ß-lactam inhibition.

Via acquisition of one of two related gene clusters VanA and VanB, which code for enzymes that modify peptidoglycan precursor, reducing affinity to vancomycin.

Antimicrovial disrupt essential structures or processes in Sports training adaptations. This in turn either kills the bacteria or stops them Antimicrobial protection mechanisms multiplying. Bacteria have in turn Antimicrobial protection mechanisms Antimicrobiak antibiotic Antimicrobkal mechanisms to withstand the actions of antibiotics. Over time bacteria have evolved many different antibiotic resistance strategies to accomplish this. Some bacteria are naturally resistant to certain antibiotics. Imagine for example an antibiotic that destroys the cell wall of the bacteria. If a bacterium does not have a cell wall, the antibiotic will have no effect. Protetion Antimicrobial protection mechanisms antibiotic Cellulite reduction massages at home Antimicrobial protection mechanisms bacteria poses a serious public health challenge worldwide. However, antibiotic resistance genes are mechanjsms confined to the clinic; instead they are widely Antimicrobial protection mechanisms in different bacterial populations in the environment. Therefore, to understand development of antibiotic resistance Antimixrobial pathogens, mechaniisms need to consider important reservoirs of resistance genes, which may include determinants that confer self-resistance in antibiotic producing soil bacteria and genes encoding intrinsic resistance mechanisms present in all or most non-producer environmental bacteria. While the presence of resistance determinants in soil and environmental bacteria does not pose a threat to human health, their mobilization to new hosts and their expression under different contexts, for example their transfer to plasmids and integrons in pathogenic bacteria, can translate into a problem of huge proportions, as discussed in this review. Selective pressure brought about by human activities further results in enrichment of such determinants in bacterial populations. Antimicrobial protection mechanisms

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