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Anti-microbial Resistance
- 1. ANTI MICROBIAL RESISTANCE KARANATAKA VETERINARY ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR VETERINARY COLLEGE HEBBAL, BENGALURU Submitted by, Rudresh Gowda B MVHK1842 Jr. M. V. Sc(Pharma)
- 3. Antimicrobial Resistance • Microorganisms, not inhibited by usually achievable systemic concentration of an Antimicrobial agent with normal dosage schedule fall in MIC range (WHO) • A species- subjected to Chemical warfare, threatens its extinction; evolves mechanisms to survive under stress -> Development of resistance • “Survival of the fittest” - immense genetic plasticity of bacterial pathogens • Evolution and Clinical/ Environmental practices
- 4. Statistics • Annual deaths- anticipated to rise to 10 million worldwide by 2050 • Two million deaths are projected to occur in India due to AMR by the year 2050 • Annually, >50,000 newborns estimated to die from sepsis due to pathogens resistant to first-line antibiotics • India spends only 4.7% of its total Gross Domestic Product on health, with government share only one-fourth (1.15%) • 33000 people die each year- direct consequence of an Antimicrobial resistance (ECDC., 2018) • GLASS- being developed to support global action plan on antimicrobial resistance Avika et al., 2019
- 5. (Nat Chem Bio., 2019)
- 6. DRUG RELATED FACTORS Over the counter availability of Drugs Counterfeit and substandard drug causing suboptimal blood concentration Irrational and non judicial use of Antimicrobials PATIENT RELATED FACTORS _ Poor adherence of dosage Regimens _ Poverty _ Lack of Education _ Self Medication PRESCRIBER RELATED FACTORS _ Inappropriate use of Antimicrobials _ Increased empirical poly-antimicrobial use _ Lack of current knowledge and training ENVIRONMENTAL FACTORS _Huge population and overcrowding _Poor Sanitation _Increase in community acquired resistance _Release of large quantities of antibiotics into environment Factors affecting Antimicrobial Resistance
- 7. • Lack of affinity of drug for bacterial target • Inaccessibility of drug into bacterial cell • Extrusion of drug by chromosomally encoded active exporters • Innate production of enzymes that inactivate drug Intrinsic Resistance (Dever., 1991)
- 8. ACQUIRED RESISTANCE Biochemical Mechanisms Production of Antibiotic inactivating enzymes Preventing drug accumulation within bacterium Modifying /protecting target site Use of alternating pathways for Metabolism/Growth Biofilms Genetic Methods # Chromosomal Methods- Mutations # Extra-Chromosomal Methods- Plasmids - Transfer of r-Genes from one Bacterium to another- ϴ Conjugation ϴ Transduction ϴ Transformation - Transfer of r-Genes between plasmids within the bacterium- ϴ Transposons ϴ Integrons
- 9. Mutation • Stable and heritable gene change, occurs spontaneously and randomly among microorganisms • Insertion- Deletion- Substitution of one or more nucleotides • Occurs once in 10^8 cell divisions • Single step Mutation • Multistep Mutation
- 10. Inactivation of Antibiotics by enzymes • Subclasses of enzymes can degrade different antibiotics within same class (β-lactams and β-lactamase) • KPC, IMP, VIM, OXA, NDM • bla CTX M-15, bla NDM 1 (Blair et al., 2015)
- 11. Inactivation of Antibiotics by transfer of Chemical group • Chemical groups to vulnerable sites on antibiotic molecule by bacterial enzymes • Acyl, phosphate, nucleotidyl and ribitoyl groups • Aminoglycoside- AAC, ANT, APH • Rifamycin- RAE (Munita et al, 2016) (Blair et al., 2015)
- 12. Reduced permeability • Prevent antibiotic reaching its intracellular/ periplasmic target by decreasing uptake of antimicrobial molecule (usually G-ve bacteria) • Hydrophilic molecules- affected by changes in permeability of outer membrane • i) Shift in type of porins expression • ii)Change in level of porin expression • iii)Impairment of porin function • Aberrant production of OprD in P. aeruginosa • OmpF and OmpC in E.Coli (Munita et al., 2016)
- 13. Bacterial Efflux Pumps • Cell membrane protein channel selectively admits or excludes chemicals from cytoplasm • Transport specific substrates, many are transporters of multiple substrates • tet(K) and tet(L)– Tetracyclines (MFS) • mefA and mefE– Macrolides • AcrAB- TolC – Enterobacteriaceae (RND) • MexAB- OprM – P. aeruginosa • MsrA – Staphylococcus epidermidis (ABC) (Xian-zhi et al., 2015)
- 14. (Munita et al, 2016)
- 15. Modifying /protecting target site • erm genes – Macrolides (MLSB) resistance by mono or di-methylating an adenine residue on 16S rRNA • cfr gene – Phenicols, Lincosamides, Linezolid (S. aureus) • armA gene – Aminoglycosides (Blair et al.,2015)
- 16. Changes in antibiotic targets by mutation • Changes to target structure- prevent efficient antibiotic binding, still enable target to carry out its normal function, confers resistance • Single point mutation • Fluroquinolones - gyrA-gyrB Rifamycin – binding site at β subunit of RNA polymerase (rpoB), interrupts transcription High level Resistance- mutation results in Amino Acid substitutions in rpoB gene (Munita et al., 2016)
- 17. Use of alternating pathways for Metabolism/Growth • Evolving new targets with similar biochemical functions original target, not inhibited by antimicrobial molecule • Methicillin resistance in S. aureus - acquisition of exogenous PBP (PBP2a gene) • Vancomycin resistance in enterococci- peptidoglycan structure modification by van gene clusters • Trimethoprim- Sulphamethaxazole - Overproduction of DHFR or DHPS through mutation (Munita et al., 2016)
- 18. Biofilms • Surface-attached groups of microbial cells encased in an extracellular matrix, significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells • Microorganisms synthesize and secrete- protective matrix attaches biofilm living or non-living surface • Polymicrobial biofilms- cooperative protective effects- Protection for neighbouring non-resistant bacteria by transfer genes to other bacteria • High cell density in biofilms-increases absolute numbers of resistant mutants • Hibernation (Dennis Scott, 2013)
- 21. Superbugs
- 22. Conjugation • Transfer of chromosomal or extrachromosomal DNA from living donor bacterium to living recipient bacterium by cell-to-cell contact • Conjugative plasmid- Self-transmissible • Opportunistic G-ve infections- Urinary tract infections, wound infections, pneumonia, and septicemia E. coli, Proteus, Klebsiella, Enterobacter, Serratia, Pseudomonas (Gary kaiser, 2019) • Transfer of r-Genes from one Bacterium to another
- 24. Transduction • Transfer of enclosed plasmid DNA from one bacterium to another by a bacteriophage • Accidently phage capsid assembles around small fragment of bacterial DNA, transducing particle - injects the fragment Staphylococcus, Streptococus, Escherichia, Salmonella, Pseudomonas (Gary kaiser, 2019)
- 26. Transformation • Uptake & Incorporation of DNA fragment of dead, degraded bacterium to competent recipient bacterium • Homologous recombination, involves similar bacterial strains or strains of same bacterial species N. gonorrhoeae, N. meningitidis, H. influenzae, L. pneomophila, S. pneumoniae, H. pylori (Gary kaiser, 2019)
- 28. Transposons (Jumping genes) • DNA sequence- can change its relative position within genome, creating/reversing mutations and altering cell's genetic identity and genome size • One and twelve genes long • Unable to replicate independently • Hitch- hike (Gary kaiser, 2019) • Transfer of r-Genes between plasmids within bacterium Barbara McClintok
- 31. Gene Cassette and Integrons • Gene Cassette- Type of mobile genetic element- gene and recombination site • Integrons- Large mobile Genetic structures in bacteria- express and capable of acquiring and exchanging gene cassette • kanMX cassette- kanamycin (an antibiotic) resistance upon bacteria
- 33. Anthelmintic resistance • Benzimidazoles- Mutation at amino acid 200 in β-tubulin isotype 1 • Macrocyclic Lactones- Structural alteration in LGIC subunit a Decreased expression of subunit c (Whittaker et al., 2017)
- 34. • Nicotinic Agonists– Down regulation of nAchE receptors Decreased affinity of these receptors for drug (Whittaker et al., 2017)
- 35. Antifungal resistance Amphotericin B Azoles. . Triazoles Echinocandins 5-Flucytosine (Jose et al., 2016)
- 36. Antiviral resistance • Cytomegalovirus – Ganciclovir Mutations in viral UL97 kinase gene • Varicella Zoster Virus – Acyclovir and Penciclovir Tyrosine Kinase mutations • Herpes Simplex Virus - Viral Tyrosine Kinase Cidofovir DNA polymerase mutations (Strasfeld, 2010)
- 37. Strategies to combat Antimicrobial Resistance • Locally-developed customized antibiotic guidelines and clinical pathways • Enhance infection prevention and control • Support surveillance of antimicrobial resistance and antimicrobial consumption • Control the source of infection • Prescribe antimicrobial only when they truly required • Prescribe appropriate antimicrobial(s) with adequate dosages • Control the source of infection • Reassess treatment when culture results available • Use of shortest duration of antimicrobials • Educating staff how to use antibiotics wisely
- 38. References • JULIAN DAVIES and DOROTHY DAVIES, 2010, Origins and Evolution of Antibiotic Resistance, Micro. and molecular bio. reviews, 74( 3): 417-433. • DEVER. L, 1991, Mechanisms of bacterial resistance to antibiotics, Archives of Int. Med; 151(5): 886-895. • ALANA GYEM, 2015, "Cut-and-Paste" mechanism of transposition. • XIAN-ZHI LI, PATRICK PLÉSIAT, HIROSHI NIKAIDO, 2015, The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria, Ame. Soc for Micro; 28(2): 340. • JOSE M. MUNITAAND CESAR A. ARIAS, 2015, Mechanisms of Antibiotic Resistance; Microbiol Spectr, 4(2): 1-17. • DENNIS SCOTT, 2013, The Mechanics of Antibiotic Resistance, 1-18. • JOHN H. WHITTAKER, STEVEN A. CARLSON, MATTHEW T BREWER, 2017, Molecular mechanisms for anthelmintic resistance in strongyle nematode parasites of veterinary importance. • S. GEERTS and B. GRYSEELS, 2000, Drug Resistance in Human Helminths:Current Situation and Lessons from Livestock, Cli. micro rev, 13 (2): 213-214.
- 39. • JOSE ANTONIO REALES CALDERÓN, GLORIA MOLERO, CONCHA GIL, JOSÉ L MARTÍNEZ, 2016, The fungal resistome: a risk and an opportunity for the development of novel antifungal therapies; Future medicinal chemistry, 8 (12). • https://infectionsinsurgery.org/global-alliance-for-infections-in-surgery-best-practices-to-combat- antimicrobial-resistance-in-surgery-2. • LYNNE STRASFELD, SUNWEN CHOU, 2010. Antiviral Drug Resistance: Mechanisms and Clinical Implications. Infect Dis Clin. North Ame., 24(2): 413–437. • DR. GARY KAISER, 2019, Horizontal Gene Transfer in Bacteria, Microbiology • AVIKA, D., NEETA KUMAR, SANJIV KUMAR and VIDYASAGAR TRIGUN, Antimicrobial resistance: Progress in the decade since emergence of New Delhi metallo-β-lactamase in India. Indian. J. Community. Med., 44 (1): 4-8
- 40. Thank You...
Editor's Notes
- 1. mycoplasma- penicillins
- enzyme catalysed modification of antibiotics is a major mechanism of antibiotic resistance that has been relevant since the first use of antibiotics, with the discovery of penicillinase (a β-lactamase), Thousands of enzymes have since been identified that can degrade and modify antibiotics of different classes, including β-lactams, aminoglycosides, phenicols and macrolides 2. such as penicillins, cephalosporins, clavams, carbapenems and monobactams, are hydrolysed 3. IMP (imipenemase), VIM (Verona integron encoded metallo β-lactamase), K. pneumoniae carbapenemase (KPC), OXA (oxacillinase) and NDM enzymes in Gram-negative bacteria such as K. pneumoniae, E. coli, P. aeruginosaand A. baumannii, has underpinned the emergence of isolates that are resistant to all β-lactam antibiotic 4. causes antibiotic resistance by preventing the antibiotic from binding to its target protein as a result of steric hindrance
- rifamycin phosphotransferase that is associated with the RAE. (rif-associated-element rifamycin-resistance genes in Actinomycetes Chloromphenicol transferase
- Ex.1 vancomycin, a glycopeptide antibiotic, is not active against gram-negative organisms due to the lack of penetration through the outer membrane 2 innate low susceptibility of Pseudomonas to β-lactams (compared to Enterobacteriaceae 3 OprD- uptake of basic amino acids and imipinems 4 Shift in porin expression from OmpK35 to OmpK36 K.Pneumonea
- ___production of complex bacterial machineries capable to extrude a toxic compound out of the cell can also result in antimicrobial resistance The influx of drugs (shown as pills) through the OM occurs in one or more of the following three pathways: porin channels, specific protein channels, and the LPS-containing asymmetric lipid bilayer region. After their entry into the periplasmic space, the drug molecules can further penetrate the IM via diffusion. However, these drugs can be extruded out of the cell by efflux transporters, which exist as either single-component pumps (“singlet”; e.g., Tet pumps) or multicomponent pumps (e.g., AcrAB-TolC and MexAB-OprM tripartite efflux systems that each typically contain a pump, an OM channel protein [OMP], and an accessory membrane fusion protein [MFP]). While the singlet pumps may take up the drug from the cytosol and the periplasm and function with porins or other types of protein channels to make the efflux process effective, the multicomponent exporters capture their substrates from the periplasm and the IM and directly pump them into the medium. Lipophilic drug molecules cross OM slowly or hydrophilic drugs penetrate by slow porins, the efflux mechanism become very effective In contrast, with the less hydrophobic and smaller drug molecules that can rapidly penetrate, for example, E. coli porins, efflux is not effective to count
- small multidrug resistance ATP-binding cassette major facilitator superfamily multidrug and toxic-compound extrusion resistance nodulation division
- erm (erythromycin ribosomal methylase), which results in macrolide resistance. These enzymes are capable of mono- or dimethylating an adenine residue in position on chloramphenicol–florfenicol resistance (cfr) methyltransferase, which specifically methylates A2503 in the 23S rRNA PRPs), which bind to and protect topoisomerase IV and DNA gyrase from the lethal action of quinolones
- RIF is a rifamycin that blocks bacterial transcription by inhibiting the DNA-dependent RNA polymerase, which is a complex enzyme with a α2ββ’σ subunit structure RIF binding pocket is a highly conserved structure located in the βsubunit of the RNA polymerase (encoded by rpoB), and after binding, the antibiotic molecule interrupts transcription by directly blocking the path of the nascent RNA .High-level RIF resistance has been shown to occur by single-step point mutations resulting in amino acid substitutions in the rpoBgene FQs kill bacteria by altering DNA replication through the inhibition of two crucial enzymes, DNA gyrase and topoisomerase IV. Development of chromosomal mutations in the genes encoding subunits of the abovementioned enzymes (gyrA-gyrBand parC-parEfor DNA gyrase and topoisomerase IV, respectively) is the most frequent mechanism of acquired resistance to these compounds.
- The synthetic pathway of folate involves two major enzymes, namely i)dihydropteroic acid synthase (DHPS), which forms dihydrofolate from paraaminobenzoic acid (inhibited by SMX), and ii)dihydrofolate reductase (DHFR), which catalyzes the formation of tetrahydrofolate from dihydrofolate(inhibited by TMP). overproduction of DHFR or DHPS through mutations in the promoter region of the DNA encoding these enzymes. These mutations result in the production of increased quantities of the above enzymes, “overwhelming” the ability of TMP-SMX to inhibit folate production and permitting bacterial survival.
- Attachment Colony formation Biofilm formation Growth Release Chronic infections in biofilms have been demonstrated to be involved include periodontitis, cystic fibrosis pneumonia, and numerous infections associated with indwelling devices such as catheters, heart valves, and prostheses.
- . Schematic overview of the major antimicrobial resistance and tolerance mechanisms employed by bacterial biofilms. Biofilm cells (yellow rectangles) are embedded in a mushroom-shaped matrix (shown in green). The biofilm is attached to a surface (grey rectangle), which can be biotic or abiotic. Pictorial representations of the resistance mechanisms are numbered as follows: (1) nutrient gradient (demonstrated here as a colour-intensity gradient) with less nutrient availability in the core of the biofilm, (2) matrix exopolysaccharides, (3) extracellular DNA, (4) stress responses (oxidative stress response, stringent response, etc.), (5) discrete genetic determinants that are specifically expressed in biofilms and whose gene products act to reduce biofilm susceptibility via diverse mechanisms (ndvB, brlR, etc.), (6) multidrug efflux pumps, (7) intercellular interactions (horizontal gene transfer, quorum sensing, multispecies communication, etc.) and (8) persister cell
- 1.Genetic recombination 2. in that it possesses all the necessary genes for that plasmid to transmit itself to another bacterium by conjugation
- Step 1: In Gram-negative bacteria, the first step in conjugation involves a conjugation pilus (sex pilus or F pilus) on the donor bacterium binding to a recipient bacterium lacking a conjugation pilus. Step 2: Typically the conjugation pilus retracts or depolymerizes pulling the two bacteria together. A series of membrane proteins coded for by the conjugative plasmid then forms a bridge and an opening between the two bacteria, now called a mating pair. Step 3: Using the rolling circle model of DNA replication, a nuclease breaks one strand of the plasmid DNA at the origin of transfer site (oriT) of the plasmid. The nuclease also has helicase activity and unwinds the strand that is going to be transferred. Step 4: The nicked plasmid strand enters the recipient bacterium. The other strand remains behind in the donor cell. Step 5: Both the donor and the recipient plasmid strands then make a complementary copy of themselves. Step 6: Both bacteria now possess the conjugative plasmid and can make a conjugation pilus
- During the replication of lytic bacteriophages and temperate bacteriophages, occasionally the phage capsid accidently assembles around a small fragment of bacterial DNA-Transducing Particle
- Step 1: A bacteriophage adsorbs to a susceptible bacterium. Step 2: The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes. Bacteriophage-coded enzymes will also breakup the bacterial chromosome. Step 3: Occasionally, a bacteriophage capsid mistakenly assembles around either a fragment of the donor bacterium's chromosome or around a plasmid instead of around a phage genome. Step 4: The bacteriophages are released as the bacterium is lysed. Note that one bacteriophage is carrying a fragment of the donor bacterium's DNA rather than a bacteriophage genome. Step 5: The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium. Step 6: The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium. Step 7: Homologous recombination occurs and the donor bacterium's DNA is exchanged for some of the recipient's DNA. (Figure 3.1.13.1.1 shows the functions of the RecA proteins involved in homologous recombination.)
- 2.a recombination of homologous DNA regions having nearly the same nucleotide sequences-- Competent bacteria are able to bind much more DNA than noncompetent bacteria
- Step 1: A donor bacterium dies and is degraded.Step 2: DNA fragments, typically around 10 genes long, from the dead donor bacterium bind totransformasomes on the cell wall of a competent, living recipient bacterium.Step 3: In this example, a nuclease degrades one strand of the donor fragment and the remaining DNA strand enters the recipient. Competence-specific single-stranded DNA-binding proteins bind to the donor DNA strand to prevent it from being degraded in the cytoplasm. Step 4: RecA proteins promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA (see Figure 3.1.13.1.1 for the functions of RecA proteins). This involves breakage and reunion of paired DNA segments. Step 5: Transformation is complete.
- Transposition often results in duplication of the same genetic material T r ansposons may c ar r y one or mor e r esist anc e genes ( see below) and c an ' hit c h- hik e' on a plasmid t o a new spec ies of bac t er ium
- 2. differences in the chemical nature of the drug and the microbial membrane structures especially for those that require entry into the microbial cell in order to affect their action
- The structure consists of an integrase (Int) with the Pint and PC promoters in the 3′ end of the gene, with its associated cassette attachment or insertion site (attI). The integrase catalyzes the sequential recombination of circularized gene cassettes into the distal attachment site to create an operon-like arrangement (ant1r, ant2r, and so on) of r genes transcribed from the strong PC promoter. Three classes of integrons have been identified that differ in their integrase genes.
- BZ exert their anthelmintic activity by binding tob-tubulin, which interferes with the polymerisation of the microtubuli. Phe being replaced by Tyr (a) Illustration depicting the disruption of tubulin heterodimer treadmilling through the binding of BZ molecules to the b-tubulin isotype-1 subunit. The disruption of tubulin–microtubule equilibrium disrupts multiple cellular processes. (b) A structural alteration resulting from a SNP in the b-tubulin subunit confers reduced binding affinity by BZ molecules. Tubulin–microtubule equilibrium is not disrupted. (a) Illustration depicting 3 of 5 channel subunits (a,b,c) of an LGIC permanently activated through the allosteric binding of ML molecules. A sustained intracellular flux of Cl hyperpolarizes the nematode neuromuscular system. Subunits a and c are sensitive to ML binding, subunit b is not. (b) A structural alteration (SNP) in LGIC subunit a and the decreased expression of subunit c confer reduced binding affinity by ML molecules and reduced susceptibility to LGIC activation. The LGIC is only activated through normal ligand binding (not bound in figure) in the orthosteric site.
- (a) Illustration depicting a heteromeric nAch receptor composed of two alpha and three non-alpha nAchR subunits that is activated through NA binding at agonist binding sites between adjacent alpha and non-alpha subunits. A sustained intracellular cation flux depolarizes the nematode neuromuscular system. (b) Arrangement of nAchR subunits and agonist binding sites of NA sensitive receptor. (c) Changes in expression levels and structural alterations (truncated nAchR subunit) in nAchR subunits reduces sensitivity to NA molecules. nAchR is not activated. (d) Arrangement of nAchR subunits of receptor with reduced NA sensitivity. NA molecules are not bound to NA binding sites.
- Amphotericin B resistance is caused by the alteration of the lipid composition (ergosterol) of the cytoplasmic membrane, reducing the affinity of the antifungal for the fungal membrane. 2) Efflux pump overexpression is the most common mechanism of resistance to azoles in the entire fungal kingdom; the overexpression of the ATP-binding cassette (ABC) efflux pumps Cdr1 and Cdr2 and the major facilitator superfamily (MSF) efflux pump Mdr1, lead to azole resistance in different Candida species. The overexpression of Afr1 and Mdr1 efflux pumps is also important in Cryptococcus neoformans for azole resistance. In Aspergillus fumigatus Mdr1, Mdr3, Mdr4 and Atrf efflux pumps are very important as well. 3) Overexpression of ERG11, also known as lanosterol 14-alpha-demethylase, or their mutations in the drug-binding domain of triazoles, confer resistance to azoles in a wide number of fungal species. Aneuploidy of ERG3 or its mutation also confers resistance to azoles. The most common resistance mechanism to echinocandins is the mutation in the catalytic domain of the (1,3)-β-D-glucan synthase (FKS1) in C. albicans, C. neoformans, and A. fumigatus and of FKS1 and FKS2 in S. cerevisiae). 6) The loss of the 5-Flucytosine permease in the cell membrane and the impaired activity in the cytosine deaminase, observed in C. neoformans and C. glabrata, confers resistance to 5-Fluorocytosine. The mutations in FUR1 are responsible for the resistance in some C. albicans strains.
- the phosphorylation of ganciclovir without impairing the important functions of this kinase in viral replication TK mutations, which often result in a premature stop codon that makes the virus TK Deficient TK mutations may result in either a loss of TK activity (TK deleted or deficient virus) or, less commonly, an alteration in TK substrate specificity (TK altered virus)
- Following locally-developed customized antibiotic guidelines and clinical pathways Supporting and enhancing infection prevention and control including correct hand hygiene protocols Supporting and enhancing surveillance of antibiotic resistance and antibiotic consumption Prescribing and dispensing antibiotics only when they are truly required Identifying and controlling the source of infection Prescribing appropriate antibiotics(s) with adequate dosages i.e. administration of antibiotics according to pharmacokinetic-pharmacodynamic principles Reassessing treatment when culture results are available Using the shortest duration of antibiotics based on evidence Educating staff how to use antibiotics wisely