Antibiotic resistance

Antibiotic resistance pattern : 

Antibiotic resistance pattern -Varun C N

Slide 2: 

Varun C N 2 Declaration All the materials that is shown in the slide has been taken from various sources (Which has been sited as appropriate) . The Non cited materials have been taken from public domains. None of them involves any of my work and hence I don’t claim any intellectual property rights over any of them. The material has been prepared only for educational purpose and I don’t take any personal responsibility for the matter produced. I have however taken maximum care to make data as reliable as possible Varun C N Feb 2012

Slide 3: 

Wattal et al,Microbiology Newsletter 1999-2008;www.sgrh.com 3 Varun C N

Slide 4: 

MRSE = methicillin-resistant Staphylococcus epidermidis, MRSA = methicillin-resistant Staphylococcus aureus, NPSP = non-penicillin-susceptible pneumococci, VRE = vancomycin-resistant enterococci, VISA = vancomycin-intermediate S. aureus. http://www.medscape.com/viewarticle/4126474 4 Varun C N

Slide 5: 

5 Varun C N

Slide 6: 

Sanjay Kumar Mallick and Silpi Basak, MRSA – too many hurdles to overcome: a study from Central India. Trop Doct April 2010 vol. 40 no. 2 108-110 6 Varun C N

Slide 7: 

A Manoharan. Correlation of TEM, SHV and CTX-M extended-spectrum beta lactamases among Enterobacteriaceae with their in vitro antimicrobial susceptibility. IJMM. Year : 2011  |  Volume : 29  |  Issue : 2  |  Page : 161-164 7 Varun C N

Slide 8: 

T- D Huang etal. Rapid emergence of carbapenemase-producing enterobacteriaceae isolates in belgium. Eurosurveillance, Volume 16, Issue 26, 30 June 2011 8 Varun C N

Slide 9: 

9 Varun C N

Slide 10: 

S John, R Balagurunathan. Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii. Year : 2011 | Volume : 29 | Issue : 3 | Page : 302-304 10 Varun C N

Slide 11: 

Mahesh E, Community-acquired urinary tract infection in the elderly. BJMP 2011;4(1):a406 11 Varun C N

Slide 12: 

Frequency of ESBL-producers among K. pneumoniae isolates (January 2004-August 2006), by country JAC 2007 R R Reinert, D Low, F Rossi, X J Zhang, C Wattal, M Dowzicky .

Slide 13: 

13 Varun C N All the data shows you that antibiotic resistance has increased overtime Gram +ve bacteria Major problems…. MRSA Penicillin R pneumococci Vancomycin-R enterococci But Many new antibiotics Gram -ve bacteria Rising problems…. ESBL producers Pan R Acinetobacter & P. aeruginosa And - Few new antibiotics

Slide 14: 

Time period Resistant organism Sensitive organism % of organism Use of Antibiotics 14 Varun C N

Slide 15: 

Varun C N 15 Intrinsic mechanism: Impermeability to the drug Absence of target site Lack of enzymes to convert the Pro drug to the active form Extrinsic mechanism Alteration in anti-microbial target Reduction or nullifying cell permeability to drug penetration Decreased uptake of antibiotic by expression of efflux pumps Production of an enzyme that inactivates the drug Production of an alternate pathway that bypass the action of drug Biofilm formation Passive Resistance

Slide 16: 

Varun C N 16 Resistance to β-lactam antibiotics β-lactamase mediated destruction of the β-lactam ring. Failure to achieve optimum concentration of drug at the site of action Low affinity binding of antibiotic to the Penicillin binding proteins (PBP) Overproduction of PBP

Slide 17: 

Varun C N 17 http://www.ricercaitaliana.it/prin/dettaglio_completo_prin_en-2004038004.htm

Slide 18: 

Varun C N 18 Taken from http://lawrencekok.blogspot.com/2011/05/ib-biology-ib-chemistry-on-antibiotics.html

Slide 19: 

Varun C N 19

ß-Lactamases: Classification ( Ambler) : 

Serine enzymes Metallo (Zn) enzymes Group C Group A Group D Group B AmpC TEM / SHV /CTX-M OXA IMP/VIM Bush. Rev Inf Dis 1987;10:681; Bush et al. Antimicrob Agents Chemother 1995;39:12; Bush. Curr Opin Investig Drugs 2002;3:1284 ß-Lactamases: Classification ( Ambler) ESBLs

Slide 21: 

Varun C N 21 Taken from http://www.wales.nhs.uk/sites3/page.cfm?orgid=379&pid=13508 ESBL detection by Double disc synergy test

Slide 22: 

P / D Effects Cefotaxime-clavulanate 61% Ceftazidime-clavulanate 20% Cefepime-clavulanate 85% Varun C N 22 Deformity effect JAC(2004)54(1):134-138 Phantom effect

Slide 23: 

Varun C N 23 Results of the cefoxitin– BA method for the detection of AmpC production. A pure AmpC-producing isolate showing cefoxitin (FOX) zone enhancement of ≥5 mm with the addition of BA Shoorashetty etal. Comparison of the boronic acid disk potentiation test and cefepime-clavulanic acid method for the detection of ESBL producing Enterobacteriaceae. Indian J Med Microbiol 2011; 29:297-301. AmpC detection

Slide 24: 

Varun C N 24 Lawn of bacterial culture Sterile disc with test org Cefoxitin disc Detection of AmpC by cefoxitin disc method Mohamudha Parveen R.; Harish B.N.; Parija S.C. AmpC beta lactamases among Gram negative clinical isolates from a tertiary hospital, South India Braz. J. Microbiol. vol.41 no.3

Slide 25: 

Varun C N 25 CAM assay. AmpC-positive extracts produce a zone of growth around wells. M3D assay. AmpC-positive extracts distort the zone around the cefoxitin disk. Nasim etal. New Method for Laboratory Detection of AmpC β-Lactamases in Escherichia coli and Klebsiella pneumoniae J. Clin. Microbiol. October 2004 vol. 42 no. 10 4799-4802

Slide 26: 

Varun C N 26 A Guide to Performing the Modified Hodge Test Using Microbiologics® . User manual Metallo β-lactamase

Slide 27: 

Inhibitory effects of 2-mercaptopropionic acid (2-MPA) on IMP-1 producers and non–IMP-1 producers. Arakawa Y et al. J. Clin. Microbiol. 2000;38:40-43 Inhibitory effects of 2-mercaptopropionic acid (2-MPA) on IMP-1 producers and non–IMP-1 producers. Three CAZ-resistant strains belonging to the gram-negative bacterial species P. aeruginosa, S. marcescens, and K. pneumoniaeand producing IMP-1 metallo-β-lactamase or serine-β-lactamases (SHV-12 or AmpC) were tested. For each IMP-1 producer, a distinct growth-inhibitory zone appeared between the KB disk containing CAZ and the filter disk containing 2-MPA (left column). No change is evident around the two KB disks containing CAZ with or without 2-MPA for each serine β-lactamase producer (right column).

Slide 28: 

Varun C N 28 S John and Balagurunathan R. Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumanii; IJMM 2011, 29 (3): 302-4

Slide 29: 

Varun C N 29 Imipenem disc Imipenem with EDTA Combined disc test for MBLs

Slide 30: 

The Efflux Pump Recognizes and Pumps out Fluroquinolones / Meropenem Environment Cytoplasm Porin Efflux System Pump (MexB) Efflux System Exit Portal (OprM) Linker Lipoprotein

Slide 31: 

Pseudomonas develops resistance to FQs by increasing the number of efflux pumps Environment Cytoplasm Porin Efflux System Pump (MexB) Efflux System Exit Portal (OprM) Linker Lipoprotein Subsequent PA can acquire resistance to Meropenem fluoroquinolones, penicillins, cephalosporins, macrolides and sulphonamides.

Slide 32: 

The Efflux Pump does not recognize Imipenem because of its unique hydroxyl side-chain Environment Cytoplasm Porin Efflux System Pump (MexB) Efflux System Exit Portal (OprM) Linker Lipoprotein

Slide 33: 

Increasing the number of Efflux Pumps also does not increase resistance to Imipenem Environment Cytoplasm Porin Efflux System Pump (MexB) Efflux System Exit Portal (OprM) Linker Lipoprotein

Slide 34: 

Varun C N 34 Resistance to Aminoglycosides The aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc.) bind irreversibly to the 16S rRNA in the 30S subunit of bacterial ribosomes. It has been proposed that some aminoglycosides prevent the transfer of the peptidyl tRNA from the A-site to the P-site, thus preventing the elongation of the polypeptide chain.

Slide 35: 

Varun C N 35 Fig: Action of enzymes that cause resistance to aminoglycoside antibiotics. A, B, and C refer to different chemical groups that can be added by the bacteria to inactivate the antibiotic.

Slide 36: 

Varun C N 36 Resistance to aminoglycosides is by Enzymatic modification AAC (6’) - Mediates resistance to Tobramycin, Gentamycin, Netilimycin and Amikacin AAC (2’’) - Mediates resistance to Tobramycin, Gentamycin, Dibekacin resistance. Altered Ribosomal binding sites Single mutation of S12 protein present in 30S subunit of ribosome Altered aminoglycoside uptake

RESISTANCE TO MACROLIDES : 

RESISTANCE TO MACROLIDES The macrolides (erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, etc.) bind reversibly to the 23S rRNA in the 50S subunit.There is evidence that they may inhibit elongation of the protein by the peptidyltransferase, the enzyme that forms peptide bonds between the amino acids.

Slide 38: 

Varun C N 38 Mechanism of resistance  Production of enzyme that alter macrolide  Active efflux of antibiotic  Alteration of ribosomal target The 2 mechanism by which bacteria can alter their ribosomes and acquire resistance have been understood  High level resistance because of an altered protein component in domain V of 50S ribosome subunit. This is a single step mutation. E.g.: In M.avium, H.pylori  Plasmid mediated macrolide, lincosamide, streptogramin B (MLSB) resistance occurs when a single adenine residue within a conserved domain V of nascent 23s RNA of 50S ribosomal unit is methylated. The production of methylases is encoded by a class of genes referred as erm (Erythromycin ribosome methylation) of which more than 40 types are known.

Slide 39: 

Varun C N 39 There are 21 classes of erm known out of which 4 classes is common in pathogenic bacteria. Erm (A) - Seen in MRSA Erm (B) – Seen in streptococci and enterococci Erm (C)- Seen in MSSA Erm (F) - Usually seen in anaerobes *Erm stands for Erythromycin ribosome methylation Resistance mediated by erm can be constitutive or inducible. Bacteria with inducible MLSB resistance, on exposure to inactive methylases undergoes rearrangement that permit ribosome to translate methylases coding sequence. This cause methylation of A2058, which mediates resistance. Such resistance is usually of complex phenotype particularly with erm (B) genes. Constitutive production of methylases generally confers a high level of predictable cross resistance to all MLSB antibiotics.

Slide 40: 

Varun C N 40 Macrolide resistance associated with active efflux is caused by 2 classes of pumps. Plasmid mediated ABC (ATP- Binding Cassette) encoded by the msr (A) gene Protein of major facilitator subfamily (MFS) found in streptococci and enterococci are encoded by a mef (A) gene. A third mechanism is modification of macrolides, by enzymes such as Acetyltransferase, Esterase and Glycosylase which is found in many enterobacteriacae species.

Slide 41: 

Fig: Diagrammatic representation of the membrane topology of proton-driven drug pumps in Gram Negative Bacteria. Source:Biochem. J. (2003) 376, 313–338

Slide 42: 

Varun C N 42 Inducible MLSB isolate- D phenotype Inducible MLSB isolate- D+ phenotype Pal N, Sharma B, Sharma R, Vyas L. Detection of inducible clindamycin resistance among Staphylococcal isolates from different clinical specimens in western India. J Postgrad Med 2010;56:182-5 The D test detects inducible clindamycin resistance

Slide 43: 

Varun C N 43 Tetracyclines The tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.) block bacterial translation by binding reversibly to the 16S rRNA in the 30S subunit and distorting it in such a way that the anticodons of the charged tRNAs cannot align properly with the codons of the mRNA.

Slide 44: 

Varun C N 44 Resistance to tetracyclines: The mechanism described for tetracycline resistance is impaired influx or decreased efflux by an active transport pump  Tet (AE) efflux pump is seen in gram negative species which is resistant to all the older types. It is susceptible to Tigecycline.  Tet (K) pump is seen in staphylococci which confers resistance only to tetracycline  Tet (M) is seen in many gram positive organisms conferring resistance to older tetracyclines.

Slide 45: 

Varun C N 45 Tet (AE) efflux pump Tet (K) pump Tet (M)

Slide 46: 

Varun C N 46 Resistance to anti tubercular drugs * The Materials shown in this part is a direct copy of write up for seminar presentation.

Slide 47: 

Varun C N 47

Slide 48: 

Varun C N 48

Slide 49: 

Varun C N 49

Slide 50: 

Varun C N 50

Slide 51: 

Varun C N 51 Newer strategies Multiple drug therapy Phage therapy Newer Antibiotics such as halocins, sulfobolocins

Slide 52: 

Varun C N 52 http://www.nature.com/nbt/journal/v24/n12/fig_tab/nbt1206-1508_F1.html

Slide 53: 

Varun C N 53

Slide 54: 

Varun C N 54

Slide 55: 

Varun C N 55 www.pnas.orgcgidoi10.1073pnas.0404439101

Slide 56: 

Varun C N 56 Thank you for your kind attention You can also follow me at my blog. Varuncnmicro.blogspot.com