Antibiotic Resistance

JACK GERSTENMAIER DEPARTMENT OF PEDIATRICS

Introduction

Objectives
Mechanisms of action for commonly prescribed

antibiotics

Cell wall inhibitors penicillins, cephalosporins, vancomycin Protein synthesis disruption tetracycline, clindamycin, aminoglycosides DNA disruption metronidazole

Common mechanisms of antibiotic resistance Porin mutation Ezymatic deactivation Efflux pump Modification of binding site

Bacterial Cell Wall

Peptidoglycan composition of cell wall

History of Antibiotics

Cell Wall Inhibitors

-lactams Interfere with cell wall synthesis by preventing transpeptidation

Bind to transpeptidase enzymes preventing formation of peptide crosslinking between N-acetylmuramic acids (NAM) thus causing a weakened cell wall Cause cell lysis and death

Activate endogenous autolytic system of bacteria

Bactericidal

Penicillin
The transpeptidase enzyme

cleaves the terminal d-alanine and the amino group of the glycine then reacts with the penultimate d-alanine on a neighboring chain to produce crosslinking in the cell wall

The structural similarity

between the penicillins and dalanyl-d-alanine allows the antibiotics to act as inhibitors of the transpeptidase enzyme

Vancomycin
Vancomycin binds with

the D-ala-D-ala of the NAM-NAG peptides through hydrogen bonding This interaction prevents the transglycosylation forming the NAM-NAG complex

Protein Synthesis Inhibitors

Antibiotics bind to the ribosomal subunit preventing

the tRNA from binding with the mRNA during protein synthesis Tetracycline

Bind at 30s ribosomal subunit

Erythromycin Bind at 50s ribosomal subunit Clindamycin Bind at 50s ribosomal subunit bacteriostatic

Tetracycline
Entry of these agents into

susceptible organisms is mediated both by passive diffusion and by an energydependent transport protein mechanism unique to the bacterial inner cytoplasmic membrane Nonresistant strains concentrate the tetracyclines intracellularly. Binds reversibly to the 30S subunit blocking access of tRNA to the mRNA complex at the acceptor site

DNA inhibitor Metronidazole

Metronidazole is taken

into anaerobic cells then broken down into toxic metabolites These metabolites are then incorporated into DNA forming unstable DNA molecules

Porin Deletion/Mutation
Gram negative bacteria

have the capability to delete or change the porin size in their outer cell membrane This will prevent or decrease the uptake of antibiotics into the cell This is a general resistance mechanism, not specific to one molecule and can effectively prevent many antibiotics from entering a cell

Phenotypic and Genotypic Characterization of Enterobacteriaceae with Decreased Susceptibility to Carbapenems: Results from Large Hospital-Based Surveillance Studies in China

Study 49 Enterobacteriaceae isolates from a hospital

in China

26 K. pneumoniae, 8 E. coli, 10 Enterobacter cloacae, 2 Enterobacter aerogenes, and 3 Citrobacter freundii isolates Expression levels of the two corresponding major porins (OmpK35 and OmpK36 for K. pneumoniae and OmpF and OmpC for E. coli, E. cloacae, E. aerogenes, and C. freundii) were investigated 33 isolates had lost or lower levels of both major porins 12 isolates had lost or lower levels of one major porin Expression of both major porins was demonstrated in just 4 isolates studied

Qiwen Yang, et. al., Antimicrobial Agents and Chemotherapy, January 2010, p. 573-577, Vol. 54, No. 1

Enzymatic Inactivation
-Lactams

-Lactamase is found in both gram-positive and gram-negative bacteria

Gram negative bacteria -Lactamase is found in periplasmic space Gram positive bacteria -Lactamase is found outside cell

-Lactamase cleaves the -lactam ring causing inactivity of the antibiotic Clavulanic acid competes for binding with the -Lactamase preventing breakdown of penicillin Enzyme modifies structure of antibiotic by adding phophoryl, adenyl, or acetyl groups Prevents uptake into cell

Aminoglycosides

Enzymatic Inactiveation

P. Gingivalis Resistance
A total of 69 isolates of A. actinomycetemcomitans

and P. gingivalis were collected from subgingival samples in chronic perio patients Selected colonies of A. actinomycetemcomitans and P. gingivalis were used to evaluate the antibacterial activity of clindamycin, metronidazole, amoxicillin, moxifloxacin and amoxicillin/clavulanic acid 21.56% P. gingivalis isolates were non-susceptible to metronidazole

Carlos M. Ardila , Med Oral Patol Oral Cir Bucal. 2010 Nov 1;15 (6):e947-51.

Efflux Pump
Efflux pump actively pumps antibiotic out of

bacterial cytoplasm

Tetracycline was one of the first antibiotics studied that a resistance mechanism of efflux was discovered. This is most commonly an active transport of Ab out of the cell Genes have been discovered for efflux pumps
tetA-tetG has are most commonly associated with gram-negative bacteria tetK, tetL are most commonly gram-positive bacteria Recently it has been discovered that there are gram-negative bacteria in which gene tetL has been discovered

M.C. Roberts / FEMS Microbiology Letters 245 (2005) 195203

Efflux Pump

Target Modification
Tetracyclines Ribosomal protection is encoded by at least 11 genes

Common genes for ribosomal protection include tetM, tetO, tetQ

When tetracycline binds to the ribosomal subunit it changes the conformation causing disruption of protein synthesis The genes encode for proteins that bind to proteins inside the ribosome causing a change in the confirmation of the tetracycline binding site thus causing release of the tetracycline molecule

M.C. Roberts / FEMS Microbiology Letters 245 (2005) 195203

Target Modification
-lactams

Binding specificity of PBP is altered preventing binding of antibiotic to bacteria Very common in gram-positive bacteria Vancomycin binding site has a lactate group added preventing binding of vancomycin however allowing continuation of cell wall synthesis

NATURE REVIEWS | MICROBIOLOGY VOLUME 5 | MARCH 2007

Macrolides

rRNA methylase methylates an adenine on rRNA preventing macrolides and lincomides from binding to the ribosome Found commonly in gram-positive cocci Study found that in addition to discovery of associated genes, oral streptococci has been shown to be erythromycin resistant

A. Villedieu et. al. Antimicrobial Agents and Chemotherapy, June 2004, Vol. 48, No. 6 p. 22982301

Genetics of Resistance
Transformation Naked DNA is taken up by bacteria which then can begin to exhibit trait associated with DNA Occurs within same species of bacteria Transduction Delivery of DNA via bacterial virus called a bacteriophage How A.A. picked up its virulence factor Occurs within same species of bacteria Conjugation Transfer through a pore formed in the membranes of two bacteria

Plasmid associated with bacterial resistance Transposons associated with tetracycline resistance and macrolides, lincosamide resistance Gram-positive to gram-negative and vise versa

Can occur between different species and between groups

Viridans group streptococci: a reservoir of resistant bacteria in oral cavities

Viridans streptococci resistance to erythromycin

decreased markedly after 1 week following treatment and continued to decrease for up to 3 months however, levels of resistance at 3 months were still higher than baseline Resistance profile of some species of viridans group streptococci can be a good indicator of risk for emergence of resistance to erythromycin by S. pyogenes or S. pneumoniae Transfer of resistance by conjugation of resistance from viridans strep to S. pneumoniae
A. Bryskier, Clin Microbiol Infect 2002; 8: 6569

Emergence of Antibiotic Resistant S. sanguis in Dental Plaque of Children After Frequent Antibiotic Therapy
50 children studied 25 experimental, 25 control matched for sex and age
Experimental children completed Ab cycle for otitis media Prescribed amoxicillin trimethoprim-sulfamethoxazole, or erythromycin Control group had no antibiotics for past 24 months

Supragingival plaque was collected from all children Gradient was established using THB agar Colonies with MICs higher than that typically expected were classified as resistant

Pamela R. Erickson, Mark C. Herzberg, Pediatr Dent. 1999;21:181-185

Emergence of Antibiotic Resistant S. sanguis in Dental Plaque of Children After Frequent Antibiotic Therapy
There was no evidence of resistance in the control group Antibiotic Resistance was observed in the experimental

group

60% of isolates demonstrated resistance to at least one drug

32% amoxicillin resistant 24% penicillin resistant

Approximately of these strains demonstrate resistance to more than one antibiotic

Resistance decreased with increased age and with time

since antibiotic treatment for amoxicillin and penicillins Resistance did not change with time for trimethoprimsulfamethoxazole or erythromycin
Pamela R. Erickson, Mark C. Herzberg, Pediatr Dent. 1999;21:181-185

Antimicrobial susceptibility of 800 anaerobic isolates from patients with dentoalveolar infection to 13 oral antibiotics
800 isolates were tested for susceptibility to 13

antibiotics

Prevotella species, Fusobacterium species, Porphyromonas species and Peptostreptococcus micros Tested against amoxicillin, augmentin, cefaclor, cefuroxime, cefcapene, cefdinir, erythromycin, azithromycin, telithromycin, minocycline, levoflaxin, clindamycin, and metronidazole

Kuriyama, Oral Microbiol Immunol 2007: 22: 285288

Antimicrobial susceptibility of 800 anaerobic isolates from patients with dentoalveolar infection to 13 oral antibiotics
The majority of Fusobacterium strains were resistant to

erythromycin, azithromycin, and telithromycin, the remaining antibiotics demonstrated a high level of antimicrobial activity Porphyromonas was susceptible to all antibiotics tested Prevotella resistance to amoxicillin occurred in 34% of isolates and all of these resistant strains were found to produce -lactamase Augmentin, telithromycin, clindamycin, and metronidazole show high activity against these strains

Kuriyama, Oral Microbiol Immunol 2007: 22: 285288

Cervical necrotizing fasciitis: 8-years' experience of microbiology

152 patients were admitted for CNF Most of these patients had been on antibiotic therapy prior to admission S. milleri and Prevotella were associated most commonly with dental CNF S. aureus was found primarily in pharyngeal CNF Penicillin and clindamycin resistance were observed

in 39% and 37% of cases, respectively

Fihman, Eur J Clin Microbiol Infect Dis. 2008 Aug;27(8):691-5. Epub 2008 Mar 4

Chlorhexidine
Usage of chlorhexidine for more than one week has

been associated with increased resistance in S. mutans and S. sobrinus S mutans has resistance to chlorhexidine has been shown to be related to the Clp ATPase family, it acts as a serine protease and removes abnormal proteins that accumulate during stress conditions S mutans can adapt following pre-incubation with sub-effective doses of chlorhexidine

Deng DM, Eur J Oral Sci 2007; 115: 363370

Possible Causes of Antibiotic Resistance

Over prescribing of antibiotics

Improper usage of antibiotics Not completing dosage

Increased usage of antibiotics in food production

Changing antibiotics during treatment

Resistance in Animal Based Bacteria

Laura Brinas, Antimicrobial Agents and Chemotherapy, Oct.

2002, p. 31563163

Antimicrobials have also been used increasingly in plant biotechnology and in animals, for the treatment of bacterial disease or as growthenhancing compounds in intense husbandry Ampicillin resistance was demonstrated in bacteria isolated from foods of animal origin, commonly chicken based food products This resistance was achieved through -Lactamase enzymes

Schwarz, Tetracycline resistance in Staphylococcus spp. from

domestic animals., Vet Microbiol. 1998 Oct;63(2-4):217-27

838 staphylococcal isolates representing 19 different species were obtained from cattle, cats, dogs, ducks, guinea pigs, horses, mink, pigeons, pigs, rabbits, and turkeys 228 (27.2%) isolates were shown to be resistant to tetracycline Additional resistances to one or more antibiotics were observed in 153 (67.1%) of the tetracycline resistant isolates
Laura Brinas, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2002, p. 31563163

Take Home Message

Be judicious in prescribing antibiotics

Encourage compliance with dosage and duration

When possible use susceptibility testing to ensure

proper prescription is given Use the narrowest spectrum antibiotic that will give necessary result