Biology of Target Change Resistance: Fluoroquinolone resistance mutations Penicillin Binding Proteins Main example organism: Streptococcus pneumoniae

S. pneumoniae Normal flora: nasopharynx –30-50% of kids (more in developing world) –~10% of adults –Makes its living by transmitting between carriers –Frequent innocent bystander in abx treatment Causes otitis media, pneumonia, bacteremia, meningitis Most disease in 65, and HIV+

Reduced permeability (Efflux) Degradation Detoxification Target alteration: enzyme Target alteration: mutation Target amplification Inactivate the activator of the prodrug Biochemical mechanisms

Fluoroquinolones Ciprofloxacin Ofloxacin Levofloxacin Sparfloxacin Grepafloxacin Trovaflocacin Gatifloxacin Gemifloxacin Moxifloxacin D. Hooper (MGH) –Lancet Inf Dis 2002 Clinical practice in 1980s –Mainly for Gram-neg –More recent ones have more Gram-pos activity Approved only for adults; cartilage toxicity in children

How it works

Two targets DNA Gyrase gyrA Topoisomerase IV parC Often, ParC is more sensitive to inhibition Example (numbers made up) Selection: wt->parC ->parCgyrA EnzymeInhibited by GyrA2 ug/ml ParC0.5 ug/ml GenotypeWild type gyrAparCgyrA parC MIC0.5 2>2

Preventing mutants by higher dosing Modified from Dong et al AAC Fig 2A M. bovis

Newer FQ Broader coverage: more species of bacteria Lower MIC for the same bug = lower concentration needed to achieve kill “dual target” – if both enzymes are equally sensitive, then single-resistant mutant has no advantage: “combination therapy” from one drug

Efflux Another mechanism of resistance to FQ Pumps that move molecules across cell membrane in all bacteria Upregulation can confer low-level resistance

Epidemiology of FQ resistance: S. pneumoniae Used in adults for pneumococcal infections and for community-acquired pneumonia (often don’t know the organism) Resistance has stayed relatively rare in most countries Sporadic in most countries

Western countries: sporadic Clin Inf Dis 2005

Still relatively rare Pletz et al. AAC 2004

Sporadic + clonal Pletz et al. AAC 2004

Risk factors for FQ-R vs. FQ-S Previous use of FQ (OR=12.1) Nursing home resident (OR=12.9) Nosocomial acquisition (OR 9.9) Notice: –These are mainly adults –Previous use is major risk factor –Not the same as pen-R risk factors

Copyright restrictions may apply. Pichichero, M. E. et al. JAMA 2007;298: Nine Cases of AOM Caused By a Multidrug-Resistant Streptococcus pneumoniae Serotype 19A ST 2722 Straina

Summary: FQ-R in S pneumo Mutational resistance in GyrA and ParC Stepwise increases FQ mainly given to adults Individual treatment history major risk factor Resistance remains rare Clonal + sporadic

Penicillin resistance in S. pneumoniae

Beta-lactams Penicillin derivatives Penicillin Ampicillin Amoxicillin Amoxicillin- clavulanate Piperacillin etceteracillin Cephalosporins 1 st -4 th generation Many different ones –Different spectrum of activity Cephakillemol

Mechanism

Beta-lactamases Not important in S. pneumoniae Many other organisms –Intrinsic resistance in mycobacteria –Some species have them already but “derepress” them to acquire resistance: Enterobacter cloacae –Very often plasmid-borne: most Gram- negative bacilli (E coli, Klebsiella, Serratia, Acinetobacter)

S. pneumoniae Alterations in PBP

Stepwise changes = more resistance Trzcinski et al JID

Figure 1. Graphical presentation of amino acid substitution within penicillin ‐ binding proteins (PBPs) 1A, 2B, and 2X in Streptococcus pneumoniae serotype 6B and 9V strains constructed in the course of the study. Polymerase chain reaction products of fragments of pbp loci corresponding to these used for transformation were sequenced and analyzed. Included are the whole 2160 ‐ bp open reading frame (ORF) of the pbp1a gene, the fragment downstream from the 613 nt (codon 205) of the pbp2b ORF of 2043 bp, and the fragment downstream from the 280 nt (codon 94) of the pbp2x ORF of 2253 bp ( GenBank accession nos. DQ190413–DQ and DQ227571–DQ227582). The top sequence shows amino acids of penicillin ‐ susceptible isolate TIGR4 (position in the protein as marked above), substitutions of which were identified in isolates analyzed elsewhere [21]. Only differences between TIGR4 and strains analyzed are indicated, and dashes indicate that the amino acid at that particular position was identical with TIGR4 sequence. Strains are presented in the order: susceptible recipient, final transformant (T), and the resistant donor of the pbp gene. Asterisks show the domain's transpeptidase (TP) active sites (Sx2K, SxN, and KT(S)G, respectively). Symbols “>” and “<” mark first and last replacement within TP domain of particular pbp2x, pbp2b [6], and pbp1a gene [22]. Sequences for transformed variants were determined in isolates passed through the infant rat colonization step before use in an in vivo competition model experiment. Many mutations to make resistant PBP SSRRSRRSSRRSRR SSRRSRRSSRRSRR SSRRSRRSSRRSRR

How do we select resistant strains? Samore et al. AJE 2006 Treatment kills both S and RTreatment kills S, promotes R

Risk factors Huang et al Pediatrics Regev-Yochay et al Scand J Inf Dis

Note It’s children (by design, but also because they have the most resistance) Risk factors are mainly for transmission opportunities Recent abx use not as impressive as in the case of FQ

Geographic variation in penicillin resistance H. Goossens et al Lancet

Trends (US): increasing until ~ 2001 DM Johnson et al. Diagn Mic Inf Dis 2006

Vaccine introduction reduced resistance disproportionately -- for a time 39.0% 37.4% Kyaw et al. 2006, NEJM

Resistance is now creeping upwards within NVT Kyaw et al. 2006, NEJM Based on Kyaw et al. 2006, NEJM

Resistance is highly clonal Global disseminationA few clones account for much of drug resistance. Eg US sample of PRSP 38% Spain 23F % Spain 9V % from 8 other clones Corso A 1998 Micro Drug Res Summarized by Klugman 2002 Klugman 2002 J Antimicrob Chem

Summary: Pen-R S pneumo Alterations in PBPs Stepwise increase in MIC with more alterations Major genetic changes required Global clones Increasing frequency in most countries though much variation Geographically, use associated with resistance, but not so strongly at individual level

Why the difference between FQ-R and Pen-R? FQ given mainly to adults Mutational resistance Hence individual use a major risk factor Hence children not affected, less spread Pen to all ages DNA import required Hence individual use less likely to lead to R Children are the source of SP for all of us – more spread