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LLüüttichttich 20042004 11

Dr. Dr. MaritaMarita LangewischeLangewische

May 13, 2004May 13, 2004

Development of bacterial resistance inDevelopment of bacterial resistance inlivestock farming after introduction of livestock farming after introduction of

fluoroquinolones fluoroquinolones -- potentials and limitspotentials and limitsof diagnosticsof diagnostics


Table of contentTable of content

Subdivision into the following chaptersSubdivision into the following chapters•• Chemical structure of (Chemical structure of (fluorofluoro))quinolonesquinolones

•• Antimicrobial effect of (Antimicrobial effect of (fluorofluoro))quinolonesquinolones

•• Development of resistance against fluorochinolonesDevelopment of resistance against fluorochinolones

•• Current situation after introduction of fluoroquinolones Current situation after introduction of fluoroquinolones into livestock farminginto livestock farming

•• Summary and conclusionsSummary and conclusions


Chemical structure of (Chemical structure of (fluorofluoro))quinolonesquinolones

Nalidixic Nalidixic acidacid•• NalidixicNalidixic acid (1962) first quinolone (acid (1962) first quinolone (gyrasegyrase inhibitor)inhibitor)•• Synthetic drugSynthetic drug•• Antimicrobial effect mainly on some gram negative Antimicrobial effect mainly on some gram negative

bacteriabacteria•• Used for infections of the urinary tractUsed for infections of the urinary tract

N N

O

C2H5

CH3

COOH



NorfloxacinNorfloxacin•• NorfloxacinNorfloxacin (1982) first quinolone with fluoride on (1982) first quinolone with fluoride on

position 6 of the aromatic ringposition 6 of the aromatic ring•• ⇒⇒ FluoroquinoloneFluoroquinolone•• Antimicrobial effect mainly on Antimicrobial effect mainly on NeisseriaNeisseria and and

HaemophilusHaemophilus sspssp. and some . and some EnterobacteriaceaeEnterobacteriaceae, , for for example example E.E. colicoli, , Klebsiella pneumoniaeKlebsiella pneumoniae, , Proteus Proteus mirabilismirabilis, and , and ShigellaShigella sspssp..

•• Used for infections of the urinary tract, enteritis, and Used for infections of the urinary tract, enteritis, and gonorrhea in humansgonorrhea in humans

NNH

N

OCOOH

C2H5

F



Development of further fluoroquinolones with Development of further fluoroquinolones with increasingincreasing antimicrobialantimicrobial effectseffects

• Group I Norfloxacin, Perfloxacin; mainly used against infections of the urinary tract

• Group II Ciprofloxacin (Ciprobay®), Enrofloxacin (Baytril®),Ofloxacin, Fleroxacin, Enoxacin (Gyramid®); usage for several indications

• Group III Levofloxacin, Sparfloxacin; fluoroquinolones with improved activity against gram positive cocci

• Group IV Gatifloxacin, Moxifloxacin (Avelox®); comparable to group 3, but with improved activity against anaerobic bacteria


Chemical structure of (Chemical structure of (fluorofluoro))chinoloneschinolones

Antimicrobial activity of fluoroquinolonesAntimicrobial activity of fluoroquinolonesAntimicrobial activity of fluoroquinolonesPathogen Group I Group II Group III Group IV1. gram positive cocciStaphylococcus aureus + ++ ++ +++Staphylococcus epidermidis + ++ + ++Streptococcus pyogenes (group A) + + + ++Streptococcus pneumoniae - + + +++Enterococcus faecalis - + + ++

2. EnterobacteriaceaeEscherichia coli ++ +++ +++ +++Klebsiella pneumoniae ++ +++ +++ +++Enterobacter spp. + +++ +++ +++Serratia marcescens + +++ ++ ++Proteus mirabilis ++ +++ +++ +++Shigella spp. ++ +++ +++ +++Activity: very good +++; good ++; medium +; no -



Antimicrobial activity of fluoroquinolonesAntimicrobial activity of fluoroquinolonesAntimicrobial activity of fluoroquinolones (cont.)Pathogen Group I Group II Group III Group IV3. Other gram negative bacteriaPseudomonas aeruginosa - ++ + +Neisseria meningitidis +++ +++ +++ +++Neisseria gonorrhoeae +++ +++ +++ +++Haemaphilus influenzae +++ +++ +++ +++Legionella spp. + +++ +++ +++

4. Anaerobic bacteriaPeptostreptococcus spp. - - + ++Bacteroides fragilis - - + ++

5. Other pathogensMycobacterium tuberculosis - + ++ ++Mycoplasma pneumoniae - + ++ ++Chlamydia trachomatis - + + +++Uroplasma spp. - - + ++Activity: very good +++; good ++; medium +; no -


Antimicrobial effect of (Antimicrobial effect of (fluorofluoro))quinolonesquinolones

Mechanism ofMechanism of antimicrobialantimicrobial effect of effect of fluoroquinolonesfluoroquinolones

• Bacterial chromosome consists of a circular, approximately 1300 µm long, double stranded DNA

• Size of bacterial cell is approximately 1000 fold smaller• ⇒ DNA has to be folded functionally to fit into the

bacterial cell• Unfolded only partially for transcription and replication• Introduction of negative supercoils into the DNA helix




• Folding and unfolding of DNA by Topoisomerase II (Gyrase) and Topoisomerase IV

• Topoisomerase II is a tetramer, consisting of each two equal A and B subunits




• Subunit A is ATP independent, cleaves and binds DNA double strains

• Subunit B is ATP dependent, twists cleaved DNA double strains


Antimicrobial effect of (Antimicrobial effect of (fluoro)quinolonesfluoro)quinolones

Mechanism of antimicrobial effect of Mechanism of antimicrobial effect of fluoroquinolonesfluoroquinolones

• Fluoroquinolones interact with the Topoisomerases and inhibit activity of these enzymes

• Lack of Topoisomerase activity is lethal for the bacteria

• Bacteriocide antimicrobial effect• Further bacteriocide effects by yet unknown

mechanisms are known



Negative influence of antimicrobial effect of Negative influence of antimicrobial effect of fluoroquinolonesfluoroquinolones

• Anaerobic milieu• Retarded bacterial growth, extended generation

time• Antagonism by bacteriostatic protein- and RNA

synthesis inhibitors• Di- and trivalent metal ions (formation of chelates)• Acid milieu (pH < 6.0)



Indications for Enrofloxacin in livestock farmingIndications for Enrofloxacin in livestock farming• Fowl, turkey: Mycoplasma, Haemophilus paragallinarum

(Coryza contagiosa avium), Pasteurella, E. coli, Salmonella, Erysipelothrix rhusiopathiae

• Pig: E. coli, Pasteurella ssp., Mycoplasma hyopneumoniae• Cattle: E. coli, Haemophilus spp., Pasteurella spp.,

Mycoplasma bovis⇒ Bacterial diseases of respiration tract and gastrointestinal

tract• Use only after determination of bacterial strain and

susceptibility testing (according to the manufacturer)


Development of resistance against fluorochinolonesDevelopment of resistance against fluorochinolones

Overview of acquired bacterial resistanceOverview of acquired bacterial resistance• Mutation of chromosomal bacterial genes• Plasmid uptake (extra chromosomal replicable

circular DNA)• Transposons (jumping genes)• Integrons and gene cassettes (highly

conserved gene sectors)



Bacterial fluoroquinolone resistanceBacterial fluoroquinolone resistanceGene cluster Affected protein Resistant pathogen MechanismgyrA Topoisomerase II

and IVSalmonella1, E. coli,Campylobacter ssp.2, 3

gyrB Topoisomerase IIand IV

High resistantSalmonella strains, likeDT 204c4

parC Topoisomerase IIand IV

Multi step mutations inSalmonella5

Point mutations leadto altered proteins,which do not interactwith quinolonessufficiently

qnr Yet unknown K. pneumoniae, E. coli6 Plasmid encodedresistance, yetunknown mechanism

1 Heisig P (1993) J. Antimicrob. Chemother. 32:367-377.2 Hakanen A et al. (2002) Antimicrob. Agents Chemother. 46:2644-2647.3 Lucey B et al. (2002) Vet. Rec. 151:317-320.4 Hakanen A et al. (2002) Antimicrob. Agents Chemother. 46:2644-2647.5 Heisig P (1999) Biospektrum Sonderband, pp. 42-46.6 Wang M et al. (2003) Antimicrob. Agents Chemother. 47:2242-2248.


Development of resistance against fluorochinolonesDevelopment of resistance against fluorochinolonesInfluence of different mutations on MIC of fluoroquinolonesInfluence of different mutations on MIC of fluoroquinolones

Genotype MIC1 Nalidixic acid(µg/ml)

MIC Ciprofloxacin (µg/ml)

Wild type (Salmonella) ≤ 4 0.015

GyrA, substitution for Ser83 ≥ 128 0.125 - 4

GyrA, substitution for Asp87 ≥ 128 0.125 - 2

GyrA, substitution for Ser83+ Asp87

≥ 128 4 - 8

GyrA + GyrB substitutions ≥ 128 32 - 128

⇒ Stepwise increase of resistance by mutations⇒ Nalidixic acid resistance is like an alarm bell1 MIC (minimum inhibitory concentration): Lowest concentration of an antimicrobialdrug, resulting in bacteriocide or bacteriostatic effect on bacteria culture



Testing of susceptibilityTesting of susceptibility• Sample drawing of bacteria• Isolating of strains• Identification and differentiation of strains• Determination of MIC• Determination of genotypes of the strains


Development of resistance against fluorochinolonesDevelopment of resistance against fluorochinolonesDifferent methods for determination of MIC in fluoroquinolonesDifferent methods for determination of MIC in fluoroquinolones• Agar diffusion method⇒ Zones of bacterial growth inhibition (in mm) are used for

classification in susceptible, intermediate or resistant• Broth microdilution method⇒ Determination of MIC50 and MIC90 by using log2 dilutions• So far evaluation according to NCCLS (National Committee

for Clinical Laboratory Standards), DIN (Deutsche IndustrieNorm), DANMAP (Danish Integrated Antimicrobial Resistance Monitoring and Research Programme), and BSAC (British Society for Antimicrobial Chemotherapy) with different break points


Development of resistance against fluorochinolonesDevelopment of resistance against fluorochinolonesDifferent methods for determination of MIC in fluoroquinolonesDifferent methods for determination of MIC in fluoroquinolones

Ciprofloxacin resistance (µg/ml) Reference

≥ 4 NCCLS M100-S11

≥ 2 DIN 58940-4

≥ 2 DANMAP 2000

≥ 1 BSAC 1996


Development of resistance against fluorochinolonesDevelopment of resistance against fluorochinolonesDifferent methods for determination of MIC in fluoroquinolonesDifferent methods for determination of MIC in fluoroquinolones• Better conclusions of data in a so called Finland-o-gram

(according to Maxwell Finland) as percentage of susceptibility against the MICs (mg/l) of the different tested strains

• Determination of worldwide consistent break points⇒ In case of fluoroquinolones first screening against Nalidixic

acid resistance⇒ Then determination of Ciprofloxacin MIC, break point ≥ 0.1251

µg/ml• Better: Determination of mutations in gyrA, gyrB or parC by

PCR• 1 Møller-Aarestrup F et al. (2003) Antimicrob. Agents

Chemother. 47:827-829.


Current situation after introduction of Current situation after introduction of fluoroquinolones into livestock farmingfluoroquinolones into livestock farming

Fluoroquinolone resistance in Fluoroquinolone resistance in CampylobacterCampylobacter• Increasing resistance in Campylobacter ssp. in fowl in the

Netherlands from 0 % in 1982 to 14 % in 1989, and from 0 % to11 % in the farm workers themselves (Endtz HP et al. (1991)J. Antimicrob. Chemother. 27:199-208.)

• In vitro experiments showed, that induction of resistance in Campylobacter occurs soon after chickens are exposed to fluoroquinolones

⇒ Increase of MIC from 0.25 µg/ml to 32 µg/ml within 5 days! (McDermott PF et al. (2002) J. Infect. Dis. 185:837-840.)

• Spread of Campylobacter by fowl is one of the main ways of contact with humans (Hamer DH & Gill CJ (2002) Nutr. Rev. 60:261-264.)


Current situation after introduction of Current situation after introduction of fluoroquinolones into livestock farmingfluoroquinolones into livestock farming

Fluoroquinolone resistance in Fluoroquinolone resistance in CampylobacterCampylobacter• Fluoroquinolone resistent Campylobacter strains become a

serious problem in human infections in USA, Spain, Portugal, andThailand. (Hakanen A et al. (2003) Emerg. Infect. Dis. 9:267-270; Nachamkin I et al. (2002) Emerg. Infect. Dis. 12:1501-1503)

• The FDA suggested to withdraw the usage of fluoroquinolones in fowl because of the increasing incidence of resistant Campylobacter spp. (Lees P. & Shojaee Aliabadi F. (2002) Int. J. Antimicrobial Agents 19:269-284.)

• Examinations in Germany showed, that 45.1 % of Campylobacterisolates of human feces revealed a Ciprofloxacin resistance (Wagner J et al. (2003) Antimicrob. Agents Chemother. 47:2358-2361.)


Current situation after introduction of Current situation after introduction of fluoroquinolones into livestock farmingfluoroquinolones into livestock farmingFluoroquinolone resistance in Fluoroquinolone resistance in SalmonellaSalmonella

• Examination in Germany by BfR (Federal Institute for Risk Assessment) showed, that 11.4 % (2001) respectively 27 % (2003) of the examined Salmonella isolates from fowl revealed a fluoroquinolone resistance, leading to a suggestion to a restrictive usage of these drugs in fowl

⇒ Danger of development of multi resistant Salmonella strains• Highly resistant Salmonella strains (DT 104) with five resistances

led to human diseases (25 cases) in Denmark in 1998 after consumption of pork, two patients died (Mølbak K et al. (1999) N. Engl. J. Med. 341:1420-1425.)


Current situation after introduction of Current situation after introduction of fluoroquinolones into livestock farmingfluoroquinolones into livestock farmingFluoroquinolone resistance in Fluoroquinolone resistance in SalmonellaSalmonella

• According to EMEA/CVMP/342/99-corr-Final 2820 patients in Great Britain revealed an infection with Salmonella DT 104 by consumption of animal products in 1997Further fluoroquinolone effects on bacteriaFurther fluoroquinolone effects on bacteria

• Ciprofloxacin induces increased release or production of vero or shiga-like toxins in E. coli. (Walterspiel JN et al. (1992) Infection 20:25-29.)


Current situation after introduction of Current situation after introduction of fluoroquinolones into livestock farmingfluoroquinolones into livestock farmingMonitoring Monitoring programs against bacterial resistanceprograms against bacterial resistance

• Necessity of implementation of an European monitoring against bacterial resistance

• Surveillance of at least three parameters:1. Pathogens with potential of zoonosis with a high aspect of

consumer protection (e. g. Salmonella spp., Campylobacter spp., E. coli)

2. Indicator bacteria (e. g. E. coli, Enterococcus ssp.) as an origin for resistance genes

3. Pathogens of veterinary medicine (Staphylococcus spp., Streptococcus spp., Pasteurella spp., Haemophilus spp.) with relationship to therapeutic use of antimicrobial drugs



• So far, several national programs have been implemented:

• SVARM (Swedish Veterinary Resistance Monitoring), Sweden

• DEFRA (Department for Environment, Food & Rural Affairs), Great Britain

• DANMAP (Danish Integrated Antimicrobial Resistance Monitoring and Research Programme), Denmark

• NORM/NORMVET, Norway• FINNRES, Finland



• NETHMAP/MARAN, the Netherlands• Different monitoring studies in Germany by the BfR

(Federal Institute for Risk Assessment) and BVL (Federal Office of Consumer Protection and Food Safety)

• The estimations about fluoroquinolone usage in livestock farming reach from banning to a relatively low hazard

• However, antimicrobial drug usage should be prudent


Summary and conclusionsSummary and conclusions• Fluoroquinolones are synthetic antimicrobial drugs with

bacteriocide activity• Indications in livestock farming are bacterial diseases of the

respiration and gastroinestinal tracts• Multistep mutations in bacterial chromosomal genes lead to

development of fluoroquinolone resistance• The susceptibility testing for fluoroquinolone resistance has to be

proven• Fluoroquinolone resistant bacteria in livestock farming lead to a

serious problem in human health• An European monitoring should help to recognize and prevent

increasing resistance• Banning or restriction of fluoroquinolone usage in livestock

farming will be further evaluated and discussed in the future

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