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Resistance of anaerobic

Can we find any clinical impact ?

Szeged ESCMID School 2005

L. Dubreuil Faculté de Pharmacie Université de Lille II, France resistance among anaerobes

Intrinsic resistance

all anaerobes aminoglycosides, quinolones, ,trimethoprim, species -dependant : : Propionibacterium & Actinomyces rifampicin : F. necrophorum, F. mortiferum : C. difficile & : C. innocuum Bacteroides fragilis group : C1G, Fusobacterium : macrolides Acquired resistance Antibiotic resistance among anaerobes

Acquired resistance : ß-lactams : ß -lactamase (butyricum, + RIC group) Prevotella, Fusobacterium B. fragilis gene + promotor cfiA carbapenemase, cfxA Hyperproduction of the chromosomal enzyme lack of porin & PBP modifications Antibiotic resistance among anaerobes

Acquired resistance : MLSb erm genes Metronidazole nim genes Fluroquinolones gyrase, topoisomerase mutations resistance to Moxifloxacin Chloramphenicol cat genes The situation in Europe

Gram positive cocci susceptible > cephalosporins glycopeptides, linezolid, rifampicin acquired resistance clindamycin > 20% metronidazole < 5% variable fluoroquinolones The situation in Europe

Actinomyces susceptible penicillins > cephalosporins glycopeptides, linezolid, rifampicin acquired resistance tetracycline macrolides (rare) variable fluoroquinolones The situation in Europe

Propionibacterium susceptible penicillins > cephalosporins fluoroquinolones glycopeptides, linezolid, rifampicin

acquired resistance tetracyclin macrolides & clindamycin increasing The situation in Europe

C. perfringens susceptible penicillins > cephalosporins, metronidazole, glycopeptides, linezolid, rifampicin most fluoroquinolones acquired resistance tetracyclins macrolides & clindamycin rare

One strain resistant to metronidazole Faris & Poxton 1999 The situation in Europe

C. difficile susceptible penicillins, metronidazole, glycopeptides, fusidic acid, acquired resistance tetracyclins, chloramphenicol macrolides & clindamycin > 60%

emerging ? Vancomycin & metronidazole The situation in Europe

Other clostridia (with exception), susceptible penicillins > cephalosporins glycopeptides linezolid, metronidazole acquired resistance tetracyclins macrolides & clindamycin cefoxitin & cefotetan chloramphenicol glycopettides :C. boltae,C. hathewayi (Van B2) The situation in Europe clostridia C. butyricum : penicillinase inhibited by RIC group C. clostridioforme broad cephalosporinase low-level resistance to , , ramoplanin C. ramosum broad cephalosporinase not inhibited by clavulanic acid low-level resistance to vancomycin, linezolid, daptomycin C. innocuum intrinsic resistance to cefoxitin and cefotetan low-level resistance to vancomycin & daptomycin And now ?

Gram positive cocci Non-sporulated gram positive Clostridia No major clinical problems except for individual patients low rate of antibiotic diffusion (brain, bone,) be aware of RIC group glycopeptide resistance in clostridia. What about gram negative anaerobes?

Prevotella 2/3 ß-lactamase production + acquired resistance clindamycin 5% metronidazole rare variable fluoroquinolones

Porphyromonas Fusobacterium penicillinase < 10% Les mousquetaires anaérobies des infections pulmonaires et O.R.L

QuickTime™ et un décompresseur TIFF (non compressé) sont requis pour visualiser cette image. Prevotella : MIC 50/90%

ß-lactamase - ß-lactamase + 0,12 0,25 8 >64 Amox +clavu 0,06 0,06 0,06 2 0,25 1 16 >64 0,12 1 8 >64 0,25 1 16 >64 0,12 0,5 4 >64 0,12 0,5 4 >64 Prevotella : MIC distribution

Nb of strains 25

Amoxycillin 20

15

10

5

0

MIC in mg/l Prevotella : distribution des CMI

Nb of strains 12 Cephalotin 10

8

6

4

2

0 ß-lac + ß -lac -

MIC in mg/l Prevotella : distribution des CMI

Nb of strains 14

Cefuroxime 12

10

8

6

4

2

0 ß-lac + 0.03 0.06 ß lac - 0.12 0.25 0.5 1 2 4 8 16 32 64 128 >128 MIC in mg/l Prevotella : distribution des CMI

Nb of strains 14

12 Cefpodoxime

10

8

6

4

2

0

ß-lac +

ß -lac -

MIC in mg/l Antibiotic susceptibility in Porphyromonas

Antibiotic MIC range MIC 50% MIC90%

Amoxicillin 0,03-0,5 0,5 0,5 Co-amoxiclav 0,03-0,25 0,25 0,25 0,06-1 1 1 Clindamycin 0,03-0,25 0,06 0,25 Metronidazole 0,03-0,25 0,125 0,25 Roxithromycin 0,25 Pristinamycin 0,25 FQ susceptibility of Prevotella & Fusobacterium

Antibiotic Prevotella Fusobacterium MIC 50% 90% 50% 90%

Ciprofloxacin 1 4 2 4 Ofloxacin 2 8 1 4 Levofloxacin 2 4 1 2

Moxifloxacin 0,12 0,5 0,12 1 Gatifloxacin 0,25 2 0,5 4 Trovafloxacin 1 2 0,25 0.5 Anaerobic lung . High rate of failures associated with penicillin-resistant Prevotella melaninogenica

10/47 resistant strains 5 patients received benzyl-penicillin 2 M every 4 hours all failed

Failure penicillin 8/18 vs 1/19 clindamycin

Gudiol Arch inter Med 1990 150 :2525 Role of ß-lactamases from gram neg anaerobes

Nord-Brook

ß-lactamases excreted by Prevotella or Fusobacterium caused clinical failures when patients were treated by penicillins otitis - sinusitis-dental abcess-lung abcess-recurrent tonsillitis Anaerobic infections

Bacteroides fragilis group + : intra-abdominal gyneco-obstetrics, biliary infections Perforated appendicitis Decubitus ulcers Last but not least the B. fragilis group

Chromosomal cep A ß-lactamase AMX + C1G+ C3G+C4G + overproduction TIC + PIP + lack of porin AMC, TTC, PTZ ? carbapenemase All ß-lactams

MLSb Clindamycin (20-40%) nim genes Nitro-imidazole AMX TIC PIP CTX IMP MTZ

CFT CFX CTT ERY CLN

AMC TCC PTZ TE CM Resistance to the combinations of ß- lactams and ß-lactamase inhibitors

IS 1224 upstream the cep A gene increases the production of the chromosomal cephalosporinase Rogers et al.J Bacteriol 1994;176:4376-4384

High level production of the cephalosporinase alone or associated with modifications in the permeability barrier (defect of porin) Phenotypes of resistance among - susceptible strains of the B. fragilis group

Antibiotic wild hyperproduction of ß-lactamase or/and lack type of porin or silent carbapenemase % (72) (19) (6) (2) (1) S R R R R Coamoxiclav S S S/I I/R R Ticar + clavu S S S S I/R Pipera+tazo S S S S I Cefotetan S/I I/R R R R Figure 1 : Antibiotic resistance rates among the B. fragilis group (256 strains) in 2003

30 27,5 28 Antibiotic resistance rates (%)

25

20

15

10 6,2 4,3 1,9 1,9 5 0 0,8

0 Metronidazole Imipenem Ticar+ clavu Pipéra +tazo Co-amoxclav Cefoxitin Ticarcillin Clindamycin Activity of fluoroquinolones against the B. fragilis group (130 strains)

60 50 40 30 CIP 20 LEV 10 MOX 0 GAR 0.06 0.25 1 4 16 64 GAR MOX LEV CIP - B. fragilis group Are antibiotic resistance rates increasing ?

Antibiotic resistance rates (%)

35 MTR 30 25 AMC

20 CLN 15 10 5 0 1996 1998 2000 2003

1996 1998 2000 2003 MTR 0,4 1,9 4,7 4,3 AMC 4 3,8 5,6 4,3 CLN 20 29 32 28 Metronidazole resistance nim on mobilisable plasmids nim A B.vulgatus nim C B. thetaiotaomicron nim D B. fragilis nim B chromosomal B. fragilis, P. acnes, A. odontolyticus, C. bifermentans nim A in Prevotella bivia nim G B. fragilis B. fragilis S or Intermediate to metronidazole B. fragilis metronidazole-R

Metronidazole resistance

Gal & Brazier, JAC 2004,54:109-116

15/ 206 isolates were found to possess nim genes and these had MICs of MTZ from 1.5 to >256 mg/L with 11.6% above the therapeutic breakpoint of 16 mg/L

Disc diffusion 28-30 mm test on FAA with a 5 µg MTZ disc. 70% showed reduced zone of susceptibility or no zone 30% with no reduced zone MIC 1.5 to 4 mg/L No zone for 7 strains without nim gene. M 17 22 23 29 31 39 ND Results (2) E c o Ø Bacteroides fragilis group R (PCR-RFLP) V Strain N° 17: nimB 22: nimE 23: nimD 29: nimD F 31: nimB o 39: nimA k Strain N° 40: nimA (sequencing) I

Ø Veillonella sp. Strain N° 18 : nimE (sequencing) H i n f I Results (3)

MTZ /R MTZ /R MTZ /I

2 copies of nimA gene in the genome of:

- MTZ Resistant strain (MIC = 64 mg/L)

- MTZ intermediate strain (MIC = 16 mg/L)

nimA SfiI nimA EcoRI nimA EcoRI Clinical significance of intermediate or resistant metronidazole clostridia ? Elsaghier , Brazier et al. JAC 2003

Failure of treatment due to Bacteroides fragilis with reduced susceptibility to metronidazole.

Pelvic collections grew mixed coliforms susceptible to cefuroxime Patient treated by Cefuroxime +metronidazole +gentamicin.

Pyrexia after four days of Tt. WBC 16000/mm3 B. fragilis MTR-R cultured from blood cultures (disk diffusion)

MIC by E test 6 mg/L. Metronidazole resistance in France

French resistant strains MTZ-R Prevotella buccae et 3 souches de P. loescheii CMI = 16 mg/L

B. fragilis MIC = 64 mg/L

H. Marchandin et al. Anaerobes 2002;8:137

Veillonella nim E in a metronidazole-susceptible Veillonella sp. strain MIC = 4 mg/L

H. Marchandin, Dubreuil, AAC 2004 48:3207 Bacteremia caused by Prevotella sp. with reduced susceptibility to metronidazole Mory et al.ECC Paris december 2004

78 old patient with sepsis Cefotaxime (3g)+ ofloxacin (400mg) E. coli S + anginosus S Patient pyrexial new blood cultures Teicoplanin 400mg and metronidazole 500mg tid added Day 7 Prevotella AMX, CTX, OFL-R, MTR S but colonies appearing later in the inhibition zone MIC 64 mg/L. Day 16 persistance of fever + 12g + Cipro 800 mg. Apyrexia in 48h Metronidazole resistance

B. fragilis & B. ovatus from appendicetomy MIC >32 mg/l : failure in Koweit Rotimi et al. Clin Microbiol Infect 1999 ; 5:166-169

B. fragilis MIC =256 in New Delhi with clinical failure

Chauldry, Emerging Infect Dis 2001,7,485-486 B. fragilis MIC >32 mg/L in Seattle

Shapiro et al. J Clin Microbiol 2004 ;42:4127-4129 B. fragilis in Hungary (Nagy et al.), Poland (Wojcik)

Prevotella loescheii MIC 12 mg/L subdural empyema in Cardiff Sandoe JAC 2001;47:366-367. Metronidazole resistance

C. difficile MIC =16 mg/L in Hong Kong, Madrid and Cardiff

Wong et al.Diagn Microbiol Infect Dis 1999 ;34:1-6 Pelaez AAC 2002,46:1647-1650 Brazier et al. JAC 2001;48:471-472 Equine isolates in Sacramento MIC = 32 mg/l

Jang et al. Clin Infect Dis 1997, S266 -267

Finegoldia magna MIC >128 mg/L Cape Town

Theron et al. JAC 2004; 54 :240-242

C. perfringens in Edinburgh MIC >32 mg/L

Faris et al. J Infect 1999,2:164-165. In vivo veritas

Many variables in addition to in vitro susceptibility testing Site of Surgical procedures Type of underlying disease Presence of other (mixed infections) Type of therapy used Correlation of clinical outcomes in patients with B. fragilis group infections treated with cefoxitin

Snydman et al AAC 1992 36 540

Patients in the treatment failure group were more likely to have - a longer cefoxitin dosing interval (p=0.019) -a shorter duration of cefoxitin (p = 0.051) -a longer duration of hospitalization -a higher observed maximum leucocyte count Plot of clinical cure or treatment failure versus MIC and cefoxitin dosing interval Time above the MIC

MIC mg/L cured failed 64

32

16

8

4 6 8

Dosing interval (h) PK/PD concentration

T > MIC MIC

T > MIC Time Antibiotic Susceptibility Testing pro or con ?

Wilson CID 1995 suppl 2 S251 Chow 1974 appropriate antibiotic therapy of Bacteroides bacteremia is associated with a better outcome than inappropriate therapy but

Surgeons advocated (MC Namara 1993) « routine practise of obtaining cultures in patients operated on for acute and complicated appendicitis should be abandoned »

« No benefit from susceptibility data » (Mosdell 1991) AST pro or con in peritonitis ?

Mosdell only 8.8% of patients who had inadequate empirical therapy was an appropriate change in antibiotic treatment

Dougherty « culture results appeared to influence antibiotic therapy for only 7% of these patients »

No benefit from susceptibility data (Mosdell 1991) Worrisome disjunction between surgeon and microbiologist AST pro or con in peritonitis ?

Worrisome disjunction between surgeon and microbiologist BUT Berne In vitro susceptibility was significantly associated with infectious postoperativc outcome «89% of patients in whom primary therapy failed had resistant strains compared with 46% in whom therapy succeeded. So..... The concept persists that in patients with polymicrobial infection adequate surgical treatment and antibiotic at least cover aerobic component of the flora will suffice AST pro or con in peritonitis ?

Hopkins 1993 Study of 175 patients with intra-abdominal infections « both aerobic and anaerobic bacteria susceptibility was statistically correlated with outcome »

26% of resistant B. fragilis from those who had an infection vs 3% those who have not

Patients with resolution of the IA infection 44% of resistant isolates vs 82% if complications Retrospective study of anaerobic bacteremia

Blood cultures positive for anaerobes 81 patients

Clinically significant bacteremia Clinically insignificant bacteremia 57 patients 24 patients

Initial tt ineffective Changed to effective 32%

Initial tt effective 49% Initial tt ineffective Not changed 19% 17% died

5% died 55% died

Salonen CID 1998 26:1413 Antimicrobial resistance and clinical outcome of Bacteroides bacteremia

Well-designed prospective study with stratification of patients 81 patients treated with an antimicrobial agent to which the isolate was susceptible and 11 with a resistant isolate.

Therapy Inactive active p= Mortality 45% 16% 0.03 Clinical failure 82% 22% 0.0002 Persistence 47% 12% 0.06 Antimicrobial resistance and clinical outcome of Bacteroides bacteremia

Failure 4/6 clindamycin-resistant B. fragilis (MIC ≥ 16 mg/L) 1/13 clindamycin-susceptible B. fragilis

Piperacillin 3 failures = all resistant isolates MIC 256 mg/L One cefotetan-resistant B. caccae = failure Multivariate analysis of risk factors for death and clinical failure of treatment for patients with Bacteroides bacteremia

Outcome, risk factor Multivariate analysis OR ( 95%CI) Death APACHE II score > 15 .0002 21 (4-104) Inactive therapy .066 5 (0.9-28) Clinical failure APACHE II score > 15 .0002 16 (4-71) Inactive therapy .001 38 (4-337) Liver disease .68 5 (1-29) Prediction of patients’ response to therapy

Specificity 97%

Positive Predictive value 82%

Sensitivity 63% Principles of antibiotic treatment in community-acquired peritonitis

•To be started as soon as the diagnostic is done and surgery is decided –Rapid treatment –IV route •Drugs may be different from those used in prophylaxis directed towards Enterobacteriaceae and anaerobes cause of mortality and abscesses formation

Bartlett JG. Med Clin North Am. 1995;79:599-617 Pathophysiology of intra-abdominal infections

Role of Enterobacteriaceae and anaerobes 100 Mortality

75 Abscesses

50 observed observed % 25

0 E. coli E. faecalis B. fragilis E. coli E. coli E. faecalis + + + E. faecalis B. fragilis B. fragilis

Onderdonk. Infect Immun. 1976 Pathophysiology of intra-abdominal infections

Role of Enterobacteriaceae and anaerobes % 100

Mortality 75 Abscesses

50

25

0 Control Gentamicin Clindamycin Clindamycin untreated + gentamicin

Weinstein. JID. 1975 INVANZ® () in communauty acquired peritonitis

Background

relative influence of the outcome •surgery à 1 •ICU à 2 •antibiotic therapy à 3 Numerous identical protocols demonstrating the equivalence of antibiotics (cf. Consensus) Impact of an initial adequate antibiotic treatment ? Community-acquired peritonitis : pronostic

100 0

90 10

80 Success 20

70 30

60 40

50 50 Success (%) Mortality (%) 40 60

30 70 Death 20 80

10 90

0 100 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 Mortality

Appropriate surgical procedure + adequate ATB 6 %

Appropriate surgical procedure + inadequate ATB 71 %

Inappropriate surgical procedure + adequate ATB 90 %

Inappropriate surgical procedure + inadequate ATB 100 %

Carlet J . L’infection en réanimation. Masson Paris 1986 pp 126-138 Inadequate antimicrobial therapy in peritonitis

Effect on mortality

92 patients patients with secondary peritonitis 65 patients (71%) received an inappropriate antibiotic therapy - initially - or in the course of therapy

94 % of the patients who died received an o inappropriate therapy

Koperna. Arch Surg 1996;131:180 A1

Effect of inadequate initial antibiotic therapy in peritonitis (2) Financial implications Canada (1993-2001) Initial therapy Adeqate Inadequate 8 hospitals n=383 n=51 (14 %)

n=3 n=2 Ampi + Genta + Metro 122 5 (4 %) (0.9%) (3.9%) 100 n=46 Cefoxitine 88 14 (16 %) (13.9%) n=21 Genta + Metro 52 9 (17 %) 80 41.2%) + Métro 31 6 (19 %) n=282 60 (85.2%) Cipro + Metro 23 6 (26 %) Ssucces Ssucces %sof treatment 40 Other< 5% of cases - - n=28 (54.9%) 20 Mean hospital cost + 65 % Mean antibiotic cost + 34 % 0 Adequatre Inadequate Mean biochemistry cost + 158 % n=331 n=51 Mean microbiology cost + 107 % Success failure others Chabot I. et al. IDSA 2002. P 604 Folie 65

A1 Insert white background for graph. White fonts Admin; 08.05.2003 Infections including B. fragilis ?

YES NO If efficacy against B. fragilis Most anaerobes are cured

Consider Prevotella & Fusobacterium and the ß-lactamase production Conclusion

Need for : antibiotic susceptibility testing ß-lactamase production Detection of a carbapenemase Detection of resistance and decreased susceptibility to metronidazole

Clindamycin empiric treatment on anaerobes not warranted