Urinary Bactericidal Activity of Oral Antibiotics against Common Urinary Tract Pathogens in an ex vivo Model

Microbiology Chemotherapy 2006;52:293–297 DOI: 10.1159/000095969

Received: February 7, 2005 Accepted after revision: December 13, 2005 Published online: September 27, 2006

Urinary Bactericidal Activity of Oral Antibiotics against Common Urinary Tract Pathogens in an ex vivo Model Branka Bedenic a Marijana Topic b Ana Budimir c Marina Bubonja d a Department of Microbiology, Medical School, University of Zagreb, b Laboratory for Quality Control, PLIVA, and c Department of Clinical and Molecular Microbiology, Clinical Hospital Center, Zagreb, and d Department of Microbiology, Medical School, Rijeka, Croatia

Key Words Bactericidal activity, urinary

-Lactam antibiotics
Fluoroquinolones
Linezolid

Abstract Background: In this investigation, the urine samples obtained in a single oral-dose pharmacokinetic study were examined for their bactericidal activity against a range of relevant urinary tract pathogens. Methods: Six healthy volunteers received a single oral dose of ten oral antibiotics available in Croatia. Urine samples were taken every 2 h during the whole dosing interval of the particular antibiotic. The urinary bactericidal activity was tested by determination of urinary bactericidal titers. Results: All antibiotics showed a significant urinary bactericidal activity against non-extended spectrum
-lactamase Escherichia coli and Proteus mirabilis. Fluoroquinolones displayed high and persisting levels of urinary bactericidal activity against all gram-negative bacteria and Staphylococcus saprophyticus. Conclusions: Average urinary bactericidal activity can be predicted from in vitro susceptibility testing, but we expect that there will be patients with a low level of urinary bactericidal activity. Copyright © 2006 S. Karger AG, Basel

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Introduction

In the assessment of an antibiotic, the bactericidal activity of plasma, urine and other body fluids is a relevant pharmacodynamic parameter, being an integration of pharmacokinetic properties with in vitro activity [1, 2]. In routine bacteriological laboratories the antibacterial activity of antibiotics is determined by in vitro testing, usually by the disk diffusion test. However, in vitro antibiotic susceptibility tests cannot reflect the situation in vivo. In vitro the bacteria are exposed to fixed concentrations of antibiotics whereas in vivo there is a more gradual decrease in antibiotic levels depending on the elimination half-life of the antibiotic. In this investigation, the urine samples obtained in a single oral-dose pharmacokinetic study were examined for their bactericidal activity against a range of relevant urinary tract pathogens. In vitro susceptibilities of urinary tract pathogens to oral antibiotics have been extensively studied, but there are no published reports on ex vivo urinary bactericidal activity of most oral antibiotics except fluoroquinolones [3] and linezolid [4]. Presented at the 1st World Conference on Magic Bullets – Ehrlich 2004, Nürnberg, Germany, September 9–11, 2004.

Branka Bedenic Department of Microbiology Medical School, University of Zagreb, Salata 3 HR–10 000 Zagreb (Croatia) Tel. +385 1 4920 026, Fax +385 1 4590 130, E-Mail [email protected]

Methods

Results

Volunteers Ten oral antibiotics available on the Croatian market were tested: amoxycillin, amoxycillin/clavulanate (co-amoxiclav), cephalexin, cefuroxime, cefadroxil, ceftibuten, norfloxacin, ciprofloxacin, sulfamethoxazole/trimethoprim (co-trimoxazole) and linezolid. Six healthy volunteers (females, age range 40–60 years) received a single oral dose of amoxycillin/clavulanate (Klavocin) 875/125 mg, cephalexin (Ceporex) 500 mg, cefuroxime (Novocef) 500 mg, cefadroxil (Duracef) 500 mg, ceftibuten (Cedax) 400 mg, norfloxacin (Nolicin) 400 mg, ciprofloxacin (Cipromed) 500 mg, co-trimoxazole (sulphamethoxazole/trimethoprim) (Sinersul) 400/80 mg and linezolid (Zyvoxid) 600 mg, respectively. The dosing of each drug was recommended by the manufacturer. Bacteria Experiments were performed on bacteria isolated from urinary tract infections in 2002–2003 at the Zagreb Clinical Hospital Center: Escherichia coli 2080/79 non-extended spectrum
-lactamase (non-ESBL), E. coli 4199/198 ESBL (TEM type), Klebsiella pneumoniae 287/286 non-ESBL, K. pneumoniae 1951 ESBL (SHV-5), Proteus mirabilis 4335/334, Serratia marcescens 4920/9199, Enterobacter cloacae 1211, Acinetobacter baumannii 4473/472, Pseudomonas aeruginosa 3579/578, Enterococcus faecalis 2252/251, Enterococcus faecium 162/161 and Staphylococcus saprophyticus 582. The isolates originated from the hospitalized patients with diagnosed urinary tract infection. P. aeruginosa, A. baumanii and ESBL producers were isolated from nosocomial infections. Reference ATCC strains of the respective species were used as the quality control strains. Susceptibility Testing Disk diffusion and broth microdilution tests were performed according to the NCCLS guidelines [5, 6]. Determination of Urinary Bactericidal Titers Urinary bactericidal titers (UBTs) of oral antibiotics were determined by the method described previously [7]. Urine samples containing antibiotics were double diluted in urine taken from the same volunteer before antibiotic administration, from 1:2 to 1:2,048 in 96-well microtiter trays. Plates were incubated at 37 ° C for 24 h before examination. The number of colonies was counted, and the urine dilution which demonstrated 99.9% killing was taken as the bactericidal dilution. A titer of 61:8 was taken as clinically relevant since it has been shown to predict a successful therapeutic outcome for the fluoroquinolones [7]. There is no recommendation in the references for the
-lactam antibiotics which are time-dependent antibiotics in contrast to the fluoroquinolones which are concentration dependent. Statistical Analysis UBTs obtained for different time-dependent antibiotics (
-lactams) were compared (for example, ceftibuten vs. cephalexin), as well as those obtained for concentration-dependent antibiotics (fluoroquinolones), which were also compared (ciprofloxacin vs. norfloxacin). UBTs of various antibiotics for particular bacterial species were compared using the paired t test. For computing reasons the UBTs had been previously transformed into ordinal data using a scale from 1 (UBT = 0) to 12 (UBT 62,048).

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In vitro Susceptibility Testing E. coli non-ESBL was susceptible to all antibiotics tested, and K. pneumoniae non-ESBL to all except amoxycillin. ESBL-positive strains were resistant to all
-lactams except ceftibuten. P. mirabilis was resistant to co-trimoxazole. A. baumanii was resistant to all antibiotics and P. aeruginosa to all apart from fluoroquinolones. E. cloacae and S. marcescens were susceptible to fluoroquinolones and ceftibuten. Enterococci were resistant to all cephalosporins as expected and co-trimoxazole. E. faecalis was susceptible to amoxycillin, co-amoxiclav, ciprofloxacin and to co-trimoxazole in contrast to E. faecium, but both were susceptible to linezolid. Determination of UBTs UBTs are shown in figures 1 and 2. Significant differences in UBTs between amoxycillin and co-amoxiclav were found for ESBL-producing E. coli and K. pneumoniae during the whole dosing interval. UBTs for ceftibuten were significantly higher than for the older cephalosporins for both ESBL-positive and ESBL-negative E. coli and K. pneumoniae in the later time intervals (after 6 h). Ceftibuten showed markedly higher UBTs for S. marcescens and E. cloacae as well. P. aeruginosa displayed significantly higher UBTs for ciprofloxacin in comparison to norfloxacin.

Discussion

According to the results of ex vivo experiments, amoxycillin could be recommended only for the therapy of infections caused by non-ESBL E. coli, P. mirabilis and E. faecalis strains. Amoxycillin combined with clavulanate showed a broader spectrum and could be considered as an option for the therapy of infections caused by ESBLnegative E. coli, K. pneumoniae, P. mirabilis, S. saprophyticus and E. faecalis. Older cephalosporins had high titers against non-ESBL E. coli, K. pneumoniae, P. mirabilis and S. saprohyticus strains in the first 4–6 h as expected since they achieve high concentrations in urine. Their main drawback is short t1/2 in urine resulting in a rapid decrease of UBTs. Ceftibuten as third generation cephalosporin was the only
-lactam displaying high and persistent activity against ESBL-producing E. coli and K. pneumoniae in urine during the whole dosing interval (24 h) due to its long elimination half-life in urine. Fluoroquinolones are the antibiotics of choice for the treat-

Bedenic /Topic /Budimir /Bubonja

Escherichia coli 2080/79 non-ESBL

2,500

Amoxycillin Co-amoxiclav

2,000

Cephalexin

1,500

Cefuroxime Cefadroxil

1,000

Escherichia coli 4199/198 ESBL

2,000 Titers

Titers

2,500

Ceftibuten

Cephalexin Cefuroxime

1,500

Cefadroxil 1,000

Ceftibuten

Co-trimoxazole

Co-trimoxazole 500

Norfloxacin Ciprofloxacin

Norfloxacin Ciprofloxacin

Time (h)

Amoxycillin

Klebsiella pneumoniae 287/286 non-ESBL

2,500

Co-amoxiclav 2,000

Cephalexin

1,500

Cefuroxime Cefadroxil

1,000

Ceftibuten

Amoxycillin Co-amoxiclav

2,000 Titers

Titers

8– 10 10 –1 2 12 –2 4

Time (h)

Escherichia coli ATCC 25922

2,500

6– 8

0– 2

4– 6

0

12 –2 4

10 –1 2

6– 8

8– 10

4– 6

2– 4

0– 2

0

2– 4

500

Cephalexin Cefuroxime

1,500

Cefadroxil 1,000

Ceftibuten

Co-trimoxazole

Co-trimoxazole 500

Norfloxacin Ciprofloxacin

Norfloxacin Ciprofloxacin

Cefadroxil

800

Ceftibuten

600

Co-trimoxazole

400

Norfloxacin

200

Ciprofloxacin 4

2

–2 12

10

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8

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12

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2

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4 –2

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10

Ciprofloxacin

Co-trimoxazole

10

Norfloxacin

8

500

Ceftibuten

8–

Co-trimoxazole

Cefadroxil

6–

Ceftibuten

1,000

Cefuroxime

6

Cefadroxil

Cephalexin

4–

1,500

Titers

Cefuroxime

4

Cephalexin

2,000

Amoxycillin Co-amoxiclav

Enterobacter cloacae 1211

900 800 700 600 500 400 300 200 100 0 2–

Amoxycillin Co-amoxiclav

Serratia marcescens 4920/919

6

8–

Time (h)

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6

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4

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Proteus mirabilis 4335/334

4

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Cephalexin

Co-trimoxazole

0–

12

Amoxycillin Co-amoxiclav

Ceftibuten

1,000

2

10

1,600

Cefadroxil

0–

10

1,800

2–

1,500

Amoxycillin Co-amoxiclav

Titers

Titers

2,000

Titers

8

Time (h)

Klebsiella pneumoniae 1951 ESBL

2,500

8–

6

Time (h)

6–

4–

0–

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4

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4

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–2

2

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2,500

Amoxycillin Co-amoxiclav

Time (h)

Fig. 1. Median UBTs of various antibiotics against Enterobacteriaceae.

Ex vivo Urinary Bactericidal Activity of Oral Antibiotics

Chemotherapy 2006;52:293–297

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Pseudomonas aeruginosa 3579/578

Pseudomonas aeruginosa ATCC 27853

120

300

Amoxycillin Co-amoxiclav

Cephalexin

250

Cephalexin

80

Cefuroxime

200

Cefuroxime

60

Cefadroxil Ceftibuten

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Norfloxacin

Norfloxacin Ciprofloxacin 0– 2 2– 4 4– 6 6– 8 8– 10 10 –1 2 12 –2 4

0

8– 10 10 –1 2 12 –2 4

Time (h)

Time (h) Acinetobacter baumannii 4473/472

Enterococcus faecalis 2252/251

Ciprofloxacin

Linezolid

Amoxycillin Co-amoxiclav

900

Co-amoxiclav

Cephalexin

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8

20

Ceftibuten

400

6–

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Cefadroxil

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6

30

Ceftibuten

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Cefuroxime

600

4

Cefadroxil

4–

40

Titers

Titers

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Enterococcus faecalis ATCC 29212

2–

Enterococcus faecium 162/161

50

Time (h) Staphylococcus aureus ATCC 29213 900

Staphylococcus saprophyticus 582

700

Cephalexin

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600

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500

Cefadroxil

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Cefadroxil

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Ceftibuten

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Norfloxacin

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Cefadroxil

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Cefuroxime 80

6

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Amoxycillin Co-amoxiclav Cephalexin

100

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Cephalexin

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120

2–

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Amoxycillin Co-amoxiclav

Titers

Titers

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6– 8

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50

Ciprofloxacin

0

Ceftibuten 100

Co-trimoxazole

20

Cefadroxil

150

6–

Titers

100

Amoxycillin Co-amoxiclav

Time (h)

Fig. 2. Median reciprocal UBTs of various antibiotics against nonfermentative and gram-positive bacteria.

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Bedenic /Topic /Budimir /Bubonja

ment of hospital-acquired urinary tract infections when P. aeruginosa, E. cloacae, S. marcescens, or ESBL-positive Enterobacteriaceae are isolated from urine as pathogens because they demonstrate excellent in vitro and ex vivo activity and achieve high concentrations in urine [8]. Their high titers persisted throughout the whole dosing interval, which can be attributed to their long elimination half-life in urine. Co-trimoxazole had good activity against non-ESBL E. coli and K. pneumoniae, ESBL-positive E. coli, E. cloacae, E. faecalis and staphylococci. Its UBTs did not change significantly during the dosing interval due to the long t1/2 in urine of both components. Linezolid had good urinary bactericidal activity against all gram-positive cocci including E. faecium resistant to amoxycillin and fluoroquinolones. Urine itself has a significant effect on the bactericidal activity of antibiotics. MICs are usually elevated if they are determined in urine instead of the standard medium [9, 10]. The susceptible

bacteria had significantly higher titers than resistant bacteria. Discrepancies were found when ESBL-producing K. pneumoniae and E. coli were exposed to older cephalosporins and co-amoxiclav. In spite of the fact that in vitro tests revealed a resistance to these antibiotics, high UBTs were observed for cephalosporins and moderate UBTs for co-amoxiclav particularly at the beginning of the dosing interval. Furthermore, a high range of UBTs (data not shown) was observed, which is attributable to a variable urinary antibiotic concentration. In spite of the fact that median UBT was above 1:8 during the whole dosing interval for most susceptible strains, in some urine samples it dropped bellow this value before the end of the dosing interval. As a consequence, in most of the patients a positive therapeutic outcome can be predicted if in vitro tests show susceptibility, but some who demonstrate low titers and thus probably achieve lower urinary antibiotic concentrations are likely to have therapeutic failure.

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© Free Author Copy - for personal use only

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Ex vivo Urinary Bactericidal Activity of Oral Antibiotics

Chemotherapy 2006;52:293–297

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