Efficacy of ß-lactams against experimental pneumococcal endocarditis caused by strains with different susceptibilities to penicillin

Cristina Pichardo1,*, Fernando Docobo-Pérez1, Maria E. Pachón-Ibáñez1, Manuel E. Jiménez-Mejías1, Andrés García-Curiel2, F. Javier Caballero-Granado1,{dagger}, Ignacio Moreno-Maqueda1,{dagger} and Jerónimo Pachón1

1 Infectious Diseases Service, Virgen del Rocío University Hospitals, Sevilla, Spain; 2 Microbiology Service, Virgen del Rocío University Hospitals, Sevilla, Spain


* Corresponding author. Tel: +34-955012376; Fax: +34-955012377; E-mail: cristina.pichardo.exts{at}juntadeandalucia.es

Received 19 April 2005; returned 11 June 2005; revised 2 August 2005; accepted 4 August 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To compare the in vitro and in vivo activity of penicillin, cefotaxime and ceftriaxone, using three strains of Streptococcus pneumoniae with different susceptibilities to penicillin (MICs of 0.015, 0.25 and 2 mg/L, respectively).

Methods: Time–kill curves and an experimental model of endocarditis in rabbits.

Results: Penicillin was efficacious in clearing bacteria from vegetations and blood irrespective of whether infections were caused by penicillin-susceptible or penicillin-resistant strains (P < 0.01 with respect to control groups). The same efficacy was shown with cefotaxime and ceftriaxone. Comparing the results of the in vivo model with those obtained in time–kill curves, penicillin showed the best results.

Conclusions: These results confirm that penicillin is efficacious in the treatment of pneumococcal infections, including those produced by strains with MICs ≤ 2 mg/L (with the exception of pneumococcal meningitis). These results also suggest that the breakpoints to define susceptibility and resistance of S. pneumoniae to penicillin must be reviewed, as has been done with amoxicillin and third-generation cephalosporins.

Keywords: Streptococcus pneumoniae , antimicrobial resistance , antimicrobial therapy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mortality rate for pneumococcal bacteraemia remains appreciable, ranging from 23 to 45% in the case of bacteraemic pneumonia.1,2 The treatment of pneumococcal infections is controversial, due to the widespread occurrence of pneumococcal strains resistant to antimicrobials, particularly ß-lactams. The resistance to penicillin and multi-drug resistance are widely distributed in different countries.3 The resistance rates to penicillin in clinical isolates have been 43.9% in Spain4 and 23% in a multicentre study from Europe and the USA.5 In Europe,6 the highest proportions of non-susceptibility to penicillin in Streptococcus pneumoniae were found in Mediterranean countries: Greece (37.5%), Israel (35%) and Spain (33.7%); in contrast, the lowest proportions of resistant isolates (<3%) were found in the Netherlands, Germany, Malta, Austria and Bulgaria.

In spite of the current levels of resistance to ß-lactams, several clinical studies in adults and children suggest that penicillin or cephalosporins are efficacious in the treatment of pneumococcal pneumonia, including bacteraemic cases, when the MIC of penicillin is ≤2 mg/L.79 However, these are observational clinical studies and/or they are not designed to compare the efficacy of ß-lactams in infections caused by pneumococci with different levels of resistance to penicillin.

The objective of the present study was to compare the in vitro and in vivo efficacy of penicillin, cefotaxime and ceftriaxone in a bacteraemic model of experimental infection by S. pneumoniae, such as experimental endocarditis, using three strains with different susceptibilities to penicillin.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Three strains of S. pneumoniae with different susceptibilities to penicillin were used. They were identified by morphology, {alpha}-haemolysis, optochin susceptibility (Optochin; Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) and bile solubility (2% sodium deoxycholate versus saline). The serotypes were determined in the Institute Carlos III (CNMVIS, Majadahonda, Madrid, Spain). The strains were: (i) strain 110.0 (S strain), susceptible to penicillin (MIC: 0.015 mg/L), isolated from blood, serotype 5; (ii) strain 3.0 (I strain), with intermediate resistance to penicillin (MIC: 0.25 mg/L), isolated from blood, serotype 23; (iii) strain 61.0 (R strain), resistant to penicillin (MIC: 2 mg/L), isolated from peritoneal fluid, serotype 23.

Media and antimicrobials

The following media were used: Todd–Hewitt broth supplemented with 0.5% yeast extract (THY) (Todd–Hewitt broth, Becton Dickinson Microbiology Systems; yeast extract, Oxoid Unipath Ltd, Basingstoke, Hampshire, UK); Trypticase soy broth (Becton Dickinson Microbiology Systems); antibiotic agar no. 5 (antibiotic medium 5, Difco Laboratories, Detroit, MI, USA); Columbia agar with 5% sheep blood (blood-agar Columbia, Becton Dickinson Microbiology Systems).

The standard reference powders of antimicrobials were obtained from the manufacturers: penicillin G (Antibióticos Farma, Madrid, Spain), cefotaxime (Aventis Pharma, Madrid, Spain), and ceftriaxone (Roche Farma, Madrid, Spain). Dilutions of antimicrobials were prepared as indicated by the NCCLS.10

Animals

New Zealand rabbits weighing 2–3 kg were purchased from B&K Universal, Barcelona, Spain. The study was performed in accordance with prevailing regulations regarding the care and use of laboratory animals in the European Community and was approved by the Ethics Committee of the University Hospitals Virgen del Rocío.

In vitro studies

The MICs of antimicrobials were determined by macrodilution in broth (THY) following the NCCLS criteria.10 Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used as controls. The MBCs were determined by sampling 100 µL of broth from the last tube with visible growth and from all tubes without visible growth, and plating onto blood-agar plates. The MBC was considered as the lowest antibiotic concentration that reduced the initial inoculum in 99.9%.

Bactericidal activity was studied with the time–kill methodology.11 Tubes with 20 mL of THY broth, with concentrations equivalent to 1 x MIC, 2 x MIC and 4 x MIC of each antimicrobial, were inoculated with ~5 x 105 cfu/mL of S. pneumoniae in the log phase of growth (4 h of incubation at 37°C in air with 10% CO2). As growth controls, tubes containing 20 mL of THY broth with bacterial inoculum and without antimicrobial were used, and a tube with 20 mL of THY broth with neither inoculum nor antimicrobials was used as a sterile control. The tubes were incubated at 37°C in air with 10% CO2. The number of cfu/mL was counted in the antimicrobial and control tubes at the initial time (0 h) and at different incubation times (2, 4, 6, 8 and 24 h). An antibiotic was considered bactericidal when a reduction in the number of cfu/mL ≥3 log10 was obtained during the first 8 h of incubation. The count at 24 h of incubation was not considered because of the possible influence of the autolysis of the S. pneumoniae strains. All the assays were performed in duplicate.

Pharmacokinetic study

Pharmacokinetic parameters for each antimicrobial were determined using groups of 2–5 healthy animals. The following dosages of antimicrobials were tested: penicillin G procaine 50 000 and 200 000 U/kg, cefotaxime 50 mg/kg and ceftriaxone 40 mg/kg. After a single intramuscular (im) dose of antimicrobial, blood samples were extracted from the marginal ear vein at 15, 30, 60, 120, 240 and 480 min, with an additional extraction at 720 min in the case of penicillin and ceftriaxone. The blood was centrifuged for 10 min at 3000 rpm. The plasma obtained was distributed in two aliquots and frozen at –70°C for subsequent study.

The plasma determinations were performed by bioassay, using the disc-plate method,12 in antibiotic agar no. 5 supplemented with 10 g/L of CaCl2 adjusted to pH 7 and Micrococcus luteus ATCC 9341 as the control strain. The determinations were carried out in triplicate. The intraday and interday variations of the assays were 5.19 ± 2.05% and 7.30 ± 3.48% for penicillin, 4.41 ± 1.50% and 4.00 ± 1.01% for cefotaxime, and 5.33 ± 2.94% and 3.00 ± 1.80% for ceftriaxone; the linearity (r2) of the assay was 0.92 ± 0.61, 0.92 ± 0.05 and 0.96 ± 0.01, respectively; and the lower limits of detection were 0.05, 0.1 and 0.5 mg/L, respectively.

Cmax (mg/L) was defined as the peak plasma concentration after drug administration. Terminal half-life (t1/2, h) was calculated as 0.693 divided by the elimination rate constant. The PKCALC program was used.13 Time in which plasma concentration remains above the MIC values ({Delta}T/MIC, h) was estimated by extrapolation from the regression line of serum elimination.14

Experimental model

Experimental aortic endocarditis was established in New Zealand rabbits (weight, 2–3 kg; B&K Universal, Barcelona, Spain) by the Perlman and Freedman15 method modified by Durack and Beeson.16 Animals were anaesthetized with a mixture of ketamine (30 mg/kg) plus xylazine (4 mg/kg) injected im. After exposing the right carotid artery, a sterile polyethylene catheter (PE90; Intramedic; Clay Adams, Becton Dickinson & Co., Sparks, MD, USA) was inserted into the artery through a small incision and advanced proximally across the aortic valve into the left ventricle. A pressure-sensitive monitoring device was attached to the distal end of the catheter to ensure that the catheter tip crossed the aortic valve and entered the left ventricle. At 24 h after insertion of the catheter, 1 mL of broth containing 107–108 cfu of S. pneumoniae was injected through the ear marginal vein. In order to know the size of the inoculum, serial dilutions were grown on blood-agar plates.

Treatment groups

Antimicrobial therapy was started 24 h after the inoculation. The presence of bacteraemia was confirmed by a blood culture yielding pneumococci obtained before the initiation of treatment. The rabbits infected with each strain were assigned to the following treatment groups: (i) no treatment; (ii) penicillin G procaine, 50 000 U/kg im twice daily (S and I strains) and 200 000 U/kg im twice daily (R strain); (iii) cefotaxime, 50 mg/kg im thrice daily; (iv) ceftriaxone, 40 mg/kg im twice daily. Antimicrobial therapy was given for 3 days. The dosages of penicillin, cefotaxime and ceftriaxone were selected to reach a {Delta}T/MIC at least >50% of the interval between doses for all three strains.

Analysis of efficacy of the treatment

At the end of the treatment period, 12–18 h after the administration of the last dose of antimicrobial, blood was extracted for quantitative blood cultures. Immediately, animals were sacrificed by intravenous (iv) injection of sodium pentobarbital. The chest was opened, the heart was extracted, and the aortic valve and valve vegetations were removed aseptically. After weighing and homogenizing the tissues in 1 mL of physiological serum (Stomacher Lab-Blender 80, Seward Medical, London, UK), dilutions were performed in saline serum from 10–1–10–8, and 50 µL of each dilution was grown on blood-agar plates. At 24 h of inoculation, the number of colonies was counted and recorded as log10 cfu/g of vegetation. When the cultures were sterile, we plated the total residuum of the homogenized vegetations and assigned the log10 corresponding to the sensitivity level of the method (log10 1 cfu/weight of the residuum). Survival of the animals during the treatment period was also measured.

Statistical analysis

The means of the log10 cfu/g of tissue of the different treatment groups were compared by ANOVA. If the difference was significant, the comparison among treatment groups was made using Dunnett and Tukey-Kramer post hoc tests. Frequency of sterile tissues and survival were analysed by Fisher's exact test. The statistical package SPSS version 12.0 (SPSS Inc., Chicago, IL, USA) was used.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro studies

The MICs and MBCs of the different antimicrobials for the three strains of S. pneumoniae are shown in Table 1. The three strains were susceptible to cefotaxime and ceftriaxone.


View this table:
[in this window]
[in a new window]
 
Table 1. MICs and MBCs of antimicrobials for the strains of S. pneumoniae

 
In time–kill curves for the S strain, penicillin did not show bactericidal activity with concentrations of 1 x MIC and 2 x MIC; with a 4 x MIC concentration, bactericidal activity was reached at 8 h (Figure 1). For the I strain, bactericidal activity of penicillin was observed with concentrations of 4 x MIC at 4 h (Figure 2). For the R strain, penicillin was bactericidal at 8 h with concentrations of 2 x MIC, and at 6 h with concentrations of 4 x MIC (Figure 3).



View larger version (13K):
[in this window]
[in a new window]
 
Figure 1. Bactericidal activity of penicillin, cefotaxime and ceftriaxone against the susceptible strain of S. pneumoniae, using 2 x MIC and 4 x MIC. Control, filled diamonds; penicillin 2 x MIC, filled squares; penicillin 4 x MIC, open squares; cefotaxime 2 x MIC, filled triangles; cefotaxime 4 x MIC, open triangles; ceftriaxone 2 x MIC, filled circles; ceftriaxone 4 x MIC, open circles.

 


View larger version (13K):
[in this window]
[in a new window]
 
Figure 2. Bactericidal activity of penicillin, cefotaxime and ceftriaxone against the intermediate strain of S. pneumoniae, using 2 x MIC and 4 x MIC. Control, filled diamonds; penicillin 2 x MIC, filled squares; penicillin 4 x MIC, open squares; cefotaxime 2 x MIC, filled triangles; cefotaxime 4 x MIC, open triangles; ceftriaxone 2 x MIC, filled circles; ceftriaxone 4 x MIC, open circles.

 


View larger version (14K):
[in this window]
[in a new window]
 
Figure 3. Bactericidal activity of penicillin, cefotaxime and ceftriaxone against the resistant strain of S. pneumoniae, using 2 x MIC and 4 x MIC. Control, filled diamonds; penicillin 2 x MIC, filled squares; penicillin 4 x MIC, open squares; cefotaxime 2 x MIC, filled triangles; cefotaxime 4 x MIC, open triangles; ceftriaxone 2 x MIC, filled circles; ceftriaxone 4 x MIC, open circles.

 
With regard to the cephalosporins, for the S and R strains, they did not have a bactericidal effect against these strains in the first 8 h. For the I strain, cefotaxime was bactericidal at 8 h with all concentrations, and at 6 h with concentrations of 2 x MIC and 4 x MIC, and ceftriaxone did not show bactericidal activity against this strain (Figure 2).

Pharmacokinetic study

Cmax, t1/2 and {Delta}T/MIC values are shown in Table 2.


View this table:
[in this window]
[in a new window]
 
Table 2. Pharmacokinetic/pharmacodynamic parameters of the different antimicrobials in rabbits

 
Efficacy of the treatment in experimental endocarditis

In the groups inoculated with the S strain, all the treatments were equally efficacious with respect to the controls in the clearance of log10 cfu/g of vegetation (P < 0.01), in the sterilization of vegetations (P < 0.01), and in the sterilization of blood cultures (P < 0.01) (Table 3). Both blood and vegetations were sterile for all the antimicrobials (Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. Number of bacteria in the aortic vegetations (log10 cfu/g, ±SD), percentage of sterile vegetations, and number of bacteria in the blood cultures obtained prior to sacrifice (log10 cfu/mL, ±SD), in the different therapeutic groups

 
In the groups inoculated with the I strain, all the treatments were effective with respect to the controls in the clearance of log10 cfu/g (P < 0.01) and in the sterilization of vegetations (P < 0.01) (Table 3). The blood cultures were sterile for all the antimicrobials (Table 3).

For the groups inoculated with the R strain, all the treatments were efficacious with respect to the controls in the clearance and in the sterilization of vegetations (P < 0.01), and in the sterilization of blood cultures (P < 0.01) (Table 3).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this work show that penicillin is as efficacious as cefotaxime and ceftriaxone in the treatment of experimental pneumococcal endocarditis, caused by penicillin-susceptible and -resistant strains (MIC ≤ 2 mg/L), in the clearance of bacteria from vegetations and blood. In terms of their pharmacodynamics, the efficacy of ß-lactams is time-dependent.17 In the present study we showed that penicillin and both cephalosporins reached plasma concentrations above the MIC for >50% of the interval between doses, which explains the efficacy of all the antimicrobials in this model. These results support the change in the breakpoints for susceptibility and resistance of pneumococci to third-generation cephalosporins made in 2002,18 which classify the three strains as susceptible to cefotaxime and ceftriaxone. On the contrary, with the previous criteria the strain resistant to penicillin had been classified as intermediate to these cephalosporins.19

The results of the present study are similar to those found by Fernández-Guerrero et al.20 who only used strains intermediate and resistant to penicillin. These authors also found that penicillin was useful in an experimental endocarditis model caused by pneumococcal strains with MICs of 1 and 4 mg/L; they correlate its results with the ratio of the Cmax to MIC for the strains, without estimating the time above the MIC because they only studied the Cmax and the trough levels of antimicrobials.

Endocarditis developed in all the rabbits independently of the susceptibility to penicillin. The concentration of bacteria in vegetations ({approx} 8 log cfu/g) and blood ({approx} 4 log cfu/mL), was the same with the three strains; the higher mortality found with the strain intermediate to penicillin seems to be strain-dependent, because the serotype was the same as that of the resistant strain. An additional analysis of the usefulness of penicillin in infections caused by intermediate and resistant strains is the fact that we did not find differences comparing the efficacy of penicillin against the endocarditis caused by the susceptible and resistant strains.

In an analysis of 63 cases of pneumococcal endocarditis treated in 15 Spanish hospitals over a period of 21 years,21 cure was achieved in 32 of 47 cases (68%) without concomitant meningitis. Considering the different treatments, cure was obtained in 74% (17 of 23), 63% (10 of 16) and 63% (5 of 8) of those patients treated with high doses of sodium penicillin G, third-generation cephalosporins and vancomycin, respectively. However, in the cases caused by strains with diminished susceptibility to penicillin (I + R) the cure was obtained in 100% (3 of 3), 56% (5 of 9) and 80% (4 of 5) of those treated with penicillin, third-generation cephalosporins and vancomycin, respectively. Although this is a non-randomized study, these clinical results are in accordance with the efficacy of penicillin in our experimental study.

The efficacy of penicillin in pneumonia in human beings, caused by penicillin-resistant strains, has been shown by other authors.8,22 In relation to the treatment of bacteraemic or non-bacteraemic pneumococcal pneumonias caused by strains with a penicillin MIC ≤2 mg/L, it was suggested that they may be treated with iv penicillin, with dosages between 150 000 and 250 000 U per kg/day.8 The results of a retrospective study on the evolution of community-acquired pneumonia, including cases with high and intermediate resistance to penicillin, showed a satisfactory evolution in 87% of patients receiving penicillin.22 Also, in an experimental model of pneumonia in immunocompetent rats caused by a penicillin-resistant strain of S. pneumoniae, penicillin in high doses was as effective as cefotaxime in the outcome.23

All these results, both in experimental models and in clinical studies, are in accordance with the studies of bactericidal activity of penicillin by means of time–kill curves. Comparing the results of the in vivo model from our work with the results obtained from time–kill curves, we could verify that penicillin was the most bactericidal antimicrobial of all ß-lactams. With regard to cephalosporins, the poor in vitro bactericidal activity found in this study is in accordance with the results from others,24 which showed that both penicillin and amoxicillin were more bactericidal than cephalosporins against three strains of S. pneumoniae with different susceptibilities to penicillin.

In summary, our results confirm that penicillin is efficacious in the treatment of pneumococcal infections, including those produced by strains with MICs ≤ 2 mg/L, provided that dosages sufficient to reach a plasma concentration higher than the MIC, for at least 50% of the interval between doses, are used. This conclusion excludes CNS infections, because of the low penetration of ß-lactams through the blood–brain barrier. Recently, internationally used criteria have changed the breakpoints to define susceptibility and resistance to amoxicillin and to third-generation cephalosporins in S. pneumoniae,18,25 based on pharmacokinetic/pharmacodynamic data and on clinical results. In the same way, the different experimental and clinical results, including the present study, suggest that the breakpoints to define susceptibility and resistance to penicillin must be changed, which may help physicians choose the best treatment for pneumococcal infections.


    Footnotes
 
{dagger} Present address. Infectious Diseases Section, Internal Medicine Service, Punta de Europa Hospital, Algeciras (Cádiz), Spain Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. Yu VL, Chiou CCC, Feldman C et al. An international prospective study of pneumococcal bacteremia: correlation with in vitro resistance, antibiotics administered, and clinical outcome. Clin Infect Dis 2003; 37: 230–7.[CrossRef][ISI][Medline]

2. Austrian R. Pneumococcal pneumonia. Diagnostic, epidemiologic, therapeutic and prophylactic considerations. Chest 1986; 90: 738–43.[ISI][Medline]

3. Klugman KP. Pneumococcal resistance to antibiotics. Clin Microbiol Rev 1990; 3: 171–96.[ISI][Medline]

4. Perez-Trallero E, García-de-la-Fuente C, García-Rey C et al. Geographical and ecological analysis of resistance, coresistance, and coupled resistance to antimicrobials in respiratory pathogenic bacteria in Spain. Antimicrob Agents Chemother 2005; 49: 1965–72.[Abstract/Free Full Text]

5. Goldstein FW, Acar JF, The Alexander Project Collaborative Group. Antimicrobial resistance among lower respiratory tract isolates of Streptococcus pneumoniae: results of a 1992–93 Western Europe and USA collaborative surveillance study. J Antimicrob Chemother 1996; 38 Suppl A: 71–84.[ISI][Medline]

6. EARSS (European Antimicrobial Resistance Surveillance System). Newsletter no. 4, January 2002. http://www.earss.rivm.nl/ (8 April 2005, date last accessed).

7. Caballero-Granado FJ, Palomino-Nicás J, Pachón J et al. Cefuroxime efficacy in treatment of bacteremic pneumonia due to penicillin-resistant and cefuroxime-resitant Streptococcus pneumoniae. Antimicrob Agents Chemother 1996; 40: 1325–6.[Free Full Text]

8. Pallares R, Liñares J, Vadillo M et al. Resistance to penicillin and cephalosporin and mortality from severe pneumococcal pneumonia in Barcelona, Spain. N Engl J Med 1995; 24: 474–80.

9. Rosón B, Carratalá J, Tubau F et al. Usefulness of ß-lactam therapy for community-acquired pneumonia in the era of drug-resistant Streptococcus pneumoniae: randomized study of amoxicillin-clavulanate and ceftriaxone. Microb Drug Resist 2001; 7: 75–86.

10. National Committee for Clinical Laboratory Standards. Methods For Dilution Antimicrobial Susceptibility Tests For Bacteria That Grow Aerobically—Sixth Edition: Document M7-A6. NCCLS, Wayne, Pennsylvania, USA, 1990.

11. National Committee for Clinical Laboratory Standards. Methods For Determining Bactericidal Activity Of Antimicrobial Agents: Tentative Guideline 771 E. NCCLS, Villanova, PA, USA, 1992.

12. Anhalt JP. Antimicrobial assays. In: Washington JA, ed. Laboratory Procedures In Clinical Microbiology. 2nd edn. New York: Springer-Verlag, 1985; 691–729.

13. Schumaker RC. PKCALC: a basic interactive computer program for statistical and pharmacokinetic analysis of data. Drug Metab Res 1986; 17: 331–48.

14. Fridmodt-Moller N, Bentzon MW, Thomsen VF. Experimental infection with Streptococcus pneumoniae in mice: Correlation of in vitro activity and pharmacokinetic parameters with in vivo effect for 14 cephalosporins. J Infect Dis 1986; 154: 511–7.[ISI][Medline]

15. Perlman BB, Freedman LR. Experimental endocarditis. II. Staphylococcal infection of the aortic valve following placement of a polyethylene catheter in the left side of the heart. Yale J Biol Med 1971; 44: 206–13.

16. Durack DT, Beeson PB. Experimental bacterial endocarditis. Colonization of a sterile vegetation. Br J Exp Pathol 1972; 53: 44–9.[ISI][Medline]

17. Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998; 26: 1–12.[ISI][Medline]

18. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement, vol. 22, no. 1. M100-S12. NCCLS, Wayne, PA, USA, 2002.

19. National Committee for Clinical Laboratory Standards. Performance Standards For Antimicrobial Susceptibility Testing, Methods For Dilution Antimicrobial Susceptibility Test For Bacteria That Grow Aerobically: Eleventh Informational Supplement, vol. 21, no. 1. M100-S11. NCCLS, Wayne, PA, USA, 2001.

20. Fernández-Guerrero ML, Arbol F, Verdejo C et al. Treatment of experimental endocarditis due to penicillin-resistant Streptococcus pneumoniae. Antimicrob Agents Chemother 1994; 38: 1103–6.[Abstract]

21. Martínez E, Miró JM, Almirante B et al. Effect of penicillin resistance of Streptococcus pneumoniae on the presentation, prognosis, and treatment of pneumococcal endocarditis in adults. The Spanish Pneumococcal Endocarditis Study Group. Clin Infect Dis 2002; 35: 130–9.[CrossRef][ISI][Medline]

22. Pallarés R, Gudiol F, Liñares J et al. Risk factors and response to antibiotic therapy in adults with bacteremic pneumonia caused by penicillin resistant pneumococci. N Engl J Med 1987; 317: 18–22.[Abstract]

23. Gavaldá J, Capdevila JA, Almirante B et al. Treatment of experimental pneumonia due to penicillin-resistant Streptococcus pneumoniae in inmunocompetent rats. Antimicrob Agents Chemother 1997; 41: 795–801.[Abstract]

24. Liñares J, Tubau F, Alcaide F et al. Actividad bactericida de cinco antibióticos betalactámicos frente a Streptococcus pneumoniae. Enferm Infecc Microbiol Clin 1993; 11 Suppl 1: 23–7.

25. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Tenth Informational Supplement, vol. 20, no. 1. M100-S10. NCCLS, Wayne, PA, USA, 2000.





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
56/4/732    most recent
dki304v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Pichardo, C.
Articles by Pachón, J.
PubMed
PubMed Citation
Articles by Pichardo, C.
Articles by Pachón, J.