Abstract

Objectives

To study the influence of resistance phenotypes (based on sentinel antibiotics: penicillin and amoxicillin with/without clavulanate) on the cefuroxime versus cefditoren susceptibility of Streptococcus pneumoniae and Haemophilus influenzae recovered from children with acute otitis media.

Methods

Middle ear isolates (193 S. pneumoniae and 114 H. influenzae) received in the Spanish Reference Laboratory (Instituto de Salud Carlos III) were tested. Antimicrobial susceptibility to penicillin, amoxicillin with/without clavulanate, cefuroxime and cefditoren was determined by agar dilution using Mueller–Hinton agar supplemented with 5% sheep blood for S. pneumoniae and Haemophilus Test Medium for H. influenzae. Strains were classified according to penicillin susceptibility (S. pneumoniae) or β-lactamase production (H. influenzae).

Results

The decrease in penicillin susceptibility of S. pneumoniae (from the susceptible to the resistant category) decreased amoxicillin and cefuroxime susceptibility rates from 100% to 34% and 0%, respectively. All pneumococcal strains were inhibited by 0.5 mg/L cefditoren, including those from penicillin-resistant serotypes 14, 23F, 6B and 9V with higher amoxicillin versus penicillin MICs. Susceptibility rates of β-lactamase-positive H. influenzae strains were 93.8% and 85.4% to amoxicillin/clavulanate and cefuroxime, respectively. Resistance to amoxicillin/clavulanate (MIC ≥ 8/4 mg/L) was 12.1% (8 out of 66) and 6.3% (3 out of 48) in β-lactamase-negative and -positive strains, respectively. All H. influenzae strains were inhibited by ≤0.06 mg/L cefditoren.

Conclusions

Susceptibility to sentinel β-lactams cannot predict activity of other members of the group. The addition of clavulanic acid to amoxicillin does not guarantee 100% coverage of H. influenzae, regardless of β-lactamase production.

Introduction

Acute otitis media (AOM) is increasing in frequency in parallel with early school attendance in developed countries, being the most frequent infection among children (60% to 70% of the children experience at least one episode in the first year of life, and by 7 years, the rate is as high as 93%).1 In 70% of patients with AOM, culture of middle ear fluid yields bacterial growth, with Haemophilus influenzae (25% of cases) and Streptococcus pneumoniae (40% of cases) being the most frequent species.2,3 Spontaneous cure rates are 50% in H. influenzae and 20% in Spneumoniae infections.3 Resistant S. pneumoniae is more frequent in middle ear samples and in the paediatric population.4 Owing to penicillin non-susceptibility in S. pneumoniae, high amoxicillin doses have been advocated, with amoxicillin/clavulanic acid as a second-line treatment or in recurrent otitis due to the presence of β-lactamase-producing H. influenzae.1,2,5 Troublesome strains from S. pneumoniae penicillin-resistant clones exhibiting higher amoxicillin versus penicillin MIC,6 and H. influenzae strains exhibiting mutations in the ftsI gene encoding PBP3 (rendering strains resistant to amoxicillin with or without clavulanic acid),7 are increasingly detected, reaching high rates in some European areas.8

Sentinel antibiotics (such as penicillin and/or amoxicillin with/without clavulanate) are included in surveillance studies to detect the presence of particular resistance mechanisms. This may not be enough to provide relevant information to clinicians to establish empirical therapy, because the activity of other agents may not be inferred from their activity.9

Cefditoren is a third-generation oral cephalosporin exhibiting good in vitro activity against S. pneumoniae10 and H. influenzae,11 a peak serum concentration of 4.2 mg/L after a 400 mg single dose, half-life of 1.6 h and a protein binding rate of 88%.12 Cefditoren exhibits high and rapid bactericidal activity against H. influenzae with different resistance phenotypes.13 Against penicillin-resistant S. pneumoniae, bactericidal activity is maintained in the presence of high concentrations of human serum.14 Cefditoren has shown clinical and bacteriological efficacy against S. pneumoniae and H. influenzae in the treatment of lower respiratory tract infections.15,16 We studied the influence of the resistance phenotype (penicillin resistance in S. pneumoniae and ampicillin/amoxicillin resistance in H. influenzae) on the cefuroxime versus cefditoren susceptibility of S. pneumoniae and H. influenzae recovered from children with AOM.

Materials and methods

Antibiotics

Powders of known potency of penicillin (Antibióticos S.A., Madrid, Spain), amoxicillin and lithium clavulanate (GlaxoSmithKline S.A., Tres Cantos, Madrid, Spain), cefuroxime (Sigma-Aldrich Chemical Co., St Louis, USA) and cefditoren (Tedec-Meiji Farma, Alcalá de Henares, Madrid, Spain) were used throughout the study.

Strains

One hundred and ninety-three S. pneumoniae paediatric middle ear isolates received in the Spanish Reference Pneumococcal Laboratory (Instituto de Salud Carlos III) during 1998–2000 were tested. This period prior to the introduction of the seven-valent conjugate vaccine was chosen in order to obtain a high number of penicillin-non-susceptible S. pneumoniae strains. Strains were classified according to penicillin susceptibility: 56 strains susceptible to penicillin (MICs ≤ 0.06 mg/L), 70 strains with intermediate resistance (MICs = 0.12–1 mg/L) and 67 full-resistant strains (MICs ≥ 2 mg/L), according to CLSI (formerly NCCLS) breakpoints.17 Serotyping was performed by Quellung reaction and/or dot blot assay.18

One hundred and fourteen H. influenzae clinical paediatric middle ear isolates received in the Spanish Reference Haemophilus Laboratory (Instituto de Salud Carlos III) were tested. A β-lactamase test using the chromogenic cephalosporin method (Nitrocefin™, Becton-Dickinson, Cockeysville, MD, USA) was performed. Strains were classified according to β-lactamase production: 66 were β-lactamase-negative and 48 were β-lactamase-positive.

In vitro susceptibility

Antimicrobial susceptibility to penicillin, amoxicillin, cefuroxime and cefditoren in S. pneumoniae and that to amoxicillin, amoxicillin/clavulanic acid (2:1), cefuroxime and cefditoren in H. influenzae was determined by the agar dilution method, as described by the CLSI guidelines,19 by using as culture media Mueller–Hinton agar (Difco Laboratories, Detroit, MI, USA) supplemented with 5% sheep blood (Biomedics, Madrid, Spain) for S. pneumoniae and Haemophilus Test Medium (Mueller–Hinton agar supplemented with 15 mg/L NAD, 15 mg/L haemin and 5 mg/mL yeast extract; Difco Laboratories) for H. influenzae. S. pneumoniae ATCC 6303, S. pneumoniae ATCC 49619, Escherichia coli ATCC 25922 and H.influenzae ATCC 49247 were used as quality controls.

Concentrations tested for all antimicrobials ranged from ≤0.007 to 16 mg/L. Breakpoints (susceptibility/resistance; mg/L) considered were those defined by CLSI17: [S. pneumoniae (amoxicillin ≤2/≥8; cefuroxime-axetil ≤1/≥4); H. influenzae (amoxicillin/clavulanic acid ≤4/≥8; cefuroxime-axetil ≤4/≥16)]. There are no defined breakpoints for cefditoren.

Results and discussion

Table 1 shows the MIC distribution and cumulative percentage distribution of S. pneumoniae strains by penicillin susceptibility and of H. influenzae strains by β-lactamase production.

Table 1

Distribution of MICs (mg/L) of the antimicrobials tested according to penicillin susceptibility or β-lactamase production [number of isolates inhibited (cumulative percentage) (MIC50 and MIC90 values are in bold)]

 ≤0.007 0.015 0.03 0.06 0.12 0.25 0.5 ≥16 
S. pneumoniae penicillin-susceptible strains (MIC ≤ 0.06 mg/L) (n = 56) 
 penicillin — 26 (46.9) 20 (82.1) 10 (100) — — — — — — — — 
 amoxicillin — 5 (8.9) 35 (71.4) 11 (91.1) 3 (96.4) 2 (100) — — — — — — 
 cefuroxime — 6 (10.7) 31 (66.1) 4 (73.2) 9 (89.3) 4 (96.4) 2 (100) — — — — — 
 cefditoren 25 (44.6) 16 (73.2) 9 (89.3) 5 (98.2) 1 (100) — — — — — — — 
S. pneumoniae penicillin-intermediate strains (MIC = 0.12–1 mg/L) (n = 70) 
 penicillin — — — — 9 (12.9) 15 (34.3) 18 (60.0) 28 (100) — — — — 
 amoxicillin — — — 1 (1.4) 4 (7.1) 9 (20.0) 14 (40.0) 16 (62.9) 21 (92.9) 5 (100) — — 
 cefuroxime — — — — — 3 (4.3) 13 (22.9) 14 (42.9) 17 (67.1) 22 (98.6) 1 (100) — 
 cefditoren — — 3 (4.3) 15 (25.7) 19 (52.9) 24 (87.1) 9 (100— — — — — 
S. pneumoniae penicillin-resistant strains (MIC ≥ 2 mg/L) (n = 67) 
 penicillin — — — — — — — — 56 (83.6) 11 (100) — — 
 amoxicillin — — — — — — — — 23 (34.3) 13 (53.7) 20 (83.6) 11 (100) 
 cefuroxime — — — — — — — — 1 (1.5) 28 (43.3) 34 (94.0) 4 (100) 
 cefditoren — — — — — 19 (28.4) 48 (100) — — — — — 
β-Lactamase-negative H. influenzae (n = 66) 
 amoxicillin — — — — — — 6 (9.1) 30 (54.5) 9 (68.2) 11 (84.8) 3 (89.4) 7 (100) 
 amoxicillin/clavulanate — — — — — — 5 (7.6) 35 (60.6) 12 (78.8) 6 (87.9) 3 (92.4) 5 (100) 
 cefuroxime — — — — — — 11 (16.7) 21 (48.5) 12 (66.7) 5 (74.2) 12 (92.4) 5 (100) 
 cefditoren 10 (15.2) 26 (54.5) 19 (83.3) 11 (100) — — — — — — — — 
β-Lactamase-positive H. influenzae (n = 48) 
 amoxicillin — — — — — — — — — — — 48 (100) 
 amoxicillin/clavulanate — — — — — — — 10 (20.8) 21 (64.6) 14 (93.8) 1 (95.8) 2 (100) 
 cefuroxime — — — — — — 12 (25.0) 16 (58.3) 9 (77.1) 4 (85.4) 6 (97.9) 1 (100) 
 cefditoren 9 (18.8) 17 (54.2) 15 (85.4) 7 (100) — — — — — — — — 
 ≤0.007 0.015 0.03 0.06 0.12 0.25 0.5 ≥16 
S. pneumoniae penicillin-susceptible strains (MIC ≤ 0.06 mg/L) (n = 56) 
 penicillin — 26 (46.9) 20 (82.1) 10 (100) — — — — — — — — 
 amoxicillin — 5 (8.9) 35 (71.4) 11 (91.1) 3 (96.4) 2 (100) — — — — — — 
 cefuroxime — 6 (10.7) 31 (66.1) 4 (73.2) 9 (89.3) 4 (96.4) 2 (100) — — — — — 
 cefditoren 25 (44.6) 16 (73.2) 9 (89.3) 5 (98.2) 1 (100) — — — — — — — 
S. pneumoniae penicillin-intermediate strains (MIC = 0.12–1 mg/L) (n = 70) 
 penicillin — — — — 9 (12.9) 15 (34.3) 18 (60.0) 28 (100) — — — — 
 amoxicillin — — — 1 (1.4) 4 (7.1) 9 (20.0) 14 (40.0) 16 (62.9) 21 (92.9) 5 (100) — — 
 cefuroxime — — — — — 3 (4.3) 13 (22.9) 14 (42.9) 17 (67.1) 22 (98.6) 1 (100) — 
 cefditoren — — 3 (4.3) 15 (25.7) 19 (52.9) 24 (87.1) 9 (100— — — — — 
S. pneumoniae penicillin-resistant strains (MIC ≥ 2 mg/L) (n = 67) 
 penicillin — — — — — — — — 56 (83.6) 11 (100) — — 
 amoxicillin — — — — — — — — 23 (34.3) 13 (53.7) 20 (83.6) 11 (100) 
 cefuroxime — — — — — — — — 1 (1.5) 28 (43.3) 34 (94.0) 4 (100) 
 cefditoren — — — — — 19 (28.4) 48 (100) — — — — — 
β-Lactamase-negative H. influenzae (n = 66) 
 amoxicillin — — — — — — 6 (9.1) 30 (54.5) 9 (68.2) 11 (84.8) 3 (89.4) 7 (100) 
 amoxicillin/clavulanate — — — — — — 5 (7.6) 35 (60.6) 12 (78.8) 6 (87.9) 3 (92.4) 5 (100) 
 cefuroxime — — — — — — 11 (16.7) 21 (48.5) 12 (66.7) 5 (74.2) 12 (92.4) 5 (100) 
 cefditoren 10 (15.2) 26 (54.5) 19 (83.3) 11 (100) — — — — — — — — 
β-Lactamase-positive H. influenzae (n = 48) 
 amoxicillin — — — — — — — — — — — 48 (100) 
 amoxicillin/clavulanate — — — — — — — 10 (20.8) 21 (64.6) 14 (93.8) 1 (95.8) 2 (100) 
 cefuroxime — — — — — — 12 (25.0) 16 (58.3) 9 (77.1) 4 (85.4) 6 (97.9) 1 (100) 
 cefditoren 9 (18.8) 17 (54.2) 15 (85.4) 7 (100) — — — — — — — — 

With respect to pneumococci, all penicillin-susceptible strains were susceptible to amoxicillin and cefuroxime, and all were inhibited by cefditoren concentrations ≤0.12 mg/L. In the penicillin intermediate resistant category, susceptibility rates decreased to 92.9% for amoxicillin and to 42.9% for cefuroxime, all strains being inhibited by 0.5 mg/L of cefditoren. In the penicillin-resistant category, susceptibility rates further decreased to 34% for amoxicillin and to 0% for cefuroxime. Against these resistant strains, the intrinsic activity in terms of MIC90 was cefditoren (0.5 mg/L) > penicillin (4 mg/L) > cefuroxime (8 mg/L) > amoxicillin (16 mg/L).

Table 2 shows MIC50, MIC90 and percentage of susceptibility of S. pneumoniae serotypes with 10 or more isolates. Serotypes 6B, 14 and 23F were the most troublesome with very low susceptibility rates to penicillin or cefuroxime (<33%) and low susceptibility rates to amoxicillin (ranging from 38.5% to 68.8%).

Table 2

MIC50, MIC90 (mg/L) and percentage of susceptibility of S. pneumoniae serotypes with 10 or more isolates

Serotype n Penicillin Amoxicillin Cefuroxime Cefditoren 
MIC50 MIC90 %S MIC50 MIC90 %S MIC50 MIC90 %S MIC50 MIC90 
13 0.015 0.03 92.3 0.03 0.06 100 0.03 0.03 100 0.007 0.015 
6A 10 0.06 0.5 50.0 0.25 100 0.25 80.0 0.03 0.12 
6B 32 3.1 68.8 21.9 0.25 0.5 
14 40 2.5 45.0 2.5 0.5 0.5 
19 56 0.5 32.1 96.4 67.9 0.12 0.5 
23F 13 0.0 16 38.5 0.0 0.5 0.5 
Serotype n Penicillin Amoxicillin Cefuroxime Cefditoren 
MIC50 MIC90 %S MIC50 MIC90 %S MIC50 MIC90 %S MIC50 MIC90 
13 0.015 0.03 92.3 0.03 0.06 100 0.03 0.03 100 0.007 0.015 
6A 10 0.06 0.5 50.0 0.25 100 0.25 80.0 0.03 0.12 
6B 32 3.1 68.8 21.9 0.25 0.5 
14 40 2.5 45.0 2.5 0.5 0.5 
19 56 0.5 32.1 96.4 67.9 0.12 0.5 
23F 13 0.0 16 38.5 0.0 0.5 0.5 

Forty-three penicillin-resistant pneumococci exhibited higher amoxicillin MIC when compared with that of penicillin. Of them, 51.2% of the isolates were serotype 14, 18.7% serotype 23F, 16.3% serotype 6B and 9.3% serotype 9V. Susceptibility rates to amoxicillin and cefuroxime among these isolates belonging to serotypes 14, 23F, 6B and 9V and exhibiting higher amoxicillin versus penicillin MIC were 0%, all of them being inhibited by cefditoren concentrations of 0.5 mg/L.

The results of our study show some differences when compared with those of previous studies20 with respect to cefditoren activity against penicillin-resistant pneumococci. Although no differences were found between penicillin-susceptible and penicillin-intermediate strains, or regarding MIC50 for penicillin-resistant strains, 2 dilutions lower cefditoren MIC90 values were found in the current study for penicillin-resistant strains. Other studies testing S. pneumoniae Spanish isolates from the last decade by microdilution reported values in the range of 0.5–2 mg/L: MIC90 = 1 mg/L for penicillin/amoxicillin-resistant strains and 0.5 mg/L for penicillin-non-susceptible strains.21 When comparing the results in the present study with those in a study testing by agar dilution Spanish isolates from 2005, similar MIC90 values were obtained with only 3.9% of isolates exhibiting a cefditoren MIC of 1 or 2 mg/L.22 Maybe, as with other β-lactams,23 a shift to higher MIC values occurs with microdilution versus agar dilution, mainly in strains exhibiting the highest MICs.

The resistance profile in the present study among penicillin-resistant isolates with low susceptibility rates to amoxicillin (23 of 67, 34.3%) and high rates of full amoxicillin resistance (31 of 67, 46.3%) suggests that an increase in amoxicillin doses may not be enough for a complete coverage of the entire spectrum of resistance in S. pneumoniae.

The addition of clavulanic acid to amoxicillin is advocated in recurrent otitis, because of the implication of β-lactamase-positive H. influenzae in 20% of otitis. The in vitro activity of the four β-lactams tested against H. influenzae is shown in Table 1. Cefditoren exhibited the highest intrinsic activity with MIC90 values of 0.06 mg/L against β-lactamase-positive or -negative strains. Susceptibility rates to amoxicillin/clavulanate and cefuroxime were 87.9% and 74.2% for β-lactamase-negative strains and 93.8% and 85.4% for β-lactamase-positive strains, respectively. No CLSI breakpoints were defined for amoxicillin and H. influenzae.

In Spain, amoxicillin resistance due to β-lactamase production has remained constant at a 20% to 25% rate over three successive nationwide surveillances (1996–97, 1998–99, 2001–02) of the SAUCE Program.24,25 The BLNAR (β-lactamase-negative ampicillin-resistant) phenotype was 9% and 4.5% in the second and third surveillances.24,25 The BLPACR (β-lactamase-positive amoxicillin/clavulanate-resistant) phenotype was not detected in the first surveillance and very infrequent (0.1%) in the other two.24,25

As the mechanism of resistance of BLNAR strains consists of mutations in the ftsI gene encoding PBP3, these strains should be considered resistant to amoxicillin/clavulanate and cefuroxime, in addition to ampicillin/amoxicillin.17 By applying the CLSI breakpoint for amoxicillin/clavulanate (there is no defined breakpoint for amoxicillin), 8 out of 66 (12.1%) β-lactamase-negative H. influenzae strains exhibited resistance to this antibiotic (MIC ≥ 8 mg/L), all of them inhibited by cefditoren concentrations ≤0.06 mg/L.

Three out of the 48 (6.3%) β-lactamase-positive strains exhibited amoxicillin/clavulanate MICs ≥8 mg/L (BLPACR phenotype); of them, two were intermediate resistant and one susceptible to cefuroxime and all were inhibited by cefditoren concentrations ≤0.06 mg/L.

It has been suggested that BSAC breakpoints for amoxicillin/clavulanate (susceptible: ≤1/resistant: ≥2 mg/L)26 are moreadequate to detect ftsI gene mutations7 and to predict pharmacodynamic activity13 against BLNAR and BLPACR phenotypes. Mutations in the ftsI gene have been detected in β-lactamase-negative H. influenzae strains even with ampicillin MICs of 1–2 mg/L.7 However, the rate of this type of mutations among β-lactamase-positive strains may be underestimated when only qualitative susceptibility to ampicillin and chromogenic β-lactamase detection is performed on a daily practice basis. In this sense, when using the amoxicillin/clavulanate BSAC resistance breakpoint (MIC ≥ 2 mg/L),26 26 isolates out of the 66 β-lactamase-negative and 38 isolates out of the 48 β-lactamase-positive strains exhibited MICs ≥2 mg/L, stressing the need for performing genetic surveillance and sequencing ftsI genes among β-lactamase-positive strains and β-lactamase-negative strains with ampicillin MICs of ≥2 mg/L.

Although this study was not designed as an epidemiological surveillance, the results are in accordance with other studies, showing an increase in the prevalence of these phenotypes in some geographical areas8 and in the paediatric population.27

In conclusion, susceptibility to sentinel β-lactams cannot predict activity of other members of the group against S. pneumoniae, and the addition of clavulanic acid to amoxicillin may not guarantee 100% coverage of H. influenzae, regardless of β-lactamase production.

Acknowledgements

We thank Federico Román (Spanish National Reference Haemophilus Laboratory, Instituto de Salud Carlos III) for providing the H. influenzae strains. This study was supported by an unrestricted grant from Tedec-Meiji S.A., Alcalá de Henares, Madrid, Spain.

Transparency declarations

L. A. has received a fee for speaking at sponsored symposia from Sociedad Española de Quimioterapia and GlaxoSmithKline S.A., and L. A. and M.-J. G. have received funds for research from GlaxoSmithKline S.A. and Novartis farmacéutica S.A., Barcelona, Spain.

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