INVESTIGATOR. R. Vanhoof 1, Engelandstraat 642, B-1180 Brussel.

Transcription

1 REPORT. In vitro study to investigate the antimicrobial activity of various antibiotics against noninvasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2-3 (SP3). INVESTIGATOR. R. Vanhoof 1, 1 Pasteurinstituut - Brussel, Eenheid Antibiotica-Onderzoek, Engelandstraat 642, B-118 Brussel. «No part of this report - including graphs, figures, tables may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing form from an authorized person representing the Scientific Institute of Public Health» September 3 1

2 REPORT. In vitro study to investigate the antimicrobial activity of various antibiotics against noninvasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2-3 (SP3). INVESTIGATOR. R. Vanhoof 1, PARTICIPANTS M. Carpentier 2, S. Damée 1, O. Fagnart 3, Y. Glupczynski 4, P. Goffinet 5, D. Govaerts 6, Ph. Lefèvre 7, M. Lontie 8, K. Magerman 9, I. Mans 1, F. Meunier, F. Moonens 11, I. Surmont 12, E. Van Bossuyt 1, M. Van De Vyvere 13, H. Van Landuyt 14, L. Van Nimmen 15, and R. Van Noyen 16. INSTITUTES. 1 Pasteurinstituut - Brussel, Eenheid Antibiotica-Onderzoek, Engelandstraat 642, B-118 Brussel. 2 Hôpital de la Citadelle, Laboratoire de Microbiologie, B-4 Liège. 3 Clinique Saint Etienne, Laboratoire Van Den Abbeele, B-12 Bruxelles. 4 Clinique Universitaire de Mont-Godinne, Laboratoire de Microbiologie, B-553 Yvoir. 5 Clinique St.Joseph, Laboratore de Microbiologie, B-67 Arlon. 6 C.H.U. André Vésale, Laboratoire de Microbiologie, B-61 Montignies-le-Tilleul. 7 Hôpital Princesse Paola, Laboratore de Microbiologie, B-69 Marche-en-Famenne. 8 Medisch Centrum Huisartsen, B-3 Leuven. 9 Virga-Jesseziekenhuis, Laboratorium Microbiologie, B-35 Hasselt. Hôpital de Jolimont, Laboratore de Microbiologie, B-7 Haine-St-Paul. 11 RHMS site Louis Caty, Laboratore de Microbiologie, B-7331 Baudour. 12 H. Hartziekenhuis, Laboratorium Microbiologie, B-88 Roeselare. 13 A.Z. Stuivenberg, Laboratorium Microbiologie, B-6 Antwerpen. 14 A.Z. St. Jan, Laboratorium Microbiologie, B-8 Brugge. 15 A.Z. Jan Palfijn, Laboratorium Microbiologie, B-9 Gent. 16 Imeldaziekenhuis, Laboratorium Microbiologie, B-28 Bonheiden. ADDRESS FOR CORRESPONDENCE. R. Vanhoof, Pasteurinstituut - Brussel, Eenheid Antibiotica-Onderzoek, Engelandstraat 642, B-118 Brussel. 2

3 MATERIAL AND METHODS. Serial, non-duplicated clinical isolates of S. pneumoniae, collected during winter 2-3 in 15 clinical laboratories throughout Belgium, were included in this study. Identification of the isolates was routinely performed by the participating laboratories. Each participating centre was asked to collect 3 consecutive isolates, which were kept at -7 C in Brain Heart Infusion Broth (Difco) containing % (v/v) glycerol until susceptibility testing at the Pasteur Institute - Brussels in Brussels. The following antibiotics were tested in the study and were provided as laboratory preparations with known potency: penicillin G, ampicillin, amoxicillin, cefuroxime, and clavulanic acid (GlaxoSmithKline), cefaclor (Eli Lilly), cefotaxime, levofloxacin, ofloxacin and telithromycin (Aventis Pharma), imipenem and ertapenem (Merck Sharp & Dohme), ciprofloxacin and moxifloxacin (Bayer), azithromycin (Pfizer). Clindamycin, erythromycin and tetracycline were obtained from a commercial source (Sigma). Amoxicillin/clavulanic acid was tested in a 2:1 ratio. All antibiotics were tested for 16 serial twofold dilutions (.1 32 µg/ml). The Minimal Inhibitory Concentrations (MIC) were determined by broth microdilution as recommended by the NCCLS. All isolates underwent a slide agglutination (Slidex pneumo Kit TM, BioMérieux) before MIC testing. S. pneumoniae ATCC 49619, S. pneumoniaetpn881 (internal control isolate) and Staphylococcus aureus NCTC (ßlactamase positive to validate the clavulanate component of amoxicillin/clavulanate) were included as quality control organisms in each series. Interpretation of the results was based on breakpoints provided by the NCCLS. The Chi-square test, with or without Yates correction, for two independent samples was used for the statistical evaluation of the results. The level of significance was set at.5. RESULTS. Bacterial isolates. A total of 492 isolates were collected by the different laboratories. However, 98 isolates (19.9 %) were withdrawn from the study for various reasons: 36 isolates (7.3 %) were not in agreement with the study protocol, 31 isolates (6.3 %) did not grow, 22 isolates (4.5 %) were negative on slidex agglutination and 9 isolates (1.8 %) were heavily contaminated. Finally, 394 documented isolates of S. pneumoniae were included in the study for further analysis. 3

4 Information on age was not available for one isolate. Thirty point five percent (1/393) of the isolates were from children (age 15 years) with 91/1 or 75.8 % from children under 3 years of age, while 69.5 % (273/393) were from adults with 181/273 or 66.3 % from adults with age 6 years. Age showed a bimodal distribution with a first peak between and years (28.4 %) and a second peak between 62 and 82 year (37.4 %). The mean age of the study population was 45.4 years. The age distribution differed significantly for patients from 5 laboratories. In the laboratories D, B and M, the mean patient age was 9.3, 24.7 and 26.5 respectively while in laboratories K and J the mean age was 67.1 and 67.7 respectively. Information on sample site was not available for one isolate. Isolates from sputum represented 66.7 % (262/393) of the specimens, while 15.5 % (61/393) were from nasal swab; 6.9 % from nasopharyngeal aspirate, 5.6 % (22/393) from pus, 3.5 % (14/393) from throat and 1.8 % (7/393) from sinus. Isolates from sputum (63.7 % or 167/262) were significantly more present in patients of the age group 6 years (P <.1), while the upper respiratory tract specimens (i.e. nasal swab, nasopharyngeal samples, throat and sinus) (65.1% or 71/9) were significantly more collected from patients in the age group 3 years (P <.1). Isolates from hospitalised patients represented 62.9 % (248/394) of the isolates while 36.6 % (144/394) were from ambulatory patients and.5% (2/394) from long care facility patients. Sixty one point nine percent 244/394) of the isolates were from male patients. The geographic distribution of the isolates is bases on the postal code of the patient. Isolates from the Northern part of the country represented 49. % (193/394) while 41.1 % (162/394) and 9.9 % (39/394) of the isolates came from patients from the Southern part and Brussels respectively. MIC determinations. The results of the MIC determinations for the various compounds are summarized in Table 1. The highest activity on a weight basis was found for imipenem and telithromycin (MIC 5, or MIC for 5% of the isolates tested, of.15 µg/ml) followed by amoxicillin, amoxicillin/clavulanic acid, cefotaxim and ertapenem (MIC 5 of.3 µg/ml), penicillin G, ampicillin, cefuroxime, and moxifloxacin (MIC 5 of.6 µg/ml), erythromycin, azithromycin and clindamycin (MIC 5 of.12 µg/ml), cefaclor and tetracycline (MIC 5 of.5 µg/ml) and ciprofloxacin, levofloxacin and ofloxacin (MIC 5 of 1. µg/ml). The interpretive categories for susceptibility and resistance are designated by colours in Table 1 (green is intermediate, red is resistant; isolates with MIC 1 µg/ml for ertapenem were considered as susceptible). 4

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6 Moxifloxacin, ertapenem and telithromycin were the compounds with the highest level of susceptibility at the recommended NCCLS breakpoints (99.4, 99.2 and 99. % resp.) followed by amoxicillin/clavulanic acid and amoxicillin (96.9 %), levofloxacin (96.7 %), cefotaxime (92.6 %) and imipenem (91.6%). The lowest degrees of susceptibilitiy were found for tetracycline, the macrolides and clindamycin. The aggregate rate of susceptible and intermediate isolates, i.e. the overall percentages of isolates likely to respond to increased dosage in approved clinical situations, was 99.7 for amoxicillin, amoxicillin/clavulanic acid, and moxifloxacin, 99.5 % for telithromycin, 99.2 % for ertapenem and 99. % for imipenem,. Resistance rates. Table 2 indicates the levels of susceptibility and resistance following the NCCLS guidelines while a comparison with the 1 surveillance study is summarized in Table 3. TABLE 2 : SUSCEPTIBILITY RATES FOLLOWING NCCLS CRITERIA OF 391 ISOLATES OF S. pneumoniae. (SP3) Antibiotic Susceptibility Rates (in %) 1 Susceptible Intermediate Resistant Penicillin Ampicillin Amoxicillin Amoxicillin/clavulanate Cefaclor Cefuroxime Cefuroxime-axetil Cefotaxime Imipenem Ertapenem 99.2 *.8 Ciprofloxacin Levofloxacin Moxifloxacin Ofloxacin Erythromycin Azithromycin Telithromycin Clindamycin Tetracycline See comment Table 1. 2 Cefuroxime-axetil: oral form of Cefuroxime 6

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8 The overall rate of decreased susceptibility to penicillin (MIC.12 µg/ml) was 15.7 % (62/394) with 6.6 % (26/394) of the isolates showing intermediate (.12 µg/ml MIC 1 µg/ml) and 9.1 % (35/394) showing high level resistance (MIC 2 µg/ml). The overall rate of decreased susceptibility to amoxicillin and amoxicillin/clavulanate (MIC 4 µg/ml) was 3.1 % (12/394) with 2.8 % (11/394) intermediate resistance and.3 % (1/394) high level resistance. The overall rate of decreased susceptibility to cefotaxim was 7.4 % (29/394) comprising 5.1 % (/394) with intermediate resistance (MIC = 2 µg/ml) and 2.3 % (9/394) with high-level resistance (MIC 4 µg/ml). A total of 8.4 % (33/394) of the isolates were found to be insusceptible to imipenem (MIC.25 µg/ml). Twenty nine (7.4 %) of these isolates showed intermediate resistance (.25 µg/ml MIC.5 µg/ml). Ertapenem insusceptibility (MIC 2 µg/ml) revealed to be.8 % (3/394). The rate of decreased susceptibility to ciprofloxacin (MIC 2 µg/ml) was 13.9 % (55/394) with 9.6 % (38/394) being intermediate (MIC = 2 µg/ml) and 4.3 % (17/394) being resistant (MIC 4 µg/ml). Levofloxacin insusceptibility (MIC 4 µg/ml) was 3.3 % (13/394) with an intermediate resistance rate (MIC = 4 µg/ml) of 1.8 % (7/394) and a resistance rate (MIC 8 µg/ml) of1.5 % (6/394). For moxifloxacin, one isolate (.3 %) was intermediate (MIC 2 µg/ml) and one isolate (.3 %) was resistant (MIC 4 µg/ml). Ofloxacin resistance (MIC 4 µg/ml) was 13.7 % (54/394) with 9.4 % (37/394) of the isolates showing intermediate resistance (MIC = 4 µg/ml) and 4.3 % (17/394) of the isolates showing high level resistance (MIC 8 µg/ml). The overall rate of decreased susceptibility to erythromycin (MIC.5 µg/ml) revealed to be 26.7 % (5/394) with 25.9 % (75/394) of these isolates showing resistance (MIC 1 µg/ml). Four isolates (1. %) showed resistance to Telithromycin (MIC 2 µg/ml); two (.5 %) of these isolates were intermediate resistant (MIC = 2 µg/ml). The overall rate of insusceptibility to tetracycline (MIC 4 µg/ml) was 32.7 % (129/394) with 29.7% (117/394) of the isolates being resistant (MIC 8 µg/ml). Resistance phenotypes. The distribution of the penicillin-cefotaxim-erythromycin-tetracycline-ciprofloxacin susceptibility phenotypes is shown in Table 4. The most common resistance phenotype was isolated insusceptibility to tetracycline (.4 %) followed by the combined insusceptibility to erythromycin and tetracycline (9.3 %). Isolated ciprofloxacin resistance was present in 7.4 % 8

9 of the isolates. Three- and fourfold resistance was found in 8.8 % and.5 % respectively of the isolates. TABLE 4 : DISTRIBUTION OF THE PENICILLIN-CIPROFLOXACIN- ERYTHROMYCIN-TETRACYCLINE SUSCEPTIBILITY PHENOTYPES (391 ISOLATES) Penicillin-Erythromycin-Tetracycline-Ciprofloxacin phenotype 1 Number (%) Susceptible 194 (49.6) Tet 41 (.5) Ery-Tet 38 (9.7) Cip 29 (7.4) Pen-Ery-Tet 23 (5.9) Ery 16 (4.1) Pen 14 (3.6) Cip-Ery-Tet 8 (2.) Pen-Cip-Ery-Tet 7 (1.8) Pen-Ery 7 (1.8) Pen-Tet 4 (1.) Cip-Ery 3 (.8) Cip-Tet 3 (.8) Pen-Cip 2 (.5) Pen-Cip-Tet 2 (.5) ß-lactam resistance The MIC 5 s and MIC 9 s (MICs for 9% of the isolates tested) of the various ß-lactams for penicillin-susceptible and penicillin-insusceptible isolates are shown in Table 5. TABLE 5 : MIC VALUES OF ß-LACTAMS FOR VARIOUS PENICILLIN SUSCEPTIBILITY CATEGORIES (391 isolates). ß-Lactam PEN S (n=332) PEN I/R (n=59) MIC5 MIC9 MIC5 MIC9 % S Penicillin Ampicillin Amoxicillin Amoxicillin/Clavulanate Cefaclor Cefuroxime Cefuroxime-axetil Cefotaxime Imipenem Ertapenem Oral form of cefuroxime MICs of all ß-lactams rose with those of penicillin. In general, penicillin and ampicillin were equally active against the penicillin insusceptible isolates while amoxicillin, 9

10 amoxicillin/clavulanate and cefotaxime were generally one doubling dilution more potent. Imipenem and ertapenem revealed to be 3 doubling dilutions more active. Cefuroxime and cefaclor were 2 and 3 dilutions respectively less active. Cross-resistance between penicillin and the other ß-lactams is not complete. The results indicate that 95.2 % and 8.6 % of the penicillin-insusceptible isolates remained susceptible to ertapenem and amoxicillin and amoxicillin/clavulanate respectively. Resistance characteristics There were no statistically significant differences for resistance between the various age groups except for tetracycline. Tetracycline resistance was significantly higher in the age group 15 years than in the group of more than 15years (.5 > P >.2). Table 6: % of isolates with resistance in the different age groups Age Pen Cip Ery Tet IR S Children Fig.1: Resistance rates: Chil vs Adult (3) % 3 Child Adult P C E T* IR Statistically significant differences for resistance were not found as far as samples sites (Table 7, Fig.2) and admission types (Table 8) were concerned. Erythromycin resistance was significantly higher in isolates from males than from females; i.e. 3.3 % vs 19.3 % (.2 > P >.1) (Table 8, Fig.3).

11 Table 7: Resistance rates (%) in URT and LRT isolates Pen Cip Ery Tet IR S URT LRT TOT Fig.2: Resistance rates in URT and LRT % 3 URT LRT P C E T IR Table 8: Resistance rates (%) following gender and type of isolate (3) Pen Cip Ery Tet IR S Gender Male Female Type AMB HOS LCF Fig.3: Resistance rates in males and females (3) % 3 M F P C E** T IR 11

12 The presence of isolates with decreased susceptibility to one or another antibiotic was higher in the Southern part (57.4 %; 93/162) than in Brussels (51.3 %; /39) and the Northern part (45.1 %; 87/193), although these differences were statistically not significant (Fig.4). Rates of decreased susceptibility to penicillin, ciprofloxacin and erythromycin were slightly higher in Southern isolates (19.8 %, 18.5 % and 32.1 % resp) than in Northern isolates (13. %, 11.9 % and 21.2 % resp) or Brussels isolates (12.8 %, 5.1 % and 3.8 % resp). Tetracycline resistance was 38.5 % in Brussels, 36.4 % in the South and 28.5 % in the North. However, the only statistically significant difference was found between Brussels and the Southern part for ciprofloxacin resistance (5.1 % vs 18.5 %;.5 > P >.2). There were no differences between the various provinces except for tetracycline. Tetracycline resistance was significantly lower in the province of Antwerp (11.3 %;.5 > P >.2) than in the other provinces, except East Flanders (. %) (Fig. 5 Fig.9). Fig. 4: Resistance rates by region (3) % 3 North South Bruss P C* E T IR Fig.5: Penicillin resistance by Province (3) % 5 AP BV BW BX HE LG LI LX NA OV WV 12

13 Fig.6: Ciprofloxacin resistance by Province (3) % 15 5 AP BV BW BX HE LG LI LX NA OV WV Fig.7: Erythromycin resistance by Province (3) % 3 AP BV BW BX HE LG LI LX NA OV WV Fig.8: Tetracycline resistance by Province (3) % 3 AP* BV BW BX HE LG LI LX NA OV WV 13

14 Fig.9: IR strains by Province (3) % 4 3 AP BV BW BX HE LG LI LX NA OV WV Results of capsular typing are summarized in Table 9. Table 9: Distribution of capsular types Capsular type N % 1 3, , , , , , , , NT 13,8 8 14