Impact of Antimicrobial Therapy on Nasopharyngeal Carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Branhamella catarrhalis

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1 477 Impact of Antimicrobial Therapy on Nasopharyngeal Carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Branhamella catarrhalis in Children with Respiratory Tract Infections Emmanuelle Varon, 1 Corinne Levy, 2 France De La Rocque, 2 Michel Boucherat, 2 Dominique Deforche, 2 Isabelle Podglajen, 1 Michel Navel, 3 and Robert Cohen 4 1 Laboratoire de Recherche Moleculaire sur les Antibiotiques, Université Paris VI, Paris, 2 Association Clinique et Thérapeutique Infantile du Val de Marne, Saint Maur, 3 Association Française de Pédiatrie Ambulatoire, Lyon, and 4 Département de Pédiatrie, Centre Hospitalier Intercommunal, Créteil, France We conducted a multicenter prospective study to document changes in nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Branhamella catarrhalis during antibiotic therapy. A cohort of 629 children with respiratory tract infections underwent nasopharyngeal sampling before and after antibiotic treatment. Susceptibility testing, serotyping, arbitrarily primed polymerase chain reaction, and pulsed-field gel electrophoresis were used to compare pretreatment and posttreatment strains of S. pneumoniae. A significant decrease in carriage of all 3 species (especially S. pneumoniae and B. catarrhalis) was recorded. The increase in the proportion of penicillin-resistant pneumococci (PRP; 66% vs. 44%) was due to the decreased carriage of penicillin-susceptible pneumococci (71 of 629 vs. 176 of 629). The risk of PRP carriage in a given child did not increase. None of the children was found to harbor genetically related strains with increased minimum inhibitory concentrations. Given the multiple resistance of PRP, b-lactam antibiotic therapy also increased the incidence of macrolide-resistant strains, whereas macrolides selected both macrolide- and penicillin-resistant strains. During the past 10 years, there has been an alarming worldwide increase in antibiotic resistance among bacterial pathogens of the respiratory tract, particularly Streptococcus pneumoniae [1]. This is generally attributed to extensive use of antibiotics and the selection pressure they exert on bacterial strains of nasopharyngeal flora [2, 3]. This ecosystem is the reservoir for bacterial pathogens (Haemophilus influenzae, Branhamella catarrhalis, S. pneumoniae, etc.) involved in respiratory tract infections (RTIs) [4, 5]. Before 3 years of age, children are more likely than adults to harbor these 3 species in their nasopharynx and to receive antibiotic therapy [4, 6, 7]. Carriage of the same S. pneumoniae serotype generally lasts several weeks or months, and it is therefore not surprising that the highest rates of resistance are observed in young children [7 9]. The ecological impact of antibiotic therapy, especially in terms of increasing Received 12 October 1999; revised 18 January 2000; electronically published 7 September Presented in part: 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, September Financial support: SmithKline-Beecham Laboratories, Nanterre, France. This study was approved by the local ethics committee, and written informed consent was obtained from all the parents or guardians at the time of enrollment of their children. Reprints or correspondence: Dr. Robert Cohen, Département de Pédiatrie, Centre Hospitalier Intercommunal, 40 avenue de Verdun, Créteil, France (Robert.cohen@wanadoo.fr). Clinical Infectious Diseases 2000;31: by the Infectious Diseases Society of America. All rights reserved /2000/ $03.00 resistance, is of great concern, particularly with regard to children [10]. Observational and case-control studies have shown that antibiotic use increases the carriage rate of penicillin-resistant S. pneumoniae [11, 12]. These studies do not explain why antimicrobial drug use is a risk factor for nasopharyngeal colonization by resistant strains. Prospective studies are probably more contributory in this respect, but few have been published on antibiotics prescribed for acute RTIs [13 16]. The aim of this study was to document changes in nasopharyngeal carriage of S. pneumoniae, B. catarrhalis, and H. influenzae induced by the antibiotics most frequently prescribed for community-acquired RTI in young children. Patients and Methods Study design. Between 21 October 1996 and 21 April 1997, 57 French pediatricians took part in this prospective study. The population consisted of children of both sexes, aged 3 months to 3 years, presenting with community-acquired upper or lower RTI and requiring antibiotic treatment. Pediatricians prescribed the antibiotic treatment of their choice. Then, according to the antibiotic prescribed, each patient was assigned to 1 of the following 7 groups, corresponding to the main antibiotics used in France in this setting: amoxicillin, 80 mg/kg/d in 3 divided doses; cefaclor, 40 mg/kg/d in 3 divided doses; cefuroxime-axetil, 30 mg/kg/d in 2 divided doses; cefixime, 8 mg/kg/d in 2 divided doses; cefpodoxime-proxetil, 8 mg/ kg/d in 2 divided doses; amoxicillin/clavulanate, 80 mg/kg/d of amoxicillin and 10 mg/kg/d of clavulanate in 3 divided doses; and erythromycin/sulfisoxazole, 50 mg/kg/d of erythromycin and 150

2 478 Varon et al. CID 2000;31 (August) mg/kg/d of sulfisoxazole in 3 divided doses. Each group was closed when the number of enrolled patients reached 100. Nasopharyngeal sampling was scheduled before treatment (first visit) and 2 6 days after the end of treatment. Standardized history and physical examination findings were recorded. Patients were excluded from the study if they attended a day-care facility (in order to reduce the influence of person-to-person exchange of strains), had received antibiotic treatment within 2 months before enrollment, were hypersensitive to the antibiotic prescribed, had severe underlying disease, or had previously been included in the study. Nasopharyngeal cultures. Nasopharyngeal specimens were taken with cotton-tipped wire swabs, immediately placed in transport medium (TGV; Sanofi Diagnostics Pasteur, Marnes la Coquette, France), and sent within 48 h to the Créteil Hospital bacteriology laboratory (Créteil, France). Each swab was swirled in 200 ml of brain-heart infusion, and 20 ml was smeared onto Columbia blood agar supplemented with colistin and nalidixic acid. The remaining suspension was used to inoculate selective broth. Isolates (at least 5 colonies of each) were identified on the basis of colony morphology, Gram staining, and biochemical characteristics. Susceptibility testing and serotyping. H. influenzae and B. catarrhalis b-lactamase production was detected with nitrocefin disks (Céphinase; BioMérieux, Marcy-l Etoile, France). Susceptibility of S. pneumoniae isolates to penicillin G was determined on the basis of MICs determined by the agar-dilution method. Isolates were classified as penicillin-susceptible (MIC,!0.12 mg/ml), intermediate-resistant ( 0.12 MIC 1.0 mg/ml), or highly resistant (MIC, 11 mg/ml) [17]. The S. pneumoniae serotype was determined by use of a panel of latex reagents sensitized with antibodies raised in the Centre National de Référence du Pneumocoque (Créteil, France; courtesy of Dr. Pierre Geslin). Randomly amplified polymorphic DNA (RAPD) analysis. Genomic DNA was extracted as described elsewhere [18]. Three amplification rounds were performed, each using one of the following oligonucleotide primers: ERIC-2, OPA-03, or OPA-13 [19, 20]. Each PCR run was carried out in a Progene thermal cycler (Techne, Cambridge, UK), in a volume of 100 ml containing primers (0.5 mmol), deoxynucleoside triphosphates (200 mmol each), reaction buffer containing magnesium chloride (1.5 mmol), and 0.8 U of Taq polymerase (Appligène, Illkirch, France). The conditions used were those described elsewhere [20]. Amplification products were resolved by electrophoresis on agarose gel and were detected by staining with ethidium bromide. RAPD patterns were examined and analyzed by direct visual comparison of the patterns. Pulsed-field gel electrophoresis (PFGE). Bacteria were grown to an optical density of 0.5 at 560 nm. The cells were harvested by centrifugation at 5000 g for 10 min at 4 C, washed with 1 mol of NaCl and 10 mmol of Tris hydrochloride (ph, 8.0), and embedded in low-melting-point agarose (1.5%). Lysis was performed by addition of 0.1 mol of EDTA, 10 mmol of Tris hydrochloride (ph, 8.0), 0.5% Triton X-100, and 0.2% deoxycholate, and incubation was for 2 h at 37 C. The agarose blocks were transferred to 0.25 M EDTA, 1% Sarkosyl, and proteinase K (100 mg/ml) and then were incubated overnight at 50 C. Pneumococcal DNA was digested with ApaI. The conditions used were those described elsewhere [21]. Statistical analysis. Data were analyzed with Statview II software (Editor Abacus Concepts, Inc., Berkeley, California). The x 2 test with continuity correction was used for statistical analysis. The McNemar test was used to compare carriage before and after antibiotic treatment. All tests were 2-sided, and the level of significance was set at P!.05. The effects of population characteristics (day care, type of RTI, symptoms, history of antibiotic treatment, etc.) on nasopharyngeal carriage were examined. Results Population. Seven hundred five patients were included. The antibiotics prescribed were amoxicillin ( n p 100), cefaclor ( n p 103), cefuroxime-axetil ( n p 98), cefixime ( n p 100), cef- podoxime-proxetil ( n p 99), amoxicillin/clavulanate ( n p 103), and erythromycin/sulfisoxazole ( n p 102). The indications were acute otitis media (57.7 %), lower RTI (23.3%), nonspecific upper RTI (11.9%), and pharyngitis (7.1%). More than 50% of patients had received antibiotics 12 months before their inclusion in the study. The mean ( SD) and median durations without antibiotic were months and 4 months (range, 2 23 months), respectively. The mean treatment period was 8 days (range, 5 12 days). Demographic and clinical data (table 1) showed no significant differences among the 7 groups, except for the presence of conjunctivitis in the cefixime group (P p.0001). Six hundred twenty-nine patients were assessable for the analysis of changes in nasopharyngeal carriage induced by antibiotic therapy. In all the groups the most frequent protocol violations leading to exclusion from the study were noncompliance with treatment ( n p 22), new antibiotic treatment be- fore the second visit ( n p 34), no posttreatment assessment ( n p 4), and nonadherence to the study protocol ( n p 15). Colonization rate before treatment. At inclusion, S. pneumoniae, B. catarrhalis, and H. influenzae were isolated from, 49.8% ( n p 351), 51.1% ( n p 360), and 30.3% ( n p 214) re- Table 1. Demographic and clinical data for the children with respiratory tract infections. Variable No. (%) of patients or other value Mean age, mo SE Day care Home 453 (64.3) Child-care giver 252 (35.7) Sex Male 378 (53.6) Female 327 (46.4) Siblings (39.0) (42.8) (14.7) 3 24 (3.4) Treated with antibiotic(s) within 2 mo before enrollment 424 (60.1) Associated signs or symptoms Fever ( 38.5 C) 338 (47.9) Conjunctivitis 126 (17.9) Total enrolled 705

3 CID 2000;31 (August) Impact of Antibiotics on Nasopharyngeal Carriage 479 Table 2. Carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Branhamella catarrhalis in relation to day-care provision for the children with respiratory tract infections. Pathogen No. (%) of children carrying pathogen, per day-care category Home (n p 453; 64.3%) Child-care giver (np 252; 35.7%) With siblings No siblings P a With siblings No siblings P a Any of the 3 below 315 (69.5) 138 (30.5) 115 (45.6) 137 (54.4) S. pneumoniae 175 (55.5) 45 (32.6) b (60.0) 61 (44.5) b.014 H. influenzae 111 (35.2) 23 (16.6) c (38.3) 36 (26.3) c.042 B. catarrhalis 169 (53.6) 50 (36.2) d (57.4) 75 (54.7) d.67 a 2 Per x test with continuity correction; all tests were 2-sided, and the level of significance was P!.05. b P p.042. c P p.05. d P p.002. spectively, of the overall population ( n p 705). Of the 705 chil- dren, 200 (39.7%) carried 11 of these 3 species, and 7 patients harbored 2 pneumococcal strains of different serotypes. The 4 most common pneumococcal serotypes (in descending order) were 6B, 19F, 23F, and 14, accounting for 170% of the isolates. Forty-four percent of S. pneumoniae strains had reduced susceptibility to penicillin (intermediate-resistant, 21.1%; highly resistant, 23.4%). b-lactamase producing strains of H. influenzae and B. catarrhalis accounted for 38.9% and 97.5%, respectively. The presence of siblings increased the carriage of the 3 species, particularly among children cared for at home (table 2). Carriage of H. influenzae was significantly more frequent in children with conjunctivitis (66 [52.4%] of 126 patients vs. 148 [25.6%] of 579 patients without conjunctivitis [ P p.0001]). Carriage of S. pneumoniae was increased among children with acute otitis media (218 [53.5%] of 407 patients vs. 133 [44.6%] of 298 children without acute otitis media [ P p.019]). No significant differences in carriage rates were found in relation to other clinical manifestations or antibiotic therapy received within 2 months before enrollment. Impact of antibiotics. A significant decrease in the carriage of any of the 3 species was observed, especially B. catarrhalis and S. pneumoniae (tables 3 and 4). No increase in the rate of b-lactamase producing strains was recorded for either B. catarrhalis (97% vs. 97% before therapy) or H. influenzae (30.9% vs. 38.9% before therapy). S. pneumoniae. Irrespective of the antibiotic received, the risk for a child of carrying PRP (MIC, mg/l) was not increased (after therapy, 140/629 [22.2%]; before therapy, 139/ 629 [22.1%]) (table 4). The decrease in S. pneumoniae carriage, which was more pronounced with amoxicillin, cefpodoximeproxetil, amoxicillin/clavulanate, and erythromycin/sulfisoxazole, was mainly due to a significant decrease in penicillinsusceptible S. pneumoniae carriage (71/629 [11.2%] patients after therapy vs. 176/629 [28%] before therapy). Therefore, among the pneumococcal isolates the rate of PRP increased from 44% before to 66% after treatment. S. pneumoniae strains carried at the end of treatment. Among the 211 patients carrying S. pneumoniae at the end of treatment, 50 did not harbor a pneumococcal strain at enrollment, whereas 31 carried another serotype and 130 carried the same serotype. Three of the latter patients harbored strains for which the penicillin MICs were increased (by 12 dilutions) after treatment. The genetic relatedness of each of these pairs of pneumococcal strains was investigated by RAPD and PFGE analysis, which revealed distinct patterns, demonstrating that none were genetically related. Table 3. Changes in carriage of Haemophilus influenzae and Branhamella catarrhalis after antimicrobial chemotherapy. Variable Amoxicillin Cefaclor Cefuroxime Cefixime Cefpodoxime Amoxicillin/ clavulanate Erythromycin/ sulfizoxazole Dosage, mg/kg/d 80 a 40 a 30 b 8 b 8 b 80/10 a 50/150 a No. of children given drug No. (%) of children carrying H. influenzae V1 25 (26.6) 39 (39.4) 27 (34.6) 37 (43.5) 25 (27.8) 20 (20.6) 20 (23.2) 193 (30.7) V2 21 (22.3) 25 (25.2) 27 (34.6) 37 (43.5) 18 (20.0) 13 (13.4) 8 (9.3) 149 (23.7) P c NS!.005 NS NS NS NS!.005!.005 B. catarrhalis V1 48 (51.1) 42 (42.4) 45 (57.7) 47 (55.3) 41 (45.6) 55 (56.7) 42 (48.8) 320 (50.9) V2 26 (27.7) 35 (35.4) 34 (43.6) 22 (25.8) 27 (30.0) 16 (16.5) 22 (25.6) 182 (28.9) P c!.005!.05!.05!.005!.005!.005!.005!.005 NOTE. V1, visit before antimicrobial therapy; V2, visit after the end of antimicrobial therapy. a In 3 divided doses. b In 2 divided doses. c Per McNemar test; all tests were 2-sided, and the level of significance was P!.05. Total

4 480 Varon et al. CID 2000;31 (August) Table 4. Changes in carriage of Streptococcus pneumoniae after antimicrobial chemotherapy: rates of penicillin and erythromycin resistance. Variable Amoxicillin Cefaclor Cefuroxime Cefixime Cefpodoxime Amoxicillin/ clavulanate Erythromycin/ sulfizoxazole Dosage, mg/kg/d 80 a 40 a 30 b 8 b 8 b 80/10 a 50/150 a No. of children given drug No. (%) of children carrying S. pneumoniae V1 40 (42.6) 50 (50.5) 37 (47.4) 43 (50.6) 55 (61.1) 50 (51.5) 40 (46.5) 315 (50.1) V2 24 (25.5) 43 (43.4) 29 (37.2) 38 (44.7) 30 (33.3) 26 (26.8) 21 (24.4) 211 (33.5) P c!.005 NS NS NS!.005!.005!.005!.005 No. (%) of children whose isolates were Pen I/R on V1 15 (37.5) 25 (50.0) 21 (56.8) 17 (39.5) 25 (45.5) 21 (42.0) 15 (37.5) 139 (44.1) Pen I/R on V2 17 (70.8) 23 (53.5) 22 (75.9) 21 (55.3) 21 (70.0) 23 (88.5) 13 (61.9) 140 (66.4) Ery I/R on V1 23 (57.5) 30 (60.0) 25 (67.6) 22 (51.2) 25 (45.5) 26 (52.0) 21 (52.5) 172 (54.6) Ery I/R on V2 17 (70.8) 28 (65.1) 21 (72.4) 23 (60.5) 19 (63.3) 17 (65.4) 19 (90.5) 144 (68.2) NOTE. Ery I/R, erythromycin-intermediate and -resistant; Pen I/R, penicillin-intermediate and -resistant; V1, visit before antimicrobial therapy; V2, visit after the end of antimicrobial therapy. a In 3 divided doses. b In 2 divided doses. c Per McNemar test; all tests were 2-sided, and the level of significance was P!.05. Total Discussion The bacterial flora of the nasopharynx is in a constant state of flux [5]. Factors controlling bacterial acquisition, elimination, and reacquisition in this ecosystem are poorly understood. The nasopharyngeal cultures performed before antibiotic treatment in this study confirm that B. catarrhalis is the leading respiratory tract pathogen in young children, followed by S. pneumoniae and then by nontypeable H. influenzae [5]. Furthermore, we confirmed that the presence of siblings and provision of day care by a child-care giver increase the carriage of the 3 species [4]. Last, carriage of H. influenzae was more frequent among children with conjunctivitis, whereas carriage of S. pneumoniae was more frequent among children with acute otitis media [22, 23]. Before treatment, the proportion of resistant strains was high among the 3 species, a usual observation in France for many years [24]. To our knowledge, the present study is the largest designed specifically to document the impact of antibiotics on nasopharyngeal carriage of the main pathogens responsible for upper RTIs. Children attending day-care centers were excluded from this study because attendance at such facilities increases the resistance rate and the risk of person-to-person exchanges of strains, particularly regarding S. pneumoniae [25, 26]. The effect of antibiotic treatment appeared to differ according to the drug prescribed and the bacterial species but was more marked with B. catarrhalis and S. pneumoniae than with H. influenzae. The drugs with most in vitro potency against wild-type strains of S. pneumoniae (e.g., amoxicillin and cefpodoxime) induced a drastic fall in the carriage of penicillin-susceptible strains, thereby increasing the proportion of penicillinresistant S. pneumoniae carried after treatment. However, the risk that a given child would carry PRP was not increased by treatment. Similar results have been reported elsewhere [13 16, 27]. Furthermore, b-lactam antibiotics increased not only the proportion of penicillin-resistant strains but also resistance to macrolides. Likewise, macrolides selected both macrolide- and penicillin-resistant strains. Several studies have shown that non b-lactam antibiotics are capable of selecting PRP. Selection can occur once multidrugresistant strains (mainly serotypes 6B and 23F) emerge and spread [24]. Thus, in areas where multidrug-resistant pneumococci predominate, cycling of antibiotic treatments in an attempt to control resistance seems inappropriate. The development of resistant mutants and the horizontal transfer of genetic material between related species therefore seem to be rare events that probably require more time to emerge. Nasopharyngeal carriage of B. catarrhalis was significantly decreased by the drugs most active against this species in vitro amoxicillin/clavulanate, cefixime, erythromycin/sulfisoxazole, and cefpodoxime. Despite the high percentage of strains producing b-lactamases, a major fall in the carriage rate was noted among patients receiving amoxicillin. This surprising result is reminiscent of the high rate of eradication of B. catarrhalis from middle-ear fluid in patients with acute otitis media treated with amoxicillin [28]. With the exception of erythromycin/sulfisoxazole and cefaclor, H. influenzae carriage was slightly affected by the different drugs, including cefixime and cefpodoxime, which are among the most active against this species. This is not the first study to show the poor effects of antibiotics on H. influenzae carriage. Samuelson et al. [29], who studied the turnover of nontypeable H. influenzae in the nasopharynx of otitis-prone children by using restriction-enzyme analysis of total genomic DNA, found no direct link between antimicrobial therapy and the elimination of nasopharyngeal colonization by a particular strain. These results are also in agreement with data from 2 of our previous studies and those of other investigators [13, 14, 16, 27]. References 1. Washington JA, Felmingham D. The in-vitro activities of co-amoxiclav and other oral antibiotics against Streptococcus pneumoniae isolates exhibiting intermediate susceptibility to penicillin. J Antimicrob Chemother 1998; 42:405 6.

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