Use of trans-complementation method to determine the effects of various ftsI mutations on ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains.

Haemophilus influenzae is a causative agent of serious infections, especially among children. ß-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, ß-lactam resistance is due to the presence of ß-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for ß-lactams (BLNAR-ß-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from ß-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced ß-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between - 0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased ß-lactam susceptibility in H. influenzae.

Antimicrobial resistance among Haemophilus influenzae isolates responsible for lower respiratory tract infections in Poland, 2005-2019.

Haemophilus influenzae is a human-specific pathogen responsible for respiratory tract infections, meningitis, and sepsis. The study aimed to characterize antibiotic resistance in H. influenzae strains isolated from patients with lower respiratory tract infections over 15 years in Poland. The minimum inhibitory concentrations (MICs) of clinically relevant antibiotics were determined by broth microdilution method. Screening for beta-lactam resistance was performed in all isolates following EUCAST recommendation. Finally, relevant changes in penicillin-binding protein 3 (PBP3) were detected by PCR screening. Of the 1481 isolates collected between 2005 and 2019, 12.6%, 0.2%, 17.1%, and 0.2% were resistant to ampicillin, amoxicillin/clavulanate, cefuroxime, and ceftriaxone, respectively. Among them, 74.4% (1102/1481) of isolates were categorized as BLNAS (ß-lactamase negative, ampicillin-susceptible), 13.0% (192/1481) as BLNAS with modified PBP3 (mutations in ftsI gene), 2.6% (39/1481) as BLNAR (ß-lactamase negative, ampicillin-resistant), and 0.2% had PBP3 modifications typical for high-BLNAR. Production of ß-lactamase characterized 9.7% of isolates (8.6% BLPAR-ß-lactamase-positive, ampicillin-resistant, and 1.1% BLPACR-ß-lactamase-positive, amoxicillin-clavulanate resistant). Three isolates with PBP3 modifications typical for high-BLNAR proved resistant to ceftriaxone (MIC > 0.125 mg/L). Resistance to ciprofloxacin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole was observed in 0.1%, 0.5%, 1.6%, and 24.7% of isolates, respectively. This is the first report of Polish H. influenzae isolates resistant to third-generation cephalosporins. Polish H. influenzae isolates demonstrate similar susceptibility trends as in many other countries. The substantial proportion of ß-lactam-resistant isolates and the emergence of those resistant to third-generation cephalosporins are of great concern and should be under surveillance.

Amino Acid Substitution in the Major Multidrug Efflux Transporter Protein AcrB Contributes to Low Susceptibility to Azithromycin in Haemophilus influenzae.

Clarithromycin-resistant Haemophilus influenzae strains with a nonsense mutation in acrR generally exhibited susceptibility to azithromycin, although one strain was found to be nonsusceptible; we aimed to clarify the differences. This strain had an amino acid substitution, Arg327Ser, in AcrB. Introduction of this substitution into H. influenzae Rd caused an increase in the MIC of azithromycin, suggesting that this substitution contributed to nonsusceptibility. These findings indicate that azithromycin-nonsusceptible isolates could occur through stepwise mutation in the acr region.

Ampicillin resistance of invasive Haemophilus influenzae isolates in Germany 2009-2012.

In this retrospective study covering a four-year observation period (2009-2012) the prevalence of aminopenicillin resistance of invasive Haemophilus influenzae (Hi) in Germany was analyzed. The main resistance mechanism against aminopenicillins is conferred by ß-lactamase production, which can be inhibited by clavulanate or sulbactam. Apart from that, ß-lactamase negative ampicillin resistance (BLNAR) has been reported due to mutations in the penicillin-binding protein PBP3. The prevalence of BLNAR varies considerably in different countries. Representative data from Germany have not been reported. We analyzed 704 culture positive cases with bacteraemia or detection of Hi in cerebrospinal fluid; 82 isolates (11.6%) were phenotypically resistant to ampicillin. Among these isolates, 65 (79.3%) showed ß-lactamase production, and 17 isolates (20.7%) were phenotypic BLNAR Hi. The proportion of ampicillin resistant isolates remained stable over the observation period. Analysis of the PBP3 sequences of 133 isolates with different susceptibility phenotypes including susceptible, BLNAR, and ß-lactamase positive isolates, revealed a high genetic diversity. Previously described PBP3 mutations were associated to elevated MIC values, albeit not exclusively, since few highly susceptible strains were found to be positive for the mutations. Furthermore, since ampicillin susceptible strains with elevated MIC values frequently harboured these mutations, prediction of the resistance phenotype using ftsI sequencing appears to be impossible.

Role of inter-species recombination of the ftsI gene in the dissemination of altered penicillin-binding-protein-3-mediated resistance in Haemophilus influenzae and Haemophilus haemolyticus.

OBJECTIVES: To screen the ftsI gene sequences obtained from clinical isolates of non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus for the presence of mosaic ftsI gene structures, and to evaluate the role of inter-species recombination of the ftsI gene in the formation and distribution of resistant ftsI genes. METHODS: The ftsI genes of 100 Haemophilus isolates comprising genetically defined ß-lactamase-negative ampicillin-susceptible (gBLNAS), ß-lactamase-positive ampicillin-resistant (gBLPAR), ß-lactamase-negative ampicillin-resistant (gBLNAR) and ß-lactamase-positive amoxicillin/clavulanate-resistant (gBLPACR) isolates of NTHi (n = 50) and H. haemolyticus (n = 50) were analysed in this study. Both the flanking regions and the full-length ftsI gene sequences of all study isolates were screened for mosaic structures using H. influenzae Rd and H. haemolyticus ATCC 33390 as reference parental sequences, and bioinformatics methods were used for recombination analysis using SimPlot. RESULTS: Of the 100 clinical isolates analysed 34% (34/100) harboured mosaic ftsI gene structures containing distinct ftsI gene fragments similar to both reference parental sequences. The inter-species recombination events were exclusively encountered in the ftsI gene of gBLNAR/gBLPACR isolates of both NTHi and H. haemolyticus, and were always associated with the formation of a mosaic fragment at the 3' end of the ftsI gene. There was no evidence supporting horizontal gene transfer (HGT) involving the entire ftsI gene among the clinical isolates in vivo. CONCLUSIONS: We provide evidence for the HGT and inter-species recombination of the ftsI gene among gBLNAR/gBLPACR isolates of NTHi and H. haemolyticus in a clinical setting, highlighting the importance of recombination of the ftsI gene in the emergence of altered penicillin-binding protein 3 and BLNAR-mediated resistance.

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations.

OBJECTIVES: To investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. METHODS: During a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. RESULTS: In 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a ß-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82-100%) and 92% ((67/73); 95% CI, 83-97%), respectively. CONCLUSIONS: The prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.

Haemophilus influenzae blood-stream infection and third-generation cephalosporin susceptibility testing: a comparative case study using EUCAST and CLSI guidelines.

Introduction: In this comparative case study, we discuss clinically relevant discrepancies of antimicrobial susceptibility testing (AST) interpretation for ceftriaxone against a non-typable, beta-lactamase negative, ampicillin-resistant (BLNAR) Haemophilus influenzae isolated from a blood culture. Case report: A 74-year-old man presented with a 3 day illness characterized by shortness of breath and dry cough, and was noted to be febrile and hypoxic on admission. A blood culture bottle flagged positive with Gram-negative coccobacilli, later identified as Haemophilus influenzae with the patient commenced on ceftriaxone. The isolate was beta-lactamase negative and antibiotic susceptibility testing (AST) using disc diffusion revealed the isolate resistant to ceftriaxone and ampicillin by EUCAST methodology, with the patient subsequently changed to amoxicillin/clavulanate. Further AST using the CLSI methodology in parallel demonstrated discrepant results between the two susceptibility methods. The patient recovered without complications. Conclusion: This discrepancy could lead to inconsistent reporting of susceptibilities between laboratories, and consequently antibiotic prescribing, especially for invasive isolates. As more laboratories adopt EUCAST methodologies for AST interpretation in Australia and globally, it is important for clinicians to consider the clinical implications of these methodological discrepancies.

Detection of beta-lactamase-negative ampicillin resistance in Haemophilus influenzae in Belgium.

Haemophilus influenzae, a frequent colonizer of the respiratory tract, is the causative agent of several clinically important infections. In cases that require therapeutic intervention, laboratory susceptibility testing can detect beta-lactam antibiotic resistance and guide the best treatment course. In the absence of a beta-lactamase, beta-lactam resistance may be due to an altered form of the PBP3 protein, encoded by the ftsI gene. While these so-called beta-lactamase-negative ampicillin-resistant (BLNAR) strains are of serious clinical interest, identification in the clinical laboratory is not always straightforward. In the current study, the ftsI genes of a set of phenotypic BLNAR H. influenzae isolates taken from samples collected in the UZ Brussel hospital in Belgium were sequenced and re-tested at the National Reference Laboratory (NRC). Non-silent mutations in the ftsI gene were found in 100% of the isolates. Although 30% of the isolates were classified by the NRC as beta-lactamase-negative ampicillin-sensitive (BLNAS) strains based on the EUCAST guidelines on ampicillin minimal inhibitory concentration (MIC), all isolates showed MIC values ≥1 mg/L. These relatively high MIC values indicate a decreased susceptibility to ampicillin, and suggest that sequencing of the ftsI gene should be used as part of an antibiotic susceptibility testing (AST) algorithm in the clinical laboratory. This would allow clinicians to make better informed decisions regarding patient treatment.

Single nucleotide polymorphisms in genes encoding penicillin-binding proteins in ß-lactamase-negative ampicillin-resistant Haemophilus influenzae in Japan.

OBJECTIVE: ß-Lactamase-negative ampicillin-resistant Haemophilus influenzae is a common opportunistic pathogen of hospital- and community-acquired infections, harboring multiple single nucleotide polymorphisms in the ftsI gene, which codes for penicillin-binding protein-3. The objectives of this study were to perform comprehensive genetic analyses of whole regions of the penicillin-binding proteins in H. influenzae and to identify additional single nucleotide polymorphisms related to antibiotic resistance, especially to ampicillin and other cephalosporins. RESULTS: In this genome analysis of the ftsI gene in 27 strains of H. influenzae, 10 of 23 (43.5%) specimens of group III genotype ß-lactamase-negative ampicillin-resistant H. influenzae were paradoxically classified as ampicillin-sensitive phenotypes. Unfortunately, we could not identify any novel mutations that were significantly associated with ampicillin minimum inhibitory concentrations in other regions of the penicillin-binding proteins, and we reconfirmed that susceptibility to ß-lactam antibiotics was mainly defined by previously reported SNPs in the ftsI gene. We should also consider detailed changes in expression that lead to antibiotic resistance in the future because the acquisition of resistance to antimicrobials can be predicted by the expression levels of a small number of genes.

Increasing trend in invasive non-typeable Haemophilus influenzae disease and molecular characterization of the isolates, Italy, 2012-2016.

Routine immunization of infants with conjugate vaccines against Haemophilus influenzae type b (Hib) has greatly reduced the incidence of invasive Hib disease; however changes in the epidemiology of H. influenzae disease have occurred. We describe the epidemiology of invasive H. influenzae disease and the characterization of isolates collected in Italy between 2012 and 2016. Trends in the overall incidence of invasive H. influenzae disease were calculated. Isolates were characterized by PCR capsular genotyping, antimicrobial susceptibility testing, ampicillin resistance-associated gene sequencing and MLST. Trends in incidence by serotype and serotype-specific distribution were estimated using multiple imputation of missing data. The overall incidence of invasive H. influenzae disease increased 22.5% yearly (from 0.11/100,000 in 2012 to 0.24/100,000 in 2016). Most cases (82.0%) were due to non-typeable H. influenzae (NTHi). An increasing trend in NTHi disease burden was estimated; the highest rise was among infants <12 months (40.8% annual increase). Invasive Hib disease showed a fluctuating trend with a clear increase in 2016, while we found an increasing trend for disease due to non-Hib capsulated serotypes in the elderly (32.9% annual increase). Ampicillin resistance mediated by either ß-lactamase or altered penicillin-binding proteins 3 (PBP3) increased. In spite of genetic diversity of NTHi, sequence types (STs) associated with ampicillin resistance status were identified (ST103/ST106 linked to ß-lactamase production and ST14 linked to a specific PBP3 substitution pattern). The increasing trend in invasive NTHi disease in infants is of concern underlying the need for the development of a future vaccine against NTHi.

Genotypic characterization of Haemophilus influenzae isolates from paediatric patients in Japan.

Purpose. ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae is frequently isolated from respiratory samples and is particularly problematic in Japan. The aim of this study was to characterize circulating isolates of H. influenzae genotypically by BLNAR-PCR and multilocus sequence typing (MLST), and to determine any associations between them.Methods. H. influenzae isolates (n=191) were collected from paediatric patients (1 month to 12 years old) between 2000 and 2011 for three types of infections: pneumonia (n=61), acute otitis media (AOM) (n=68) and meningitis (n=62). All were characterized for capsular type by agglutination tests, and for ß-lactam resistance by real-time PCR. The sequence types (STs) determined by MLST were analysed using eburst v3.Results. Eighty-eight out of 191 (46.1 %) H. influenzae isolates were BLNAR by PCR; 37 of 61 (60.7 %) from pneumonia; 33 of 68 (48.5 %) from AOM and 18 of 62 (29.0 %) from meningitis cases. MLST identified 40 and 44 STs among isolates from pneumonia and AOM, respectively. BLNAR were found in singletons such as ST156 in pneumonia, and ST161 and ST396 in AOM. In contrast, eight STs were identified in meningitis, of which seven were genotypically closely related, while ST54 was the most frequent (62.9 %), unlike in the MLST database registrations, where ST6 predominated.Conclusion. Non-typeable H. influenzae (NTHi), mostly derived from pneumonia and AOM, were genetically diverse, in contrast to the predominance of H. influenzae type b (Hib) among meningitis cases. The associations between certain STs and ß-lactam resistance among NTHi were confirmed.

Ampicillin resistance in Haemophilus influenzae from COPD patients in the UK.

BACKGROUND: Haemophilus influenzae is commonly isolated from the airways of COPD patients. Antibiotic treatment may cause the emergence of resistant H. influenzae strains, particularly ampicillin-resistant strains, including ß-lactamase-negative ampicillin resistance (BLNAR) strains. Genetic identification using ftsI sequencing is the optimum method for identifying mutations within BLNAR strains. The prevalence of BLNAR in COPD patients during the stable state has not been reported. We investigated the antibiotic resistance patterns of H. influenzae present in the sputum of stable COPD patients, focusing on ampicillin resistance; the prevalence of enzyme and non-enzyme-mediated ampicillin resistance was determined. A subset of patients was followed up longitudinally to study H. influenzae strain switching and antibiotic sensitivity changes. PATIENTS AND METHODS: Sputum sampling was performed in 61 COPD patients, with 42 samples obtained at baseline; H. influenzae was detected by polymerase chain reaction in 28 samples. In all, 45 patients completed the follow-up for 2 years; 24 H. influenzae isolates were obtained. RESULTS: Disk diffusion showed the highest antibiotic resistance in the penicillin antibiotic group (eg, 67% for ampicillin) and macrolides (eg, 46% for erythromycin), whereas all isolates were susceptible to quinolones. Of the 16 isolates resistant to ampicillin, 9 (56%) were ß-lactamase positive. The ß-lactamase-negative isolates were further investigated; none of these fulfilled the phenotypic BLNAR classification criteria of ampicillin minimum inhibitory concentration >1 µg/mL, and only one demonstrated an ftsI mutation. Frequent H. influenzae strain switching was confirmed using multilocus sequence typing and was associated with changes in the antibiotic sensitivity pattern. CONCLUSION: We observed an overidentification of ampicillin resistance by disk diffusion. The majority of ampicillin resistance was due to enzyme production. H. influenzae strain changes during the stable state may be associated with a change in antibiotic sensitivity; this has implications for empirical antibiotic prescribing.

ß-Lactam resistance among Haemophilus influenzae isolates in Poland.

OBJECTIVES: Haemophilus influenzae is a human-specific Gram-negative coccobacillus responsible for a significant number of respiratory tract infections and severe invasive infections such as meningitis and sepsis. The purpose of this study was to characterise the mechanisms of ß-lactam resistance among Polish H. influenzae isolates and to evaluate the resistance detection methods applied. METHODS: This study was conducted on 117 Polish H. influenzae isolates collected in 2012. Minimum inhibitory concentrations were assessed by broth microdilution. All strains were evaluated using the disk diffusion method and the algorithm proposed by the Nordic Committee on Antimicrobial Susceptibility Testing (NordicAST). To detect changes in penicillin-binding protein 3 (PBP3), PCR screening was performed, followed by ftsI gene sequencing. RESULTS: Neither ß-lactamase production nor PBP3 alterations were demonstrated in 76 isolates (65.0%). Susceptibility to ampicillin, amoxicillin, amoxicillin/clavulanic acid, cefuroxime (intravenous) and ceftriaxone was observed in 70.9%, 78.6%, 98.3%, 82.9% and 100% of the isolates, respectively. ß-Lactamase production characterised 21 isolates (17.9%). Screening PCR identified 20 isolates (17.1%) with PBP3 alterations, and according to subsequent ftsI sequencing all these strains were finally recognised as gBLNAR (genetically ß-lactamase-negative, ampicillin-resistant), among which 65.0% were ampicillin-resistant. According to molecular classification of PBP3 alterations, 95.0% of gBLNAR belonged to group II, representing four subgroups IIa-IId. CONCLUSIONS: Haemophilus influenzae resistance to antibiotics requires continuous attention, effective detection methods and a rational policy of antibiotic usage. The algorithm proposed by NordicAST can be applied in routine laboratory work, whereas sequencing of the ftsI gene may be useful in molecular epidemiology studies.

The first case report of septic abortion resulting from ß-lactamase-negative ampicillin-resistant non-typeable Haemophilus influenzae infection.

Introduction. This is the first case report of septic abortion due to ß-lactamase-negative ampicillin-resistant (BLNAR) non-typeable Haemophilus influenzae infection. In Japan, BLNAR H. influenzae is widespread and has become a clinical concern, especially in paediatrics and otolaryngology, but H. influenzae has not been previously recognized as a causative agent of obstetric or gynaecological infection. Case presentation. A 31-year-old pregnant woman presented at 17 weeks and 6 days of gestation with a high fever; she was admitted with a diagnosis of threatened premature delivery. Despite tocolytic treatment, she aborted spontaneously 2 h after admission and then entered septic shock. BLNAR H. influenzae was detected in both blood and vaginal cultures. Her condition gradually improved after several days of treatment with cefotaxime, and she was ultimately discharged without sequelae or complaints. Conclusion. Although penicillin with a ß-lactamase inhibitor is currently recommended for the treatment of septic abortion, this combination will probably lead to treatment failure in the case of BLNAR H. influenzae infection. As this study reveals, H. influenzae can cause septic abortion; hence, future efforts should be undertaken to detect and therapeutically target this pathogen during pregnancy.

Prevalence of macrolide-non-susceptible isolates among ß-lactamase-negative ampicillin-resistant Haemophilus influenzae in a tertiary care hospital in Japan.

ß-Lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae account for a large portion of H. influenzae clinical isolates in Japan. The aim of this study was to clarify the antimicrobial susceptibility of BLNAR H. influenzae clinical isolates as well as the annual changes in susceptibility. BLNAR H. influenzae isolates were collected from a tertiary care hospital from 2007 to 2012. Antimicrobial susceptibility testing was performed and resistance mechanisms were analysed. All of the isolates (n=304) had amino acid substitutions in penicillin-binding protein 3 (PBP3) and isolates were classified by these amino acid substitutions: R517H or N526K (class I); S385T and R517H (class II); and S385T and N526K (class III). Classes I, II and III represented 8.2% (n=25), 9.5% (n=29) and 81.6% (n=248) of the isolates, respectively; 2 isolates could not be classified because they had a PBP3 with a substantially mutated FtsI transpeptidase domain. All of the isolates were highly susceptible to fluoroquinolones and carbapenems. The number of clarithromycin (CAM)-non-susceptible [minimum inhibitory concentration (MIC) ≥16µg/mL] H. influenzae isolates increased significantly between 2010 and 2012. Moreover, CAM-non-susceptible H. influenzae isolates were prevalent among class II and class III BLNAR H. influenzae. Multilocus sequence typing (MLST) of the CAM-resistant (MIC ≥32µg/mL) H. influenzae isolates showed that they were not specific sequence types, suggesting that CAM resistance may occur in any isolates. These results raise concern regarding the occurrence of multidrug-resistant BLNAR H. influenzae.

Novel broad-spectrum and long-acting parenteral cephalosporins having an acyl cyanamide moiety at the C-3 terminal: Synthesis and structure-activity relationships.

A series of novel 7ß-[2-(2-aminothiazole-4-yl)-2-(Z)-(alkoxyimino)acetamido]-cephalosporins having pyridinium-linked acyl cyanamide at the C-3 position were prepared and their antibacterial activities and pharmacokinetics profiles were evaluated. Most of the compounds exhibited potent antibacterial activities against penicillin-resistant Streptococcus pneumoniae (PRSP) and ß-lactamase non-producing penicillin-resistant Haemophilus influenzae (BLNAR). Introduction of a propenyl group between the cephalospoin core and the side chains at the C-3 position improved the pharmacokinetics profile. Among these compounds, 7ß-[2-(2-aminothiazole-4-yl)-2-(Z)- (alkoxyimino)acetamido]-3-(pyridin-1-ium-1-yl)prop-1-en-1-yl)cephalosporins (32j) showed well-balanced antibacterial activity against S. pneumoniae and H. influenzae which included resistant strains and also other Gram-positive or Gram-negative pathogens. Furthermore, 32j showed a long half-life comparable to that of Ceftriaxone in mice and monkeys.

Individual-level effects of antibiotics on colonizing otitis pathogens in the nasopharynx.

BACKGROUND: Although there is evidence of an association between antibiotic consumption and resistant bacteria on a population level, the relationship on an individual level has been less well studied, particularly in terms of nasopharyngeal colonization. We have therefore analysed this association, using data from a closely followed cohort of children taking part in a vaccination trial. METHODS: 109 children with early onset of acute otitis media (AOM) were randomised to heptavalent pneumococcal conjugate vaccine (PCV7) or no vaccination. They were followed for three years with scheduled appointments as well as sick visits. Nasopharyngeal cultures were obtained at all visits. Antibiotic treatments were recorded, as were risk factors for AOM, including siblings, short breast-feeding and parental smoking. Data were entered into a Cox regression model, and the findings of Streptococcus pneumoniae and Haemophilus influenzae with reduced susceptibility to the penicillin group were related to the number of previous courses of antibiotics. RESULTS: There was evidence of an association between the amount of previously consumed betalactams and colonization with beta-lactamasenegative ampicillin-resistant (BLNAR) H. influenzae (RR 1.21; 95% CI 1.03-1.43; p = 0.03), and also with the most commonly prescribed drug; amoxicillin (RR 1.39; 95% CI 1.09-1.76; p = 0.01). There was no evidence for an association between antibiotic consumption and betalactamase producing H. influenzae or S. pneumoniae with reduced susceptibility to penicillin. Furthermore, there was no evidence of an association between resistant bacteria and AOM risk factors or PCV7. CONCLUSION: In this subgroup of children, most of whom were given several courses of antibiotics in early childhood, there was evidence of an association between betalactam/amoxicillin consumption and nasopharyngeal colonization with BLNAR strains, bacteria that have increased in prevalence during the last 10-15 years, and that are notoriously difficult to treat with oral antibiotics.