Adherence of Haemophilus influenzae to buccal epithelial cells.

The role of adherence of Haemophilus influenzae to epithelial surfaces in the pathogenesis of infection is unknown. Fluorescent-antibody and radiolabeled adherence methods were adapted to study H. influenzae adherence to human buccal epithelial cells. By the fluorescent-antibody method, 19 of 21 (90%) nontypable H. influenzae strains were found to be adherent compared with 2 of 42 (5%) type b strains (P less than 0.0001). Using a radiolabeled adherence method, we found that 9 of 12 (75%) nontypable H. influenzae strains were adherent to buccal epithelial cells whereas only 3 of 32 (9%) type b strains were adherent (P = 0.001). Results of H. influenzae adherence examined by both methods correlated significantly (P = 0.01). H. influenzae adherence to adult pharyngeal, nasal, and buccal epithelial cells was comparable. Type b H. influenzae did not adhere to the buccal epithelial cells of well children, children with H. influenzae type b disease, or children with upper respiratory infections. In contrast, nontypable H. influenzae did adhere to the buccal epithelial cells of well children and children with upper respiratory infections. These observed in vitro differences in adherence between nontypable and type b H. influenzae strains may explain differences in colonization, pathogenesis, and types of infection due to nontypable and type b H. influenzae.     

Viral-bacterial interactions and risk of acute otitis media complicating upper respiratory tract infection

Acute otitis media (AOM) is a common complication of upper respiratory tract infection whose pathogenesis involves both viruses and bacteria. We examined risks of acute otitis media associated with specific combinations of respiratory viruses and acute otitis media bacterial pathogens. Data were from a prospective study of children ages 6 to 36 months and included viral and bacterial culture and quantitative PCR for respiratory syncytial virus (RSV), human bocavirus, and human metapneumovirus. Repeated-measure logistic regression was used to assess the relationship between specific viruses, bacteria, and the risk of acute otitis media complicating upper respiratory tract infection. In unadjusted analyses of data from 194 children, adenovirus, bocavirus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were significantly associated with AOM (P < 0.05 by χ(2) test). Children with high respiratory syncytial virus loads (≥3.16 × 10(7) copies/ml) experienced increased acute otitis media risk. Higher viral loads of bocavirus and metapneumovirus were not significantly associated with acute otitis media. In adjusted models controlling for the presence of key viruses, bacteria, and acute otitis media risk factors, acute otitis media risk was independently associated with high RSV viral load with Streptococcus pneumoniae (odds ratio [OR], 4.40; 95% confidence interval [CI], 1.90 and 10.19) and Haemophilus influenzae (OR, 2.04; 95% CI, 1.38 and 3.02). The risk was higher for the presence of bocavirus and H. influenzae together (OR, 3.61; 95% CI, 1.90 and 6.86). Acute otitis media risk differs by the specific viruses and bacteria involved. Acute otitis media prevention efforts should consider methods for reducing infections caused by respiratory syncytial virus, bocavirus, and adenovirus in addition to acute otitis media bacterial pathogens.     

A longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion

We analyzed data from a 14-year longitudinal study of respiratory infections in young children to determine the relative importance of viral respiratory infection and nasopharyngeal colonization with Streptococcus pneumoniae and Haemophilus influenzae as factors influencing the occurrence of acute otitis media with effusion. The incidence of this disorder was increased in children with viral respiratory infections (average relative risk, 3.2; P less than 0.0001). Infection with respiratory syncytial virus, influenza virus (type A or B), and adenovirus conferred a greater risk of otitis media than did infection with parainfluenza virus, enterovirus, or rhinovirus. Colonization of the nasopharynx with Str. pneumoniae or H. influenzae had a lesser effect on the incidence of the disease (average relative risk; 1.5; P less than 0.01). Infections with the viruses more closely associated with acute otitis media (respiratory syncytial virus, adenovirus, and influenza A or B) were correlated with an increased risk of recurrent disease. Prevention of selected otitis-associated viral infections should reduce the incidence of this disease.     

High frequency of multiresistant respiratory tract pathogens at community level in South India

Objective: To describe the patterns of antibiotic susceptibility of outpatient strains of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in the district of Pondicherry in South India.
Methods: The antibiotic susceptibilities of 94 S. pneumoniae , 97 H. influenzae and 104 M. catarrhalis strains, collected from outpatients with respiratory tract infections, were determined by disk diffusion and Etest.
Results: Resistance or reduced susceptibility to trimethoprim-sulfamethoxazole was found in 67% of S. pneumoniae , 53% of H. influenzae and 24% of M. catarrhalis strains. Thirty-seven per cent of S. pneumoniae and 39% of H. influenzae strains were resistant or showed reduced susceptibility to tetracycline. Reduced susceptibility to penicillin was found in 6% of S. pneumoniae strains. Overall, 10% of S. pneumoniae and 38% of H. influenzae strains showed reduced susceptibility to ≥3 antibiotics. Comparisons between the antibiotic susceptibility patterns of the Indian strains and a corresponding collection of strains from Sweden indicate that the susceptibility of the native susceptible population is independent of geographic origin.
Conclusions: The findings indicate high consumption of tetracycline and trimethoprim-sulfamethoxazole in the area, which emphasizes the need for surveillance of the pattern of antibiotic susceptibility among respiratory tract pathogens at community level in developing countries and for the implementation of local guidelines for rational use of antibiotics.     

Epidemiology and sensitivity to antibiotics in paediatric respiratory infections over a 4-year period. Retrospective study of 460 H. influenzae,S. pneumoniae and M. catarrhalis strains

H. influenzae, S. pneumoniae and M. catarrhalis are the most common bacterial pathogens causing respiratory infections in children. Resistance to antibiotics may vary according to the geographical area. It is therefore important that the resistance pattern of such pathogens is determined by surveillance studies carried out both on a national scale and by individual laboratories. In this study, we determined retrospectively the prevalence of H. influenzae, S. pneumoniae and M. catarrhalis in upper respiratory tract infections involving subjects of paediatric age, with reference to the type of clinical sample (pharingeal swab and nasal swab), symptomatology and age group. Moreover, for the above micro-organisms the pattern of sensitivity to antibiotics was assessed. In the observation period (January 1996-December 1999), at the day hospital of the Paediatric Pneumology Division of the Gaslini Institute in Genova, in 476 patients between 0 and 15 years of age a total of 460 respiratory pathogens were isolated: 164 S. pneumoniae strains, 163 of H. influenzae (96 belonging to type B and 67 non-attributable to any type) and 133 of M. catarrhalis. As regards sensitivity to antibiotics, ceftriaxone and amoxycillin/clavulanic acid proved to be the most active molecules in all the studied strains.     

Branhamella catarrhalis: incidence in pulmonary infections and determination of sensitivity to 5 antibiotics

This study reports 45 cases of respiratory tract infection associated with Branhamella catarrhalis, diagnosed by bacteriological examination out of 980 sputum samples studied over a 6 months period. These infections were observed mainly in patients with chronic respiratory disease (68.9%). More than half of the isolates were found in pure culture, others were isolated from mixed infections most often with Haemophilus influenzae, Streptococcus pneumoniae or H. influenzae plus S. pneumoniae. 64.7% of Branhamella catarrhalis isolates produced beta-lactamase. In vitro antimicrobial susceptibility testing demonstrated that the B. catarrhalis isolates, including beta-lactamase producing strains, were very susceptible to clavulanic acid plus amoxycillin (MIC90:0.12 microgram/ml) as well as to doxycycline and erythromycin (MIC90:0.5 microgram/ml).     

Comparative in vitro activity of ertapenem against bacterial pathogens isolated from patients with lower respiratory tract infections

This study compared the in vitro activity of ertapenem, ceftriaxone, cefepime, ciprofloxacin and amoxicillin–clavulanate against 381 aerobic and facultative bacterial pathogens isolated from 320 patients with acute bacterial exacerbation of chronic bronchitis or community-acquired pneumonia. Streptococcus pneumoniae and Haemophilus influenzae accounted for 54.6% of the isolates. The ertapenem MIC was ≤2 mg/L for 98.4% of isolates and ≥8 mg/L for 1.0% (all methicillin-resistant Staphylococcus aureus ). Ertapenem had the most potent activity against Enterobacteriaceae, Moraxella catarrhalis , and methicillin-susceptible S. aureus , and its activity against H. influenzae and H. parainfluenzae , all strains of which were susceptible, was not altered by β -lactamase production. Only one S. pneumoniae strain, a penicillin-resistant isolate, was resistant to ertapenem. Ertapenem was highly active in vitro against pyogenic bacteria recovered from patients with community-acquired lower respiratory tract infections.     

Persistent infection of human adenovirus type 5 in human monocyte cell lines.

Adenovirus infection of human monocyte hybridoma cell lines and the fusion partner U937 was investigated. Adenovirus adsorbed poorly to these cells as well as primary human alveolar macrophages. The virus-binding experiments showed a 100-fold reduction in apparent viral binding to these cells compared to the permissive HeLa cells. Adsorption of adenovirus to these cells could be enhanced by preincubation of adenovirus with its antiserum. Following entry into the cells amplification of adenovirus DNA was detected starting at 2 days postinfection but few mature virus particles were produced. The infected cultures survived the infection and continued to grow for more than a year. In these chronically infected cultures, linear adenovirus DNA persisted up to 200 copies per cell and a small amount of mature virus was produced. Infectious center assay and cell cloning experiments showed that the majority of the cells in the chronically infected cultures harbor adenovirus genome. These results indicate that restriction of replication of human adenovirus type 5 at the late phase results in persistent infection of U937 and the human monocyte hybridoma cell lines.     

2011—2012年徐州地区儿童急性呼吸道感染中腺病毒感染特征

目的了解徐州地区儿童腺病毒感染的型别及流行特点。方法采用Real—timePCR方法对2011年1月至2012年12月共990份急性呼吸道感染患儿鼻咽分泌物标本进行检测;对腺病毒阳性的标本进行常规PCR扩增hexon基因并对扩增产物进行测序分型,同时用Hep-2进行细胞分离。结果990份标本中,Real-timePCR检测腺病毒阳性数为164（16．6％）;腺病毒感染全年散发,其高峰期为3—4月;以7岁以下儿童多见,感染阳性率为91．5％;对其中100例PCR阳性标本扩增产物进行测序分型,涵盖B＼C＼E三个亚种,以HAdVB318株（占18．O％）,HAdVC217株（占17．0％）,HAdVB716株（占16．0％）多见,混合HAdVB3和HAdVB7感染2例;134例PCR阳性标本中分离出毒株59株,分离率为44．0％。结论腺病毒是徐州地区儿童急性呼吸道感染的重要病原,徐州地区腺病毒型别种类多样化,病毒重组变异概率较高,需加强对该病毒的流行监测。     

Binding of Haemophilus influenzae to purified mucins from the human respiratory tract.

Mucins are high-molecular-weight glycoproteins and major constituents of the mucus layer which covers the airway surface. We have studied the interactions between bacteria, mucins, and epithelial cells from the human respiratory tract. Nontypeable strains of Haemophilus influenzae were found to bind to purified airway mucins in suspension and on solid phase. Mucins in suspension inhibited the attachment of these strains to nasopharyngeal epithelial cells, while mucin coating of the cells enhanced their binding. In contrast, strains of Streptococcus pneumoniae and encapsulated and other nontypeable H. influenzae strains failed to interact with mucins. These H. influenzae strains used other strategies for adherence to epithelial cells. The type b strain 770235 attached via fimbriae but also expressed a subcapsular adhesin that was detected in a capsule- and fimbria-defective mutant. Mucin pretreatment of these bacteria did not inhibit adherence, but mucin pretreatment of epithelial cells inhibited adherence, probably by shielding of the receptors for these adhesins. Non-mucin-binding nontypeable and encapsulated H. influenzae strains would, therefore, adhere only after disruption of the mucus layer and exposure of cellular receptors. Differences in tissue toxicity and invasiveness among H. influenzae strains may also be influenced by the mucin interactions of the strains.     

Fatal adenovirus type 7b infection in a child with Smith-Lemli-Opitz syndrome

Adenovirus type 7 causes worldwide respiratory tract infections, mainly in children. Severe systemic infections can occur, especially in immunocompromised patients and in patients with underlying chronic diseases. This report describes the first case of a fatal disseminated adenovirus type 7 infection in a child with Smith-Lemli-Opitz syndrome, a rare autosomal recessive disorder due to a primary enzymatic defect in cholesterol metabolism. Nasopharyngeal secretions and autopsy specimens including liver, lung, pleural fluid, and rectum were collected for viral culture. Adenovirus serotype 7 strains were obtained from all anatomic sites, except the liver. All these clinical isolates were analyzed using restriction endonuclease digestion of the genome, identifying them as genome type 7b, a virulent type. In this case, the fatal evolution could have been accelerated by the presence of an immunodeficiency although immunodeficiency is not included in the definition of Smith-Lemli-Opitz syndrome. The frequent recurrent banal infections in Smith-Lemli-Opitz syndrome could be prevented by a cholesterol supplementation regimen. Finally, this report emphasizes the need for efficient therapy for disseminated adenovirus infections, especially for virulent genome types.     

Quantitative detection of Moraxella catarrhalis in nasopharyngeal secretions by real-time PCR

The recognition of Moraxella catarrhalis as an important cause of respiratory tract infections has been protracted, mainly because it is a frequent commensal organism of the upper respiratory tract and the diagnostic sensitivity of blood or pleural fluid culture is low. Given that the amount of M. catarrhalis bacteria in the upper respiratory tract may change during infection, quantification of these bacteria in nasopharyngeal secretions (NPSs) by real-time PCR may offer a suitable diagnostic approach. Using primers and a fluorescent probe specific for the copB outer membrane protein gene, we detected DNA from serial dilutions of M. catarrhalis cells corresponding to 1 to 10(6) cells. Importantly, there was no difference in the amplification efficiency when the same DNA was mixed with DNA from NPSs devoid of M. catarrhalis. The specificity of the reaction was further confirmed by the lack of amplification of DNAs from other Moraxella species, nontypeable Haemophilus influenzae, H. influenzae type b, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus pyogenes, Bordetella pertussis, Corynebacterium diphtheriae, and various Neisseria species. The assay applied to NPSs from 184 patients with respiratory tract infections performed with a sensitivity of 100% and a specificity of up to 98% compared to the culture results. The numbers of M. catarrhalis organisms detected by real-time PCR correlated with the numbers detected by semiquantitative culture. This real-time PCR assay targeting the copB outer membrane protein gene provided a sensitive and reliable means for the rapid detection and quantification of M. catarrhalis in NPSs; may serve as a tool to study changes in the amounts of M. catarrhalis during lower respiratory tract infections or following vaccination against S. pneumoniae, H. influenzae, or N. meningitidis; and may be applied to other clinical samples.     

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.     

Streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro HMPV infection

It remains largely unknown which factors determine the clinical outcome of human metapneumovirus (HMPV) infections. The aim of the present study was to analyse whether exposure to bacterial pathogens can influence HMPV infections. From 57 children, serum samples and colonization data for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pneumoniae were collected at 1.5, 6, 14 and 24 months of age. Seroconversion rates to HMPV were determined and related to bacterial carriage. Frequent nasopharyngeal carriage (≥2 times in the first 2 years of life) of S. pneumoniae, but not of the other three pathogens, was associated with increased seroconversion rates of infants to HMPV at the age of 2 years (frequently vs. less exposed, 93% vs. 59%; p <0.05). Subsequently, the susceptibility of well-differentiated normal human bronchial epithelial cells (wd-NHBE) pre-incubated with bacterial pathogens to in vitro HMPV infection was evaluated. Pre-incubation of wd-NHBE with S. pneumoniae resulted in increased susceptibility to infection with HMPV-enhanced green fluorescent protein (EGFP), as determined by enumeration of EGFP-positive cells. This was not the case for cells pre-incubated with H. influenzae, M. catarrhalis on S. aureus. We conclude that exposure to S. pneumoniae can modulate HMPV infection.     

Bacterial adherence to bladder uroepithelial cells in catheter-associated urinary tract infection

To assess the role of bacterial adherence to uroepithelial cells in the pathogenesis of nosocomial urinary tract infection, we prospectively studied 55 patients with indwelling urinary catheters. We obtained uroepithelial cells from the bladder and urine for culture on the patients' entry into the study and every two to four days during catheterization. In all, 235 collections of uroepithelial cells from these patients were used in an in vitro adherence assay with six gram-negative bacterial strains. With uroepithelial cells from patients who did not have bacterial infections, the adherence of the bacteria used in the assay differed significantly according to species. The least adherence occurred with Escherichia coli GR12; the adherence increased with (in order) Proteus mirabilis, E. coli J96, Klebsiella pneumoniae, Serratia marcescens, and Pseudomonas aeruginosa. With cells collected just before the onset of bacteriuria, adherence of these gram-negative strains was higher in patients in whom gram-negative rod infections developed than in those with gram-positive coccal infections (P = 0.005). Analysis with the Cox proportional-hazards model demonstrated that a significant increase in bacterial adherence to uroepithelial cells in the bladder occurred two to four days before the onset of bacteriuria, but that adherence returned to base-line values with the onset of bacteriuria. These results suggest that a transient increase in the adherence of gram-negative bacteria to bladder epithelial cells may be an important early event in the development of catheter-associated bacteriuria.     

Increase in specific antibody-forming cells in human tonsils after oral stimulation with D-53, a ribosomal vaccine

Thirty subjects who had received an oral ribosomal vaccine to common bacteria of upper respiratory tract infections, and ten controls were tonsillectomized for recurrent infections or rhonchologic pathology. A three-step indirect immunofluorescence technique was used to identify and enumerate specific antibody forming cells (SAFC) in their tonsils. Plasma-cells specific of the four strains being constituents of the oral vaccine (H. influenzae, K. pneumoniae, S. pyogenes and S. pneumoniae) were observed in all samples. The numbers of SAFC were significantly higher in the subjects who had received the oral vaccine. These results support the efficiency or oral immunization in increasing local defenses, even at distance from the sensitized gut-associated lymphoid tissue. These data provide evidence for the homing phenomenon in human mucosae associated lymphoid tissue (MALT).     

The role of BK virus in acute respiratory tract disease and the presence of BKV DNA in tonsils

The significance of BKV infections relative to infections by generally tested respiratory agents was investigated in children with acute respiratory disease. Paired sera from 177 children admitted to a hospital for acute respiratory disease were tested for significant rises in antibodies. Sera from seven patients showed a seroconversion to BKV and clinical signs of acute upper respiratory tract infection were exhibited by each of these patients. BKV infections were present in 8% of the patients with upper respiratory tract disease while seroconversions to adenovirus (2%), influenza A virus (1%), parainfluenza virus (5%), RS virus (6%) and mycoplasma pneumoniae (1%) were observed in 15% of the patients with upper respiratory tract disease. BKV was isolated from the urine of one child with tonsillitis with a concomitant seroconversion to BKV. Tonsils from children with recurrent attacks of acute respiratory disease were tested for the presence of BKV DNA by hybridization with a cloned genomic 32P-labeled DNA of prototype BKV. Five of twelve tonsil DNAs showed hybridization with BKV DNA. Each tonsil showing hybridization with BKV DNA contained multiple nonintegrated copies of the BKV genome per diploid amount of host cell DNA. Attempts to recover infective BKV by transfection of primary human embryonic cells with tonsil DNAs or by co-cultivation of tonsillar cells with primary human embryonic cells were unsuccessful.     

Anti-adhesive activity of human casein against Streptococcus pneumoniae and Haemophilus influenzae

The casein fraction of human milk was found to inhibit the attachment of Streptococcus pneumoniae and Haemophilus influenzae human respiratory tract epithelial cells. The inhibitory activity for S. pneumoniae remained after heat and trypsin treatment of the casein and was found in oligosaccharides released from casein. kappa-Casein, which is the most highly glycosylated casein component, inhibited pneumococcal attachment at concentrations similar to the whole casein fraction. The results are consistent with the known recognition of GlcNAc beta 1-3Gal by S. pneumoniae, since human milk and bovine colostrum, which contain GlcNAc, inhibited attachment, but mature bovine milk lacking GlcNAc did not. The effect on H. influenzae was similar to that on S. pneumoniae in that the attachment was inhibited by human casein and bovine colostrum, but not by either mature bovine milk or by the bovine casein fraction. The kappa-casein component of human milk was a less efficient inhibitor of H. influenzae attachment than the whole casein fraction and the free oligosaccharides were inactive. This anti-microbial effect of human casein represents a new mechanism for the protection by breast-milk against respiratory tract infection.     

Dynamic Virus-Bacterium Interactions in a Porcine Precision-Cut Lung Slice Coinfection Model: Swine Influenza Virus Paves the Way for Streptococcus suis Infection in a Two-Step Process

Swine influenza virus (SIV) and Streptococcus suis are common pathogens of the respiratory tract in pigs, with both being associated with pneumonia. The interactions of both pathogens and their contribution to copathogenesis are only poorly understood. In the present study, we established a porcine precision-cut lung slice (PCLS) coinfection model and analyzed the effects of a primary SIV infection on secondary infection by S. suis at different time points. We found that SIV promoted adherence, colonization, and invasion of S. suis in a two-step process. First, in the initial stages, these effects were dependent on bacterial encapsulation, as shown by selective adherence of encapsulated, but not unencapsulated, S. suis to SIV-infected cells. Second, at a later stage of infection, SIV promoted S. suis adherence and invasion of deeper tissues by damaging ciliated epithelial cells. This effect was seen with a highly virulent SIV subtype H3N2 strain but not with a low-virulence subtype H1N1 strain, and it was independent of the bacterial capsule, since an unencapsulated S. suis mutant behaved in a way similar to that of the encapsulated wild-type strain. In conclusion, the PCLS coinfection model established here revealed novel insights into the dynamic interactions between SIV and S. suis during infection of the respiratory tract. It showed that at least two different mechanisms contribute to the beneficial effects of SIV for S. suis, including capsule-mediated bacterial attachment to SIV-infected cells and capsule-independent effects involving virus-mediated damage of ciliated epithelial cells.     

Antimicrobial susceptibility to levofloxacin and other antibacterial agents among common respiratory pathogens—a Brazilian perspective from the GLOBAL Surveillance Initiative 2001–2002

The GLOBAL (Global Landscape On Bactericidal Activity of Levofloxacin) Surveillance programme monitored antimicrobial susceptibility patterns of the key respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis collected in Brazil during 1997-1998, 1999-2000 and 2001-2002. Penicillin and azithromycin resistance among S. pneumoniae strains increased from 1997-1998, reaching 7.9% and 9.5%, respectively, in 2001-2002. Although decreasing by 4.9% since the previous study, trimethoprim-sulphamethoxazole resistance remained high at 33.7%. Concurrent resistance to penicillin, azithromycin and trimethoprim-sulphamethoxazole was seen in 2.9% of the S. pneumoniae isolates collected. Levofloxacin remained extremely active against S. pneumoniae, with 0.3% resistance reported in 1997-1998 and 0% resistance in 1999-2000 and 2001-2002. beta-Lactamase production in H. influenzae was > 10% in all three studies, with correspondingly high rates of ampicillin resistance. Trimethoprim-sulphamethoxazole was the least active agent tested against H. influenzae, with resistance rates of > 40% recorded in all three studies. All H. influenzae isolates were susceptible to cefuroxime, ceftriaxone, azithromycin and levofloxacin. Of the M. catarrhalis isolates, 98.0% in 1997-1998, 98.0% in 1999-2000 and 81.8% in 2001-2002 were beta-lactamase-positive. The continued high prevalence of antimicrobial resistance in Brazil underscores the importance of current surveillance initiatives. Levofloxacin, a fluoroquinolone prescribed widely for respiratory tract infections, continued to show potent activity against key respiratory pathogens.