Reduced lysosomal clearance of autophagosomes promotes survival and colonization of Helicobacter pylori.

Evasion of autophagy is key for intracellular survival of bacteria in host cells, but its involvement in persistent infection by Helicobacter pylori, a bacterium identified to invade gastric epithelial cells, remains obscure. The aim of this study was to functionally characterize the role of autophagy in H. pylori infection. Autophagy was assayed in H. pylori-infected human gastric epithelium and the functional role of autophagy was determined via genetic or pharmacological ablation of autophagy in mouse and cell line models of H. pylori infection. Here, we showed that H. pylori inhibited lysosomal function and thereby promoted the accumulation of autophagosomes in gastric epithelial cells. Importantly, inhibiting autophagosome formation by pharmacological inhibitors or genetic ablation of BECN1 or ATG5 reduced H. pylori intracellular survival, whereas inhibition of lysosomal functions exerted an opposite effect. Further experiments demonstrated that H. pylori inhibited lysosomal acidification and the retrograde trafficking of mannose-6-phosphate receptors, both of which are known to positively regulate lysosomal function. We conclude that H. pylori subverts autophagy into a pro-survival mechanism through inhibition of lysosomal clearance of autophagosomes. Disruption of autophagosome formation offers a novel strategy to reduce H. pylori colonization in human stomachs. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epidemiology and trends in the antibiotic susceptibilities of Gram-negative bacilli isolated from patients with intra-abdominal infections in the Asia-Pacific region, 2010-2013.

This study was conducted to investigate the epidemiology and antimicrobial susceptibility patterns of Gram-negative bacilli (GNB) isolated from intra-abdominal infections (IAIs) in the Asia-Pacific region (APR) from 2010-2013. A total of 17 350 isolates were collected from 54 centres in 13 countries in the APR. The three most commonly isolated GNB were Escherichia coli (46.1%), Klebsiella pneumoniae (19.3%) and Pseudomonas aeruginosa (9.8%). Overall, the rates of extended-spectrum ß-lactamase (ESBL)-producing E. coli and K. pneumoniae were 38.2% and 24.3%, respectively, and they were highest in China (66.6% and 38.7%, respectively), Thailand (49.8% and 36.5%, respectively) and Vietnam (47.9% and 30.4%, respectively). During 2010-2013, the rates of ESBL-producing E. coli and K. pneumoniae isolates causing community-associated (CA) IAIs (collected <48 h after admission) were 26.0% and 13.5%, respectively, and those causing hospital-associated (HA) IAIs were 48.0% and 30.6%, respectively. Amikacin, ertapenem and imipenem were the most effective agents against ESBL-producing isolates. Piperacillin/tazobactam displayed good in vitro activity (91.4%) against CA ESBL-producing E. coli. For other commonly isolated Enterobacteriaceae, fluoroquinolones, cefepime and carbapenems exhibited better in vitro activities than third-generation cephalosporins. Amikacin possessed high in vitro activity against all GNB isolates (>80%) causing IAIs, except for Acinetobacter calcoaceticus-baumannii (ACB) complex (30.9% for HA-IAI isolates). All of the antimicrobial agents tested exhibited <45% in vitro activity against ACB complex. Antimicrobial resistance is a persistent threat in the APR and continuous monitoring of evolutionary trends in the susceptibility patterns of GNB causing IAIs in this region is mandatory.

Epidemiology and antimicrobial susceptibility profiles of pathogens causing urinary tract infections in the Asia-Pacific region: Results from the Study for Monitoring Antimicrobial Resistance Trends (SMART), 2010-2013.

A total of 9599 isolates of Gram-negative bacteria (GNB) causing urinary tract infections (UTIs) were collected from 60 centres in 13 countries in the Asia-Pacific region from 2010-2013. These isolates comprised Enterobacteriaceae species (mainly Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Klebsiella oxytoca, Enterobacter cloacae and Morganella morganii) and non-fermentative GNB species (predominantly Pseudomonas aeruginosa and Acinetobacter baumannii). In vitro susceptibilities were determined by the agar dilution method and susceptibility profiles were determined using the minimum inhibitory concentration (MIC) interpretive breakpoints recommended by the Clinical and Laboratory Standards Institute in 2015. Production of extended-spectrum ß-lactamases (ESBLs) amongst E. coli, K. pneumoniae, P. mirabilis and K. oxytoca isolates was determined by the double-disk synergy test. China, Vietnam, India, Thailand and the Philippines had the highest rates of GNB species producing ESBLs and the highest rates of cephalosporin resistance. ESBL production and hospital-acquired infection (isolates obtained ≥48 h after admission) significantly compromised the susceptibility of isolates of E. coli and K. pneumoniae to ciprofloxacin, levofloxacin and most ß-lactams, with the exception of imipenem and ertapenem. However, >87% of ESBL-producing E. coli strains were susceptible to amikacin and piperacillin/tazobactam, indicating that these antibiotics might be appropriate alternatives for treating UTIs due to ESBL-producing E. coli. Fluoroquinolones were shown to be inappropriate as empirical therapy for UTIs. Antibiotic resistance is a serious problem in the Asia-Pacific region. Therefore, continuous monitoring of evolutionary trends in the susceptibility profiles of GNB causing UTIs in Asia is crucial.

Critical Role of Antimicrobial Peptide Cathelicidin for Controlling Helicobacter pylori Survival and Infection.

The antimicrobial peptide cathelicidin is critical for protection against different kinds of microbial infection. This study sought to elucidate the protective action of cathelicidin against Helicobacter pylori infection and its associated gastritis. Exogenous cathelicidin was found to inhibit H. pylori growth, destroy the bacteria biofilm, and induce morphological alterations in H. pylori membrane. Additionally, knockdown of endogenous cathelicidin in human gastric epithelial HFE-145 cells markedly increased the intracellular survival of H. pylori. Consistently, cathelicidin knockout mice exhibited stronger H. pylori colonization, higher expression of proinflammatory cytokines IL-6, IL-1ß, and ICAM1, and lower expression of the anti-inflammatory cytokine IL-10 in the gastric mucosa upon H. pylori infection. In wild-type mice, H. pylori infection also stimulated gastric epithelium-derived cathelicidin production. Importantly, pretreatment with bioengineered Lactococcus lactis that actively secretes cathelicidin significantly increased mucosal cathelicidin levels and reduced H. pylori infection and the associated inflammation. Moreover, cathelicidin strengthened the barrier function of gastric mucosa by stimulating mucus synthesis. Collectively, these findings indicate that cathelicidin plays a significant role as a potential natural antibiotic for H. pylori clearance and a therapeutic agent for chronic gastritis.

Molecular epidemiology of Clostridium difficile infection in a major chinese hospital: an underrecognized problem in Asia?

Clostridium difficile infection is almost unrecognized in mainland China. We have undertaken a study in a large Chinese teaching hospital in Changsha, Hunan, China, to identify cases of C. difficile, record patient characteristics, and define the molecular epidemiology with respect to ribotype distribution and cross-infection. Between April 2009 and February 2010, we examined fecal samples from 70 hospitalized patients with diarrhea who were receiving or had received antibiotics within the previous 6 weeks. Clinical information was collected and the samples were cultured for C. difficile retrospectively. Isolates were ribotyped, and multiple-locus variable-number tandem-repeat assay (MLVA) subtyping was performed on clusters of the same ribotype. The mean age of patients from whom C. difficile was cultured was 58 years, with only 4/21 patients aged >65 years. All patients, with a single exception, had received a third-generation cephalosporin and/or a quinolone antibiotic. Twenty-one isolates of C. difficile were recovered, and seven different ribotypes were identified, the dominant types being 017 (48%), 046 (14%), and 012 (14%). We identified two clusters of cross-infection with indistinguishable isolates of ribotype 017, with evidence of spread both within and between wards. We have identified C. difficile as a possibly significant problem, with cross-infection and a distinct ribotype distribution, in a large Chinese hospital. C. difficile may be underrecognized in China, and further epidemiological studies across the country together with the introduction of routine diagnostic testing are needed to ascertain the size of this potentially significant problem.

Structural basis of FliG-FliM interaction in Helicobacter pylori.

FliG and FliM are switch proteins that regulate the rotation and switching of the flagellar motor. Several assembly models for FliG and FliM have recently been proposed; however, it remains unclear whether the assembly of the switch proteins is conserved among different bacterial species. We applied a combination of pull-down, thermodynamic and structural analyses to characterize the FliM-FliG association from the mesophilic bacterium Helicobacter pylori. FliM binds to FliG with micromolar binding affinity, and their interaction is mediated through the middle domain of FliG (FliGM ), which contains the EHPQR motif. Crystal structures of the middle domain of H. pylori FliM (FliM(M)) and FliG(M) -FliM(M) complex revealed that FliG binding triggered a conformational change of the FliM α3-α1' loop, especially Asp130 and Arg144. We furthermore showed that various highly conserved residues in this region are required for FliM-FliG complex formation. Although the FliM-FliG complex structure displayed a conserved binding mode when compared with Thermotoga maritima, variable residues were identified that may contribute to differential binding affinities across bacterial species. Comparison of the thermodynamic parameters of FliG-FliM interactions between H. pylori and Escherichia coli suggests that molecular basis and binding properties of FliM to FliG is likely different between these two species.

Helicobacter pylori causes epigenetic dysregulation of FOXD3 to promote gastric carcinogenesis.

BACKGROUND & AIMS: Deregulation of forkhead box (Fox) proteins, an evolutionarily conserved family of transcriptional regulators, leads to tumorigenesis. Little is known about their regulation or functions in the pathogenesis of gastric cancer. Promoter hypermethylation occurs during Helicobacter pylori-induced gastritis. We investigated whether the deregulated genes contribute to gastric tumorigenesis. METHODS: We used integrative genome-wide scans to identify concomitant hypermethylated genes in mice infected with H pylori and human gastric cancer samples. We also analyzed epigenetic gene silencing in gastric tissues from patients with H pylori infection and gastritis, intestinal metaplasia, gastric tumors, or without disease (controls). Target genes were identified by chromatin immunoprecipitation microarrays and expression and luciferase reporter analyses. RESULTS: Methylation profile analyses identified the promoter of FOXD3 as the only genomic region with increased methylation in mice and humans during progression of H pylori-associated gastric tumors. FOXD3 methylation also correlated with shorter survival times of patients with gastric cancer. Genome demethylation reactivated FOXD3 expression in gastric cancer cell lines. Transgenic overexpression of FOXD3 significantly inhibited gastric cancer cell proliferation and invasion, and reduced growth of xenograft tumors in mice, at least partially, by promoting tumor cell apoptosis. FOXD3 bound directly to the promoters of, and activated transcription of, genes encoding the cell death regulators CYFIP2 and RARB. Levels of FOXD3, CYFIP2, and RARB messenger RNAs were reduced in human gastric tumor samples, compared with control tissues. CONCLUSIONS: FOXD3-mediated transcriptional control of tumor suppressors is deregulated by H pylori infection-induced hypermethylation; this could perturb the balance between cell death and survival. These findings identify a pathway by which epigenetic changes affect gastric tumor suppression.

Multiple conformations of the FliG C-terminal domain provide insight into flagellar motor switching.

Bacterial flagellar switching between counterclockwise and clockwise directions is mediated by the coupling of the chemotactic system and the motor switch complex. The conformational changes of FliG are closely associated with this switching mechanism. We present two crystal structures of FliG(MC) from Helicobacter pylori, each showing distinct domain orientations from previously solved structures. A 180° rotation of the charged ridge-containing C-terminal subdomain FliG(Cα1-6) that is prompted by the rotational freedom of Met245 psi and Phe246 phi at the MFXF motif was revealed. Studies on the swarming and swimming behavior of Escherichia coli mutants further identified the importance of the 245MFXF248 motif and a highly conserved residue, Asn216, in motor switching. Additionally, multiple conformations of FliG(Cα1-6) were demonstrated by intramolecular cysteine crosslinking. The conformational flexibility of FliGc leads us to propose a model that accounts for the symmetrical torque generation process and for the dynamics of the motor.

A multicentre surveillance study on the characteristics, bacterial aetiologies and in vitro antibiotic susceptibilities in patients with acute exacerbations of chronic bronchitis.

BACKGROUND AND OBJECTIVE: Antimicrobial resistance is a global problem and the prevalence is high in many Asian countries. METHODS: A prospective observational study of the prevalence of bacterial pathogens and their antimicrobial susceptibilities in patients with acute exacerbations of chronic bronchitis (AECB) was conducted in Indonesia, Philippines, Korea, Thailand, Malaysia, Taiwan and Hong Kong from August 2006 to April 2008. The diagnosis of AECB was based on increased cough and worsening of two of following: dyspnoea, increased sputum volume or purulence. Patients who had taken antibiotics within 72 h of presentation were excluded. All bacterial strains were submitted to a central laboratory for re-identification and antimicrobial susceptibility testing to 16 antimicrobial agents according to Clinical and Laboratory Standards Institute. RESULTS: Four hundred and seven isolates were identified among 447 patients of AECB. The most frequent organisms isolated were Klebsiella pneumoniae and associated species (n = 91 + 17), Haemophilus influenzae (n = 71), Pseudomonas aeruginosa (n = 63), Streptococcus pneumoniae (n = 32), Acinetobacter baumannii (n = 22) and Moraxella catarrhalis (n = 21). According to Clinical and Laboratory Standards Institute susceptibility breakpoints, 85.7% and >90% of these pathogens were susceptible to levofloxacin and cefepime respectively. Other options with overall lower susceptibilities include imipenem, ceftazidime, ceftriaxone and amoxicillin/clavulanate. CONCLUSIONS: Gram-negative bacteria including Klebsiella spp., P. aeruginosa and Acinetobacter spp. constitute a large proportion of pathogens identified in patients with AECB in some Asian countries. Surveillance on the local prevalence and antibiotic resistance of these organisms is important in guiding appropriate choice of antimicrobials in the management of AECB.

Molecular interaction of flagellar export chaperone FliS and cochaperone HP1076 in Helicobacter pylori.

Flagellar export chaperone FliS prevents premature polymerization of flagellins and is critical for flagellar assembly and bacterial colonization. Previously, a yeast 2-hybrid study identified various FliS-associated proteins in Helicobacter pylori, but the implications of these interactions are not known. Here we demonstrate the biophysical interaction of FliS (HP0753) and the uncharacterized protein HP1076 from H. pylori. HP1076 possesses a cochaperone activity that promotes the folding and chaperone activity of FliS. We further determined the crystal structures of FliS, HP1076, and the binary complex at 2.7, 1.8, and 2.7 Å resolution, respectively. HP1076 adopts a helix-rich bundle structure and interestingly shares a similar fold with a flagellin homologue, hook-associated protein, and FliS. The FliS-HP1076 complex revealed an extensive electrostatic and hydrophobic binding interface, which is distinct from the flagellin binding pocket in FliS. The helical stacking interaction between HP1076 and FliS suggests that HP1076 stabilizes 2 α helices of FliS and therefore the overall structure of the bundle. Our findings provide new insights into flagellar export chaperones and may have implications for other secretion chaperones in the type III secretion system.

Crystal structure of activated CheY1 from Helicobacter pylori.

Chemotaxis is an important virulence factor for Helicobacter pylori colonization and infection. The chemotactic system of H. pylori is marked by the presence of multiple response regulators: CheY1, one CheY-like-containing CheA protein (CheAY2), and three CheV proteins. Recent studies have demonstrated that these molecules play unique roles in the chemotactic signal transduction mechanisms of H. pylori. Here we report the crystal structures of BeF(3(-)-activated CheY1 from H. pylori resolved to 2.4 A. Structural comparison of CheY1 with active-site residues of BeF3(-)-bound CheY from Escherichia coli and fluorescence quenching experiments revealed the importance of Thr84 in the phosphotransfer reaction. Complementation assays using various nonchemotactic E. coli mutants and pull-down experiments demonstrated that CheY1 displays differential association with the flagellar motor in E. coli. The structural rearrangement of helix 5 and the C-terminal loop in CheY1 provide a different interaction surface for FliM. On the other hand, interaction of the CheA-P2 domain with CheY1, but not with CheY2/CheV proteins, underlines the preferential recognition of CheY1 by CheA in the phosphotransfer reaction. Our results provide the first structural insight into the features of the H. pylori chemotactic system as a model for Epsilonproteobacteria.

The TUBEX test detects not only typhoid-specific antibodies but also soluble antigens and whole bacteria.

TUBEX (IDL Biotech) is a 5 min semiquantitative colorimetric test for typhoid fever, a widely endemic disease. TUBEX detects anti-Salmonella O9 antibodies from a patient's serum by the ability of these antibodies to inhibit the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle. Herein, we report that TUBEX could also be used to specifically detect soluble O9 lipopolysaccharide in antigen-spiked buffer by the ability of the antigen to inhibit the same binding between the particles. Sensitivity of antigen detection was improved (8-31 mug ml(-1)) by using a modified protocol in which the test sample was mixed with the indicator particles first, rather than with the magnetic particles as for antibody detection. The antigen was also detectable in spiked serum and urine samples, albeit less well (2-4-fold) than in buffer generally. However, no antigen was detected from six typhoid sera examined, all of which had anti-O9 antibodies. In addition, whole organisms of Salmonella Typhi (15 strains) and Salmonella Enteritidis (6 strains) (both O9(+) Salmonella), grown in simulated blood broths or on MacConkey agar, were also detectable by TUBEX when suspended at >9 x 10(8) organisms ml(-1). Expectedly, Salmonella Paratyphi A (7 strains), Salmonella Typhimurium (1 strain) and Escherichia coli (2 strains) were negative in the test. Thus, the same TUBEX kit may be used in several ways both serologically and microbiologically for the rapid diagnosis of typhoid fever. However, validation of the newer applications will require the systematic examination of real patient and laboratory materials.

Levofloxacin-resistant Helicobacter pylori in Hong Kong.

BACKGROUND: Fluoroquinolone-resistant Helicobacter pylori emerged in 1995 and the resistance was due to point mutation in the gyrA gene. In this study we investigate the resistance mechanism and the antimicrobial susceptibilities of clarithromycin, metronidazole, amoxicillin, tetracycline and telithromycin against levofloxacin-resistant H. pylori in Hong Kong. METHODS: One hundred and ninety-one nonduplicate H. pylori isolates were collected during 2004 and 2005, and 25 isolates with levofloxacin zone sizes less than 30 mm were selected for minimal inhibitory concentration determination by agar dilution, gyrA gene amplication and sequencing the amplified gyrA gene. RESULTS: The prevalence of levofloxacin-resistant H. pylori was 11.5% (22/191). Among these levofloxacin-resistant strains, 7 (31.8%) and 10 (45.5%) were resistant to clarithromycin and metronidazole, respectively, 17 (77.3%) had point mutations in gyrA gene at amino acids 87, 91 and 130 and the most frequent mutation point was at position 91. CONCLUSIONS: Amoxicillin, tetracycline and telithromycin were active against levofloxacin-resistant H. pylori and levofloxacin resistance was mainly due to point mutation in the gyrA gene, especially at amino acid position 91.

Rapid genotyping of CTX-M extended-spectrum beta-lactamases by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (dHPLC) is a powerful technique which has been used extensively to detect genetic variation. This is the first report of the application of dHPLC for rapid genotyping of bacterial beta-lactamase genes. The technique was specifically developed to genotype members of all blaCTX-M DNA homology groups. Thirteen well-defined blaCTX-M extended-spectrum beta-lactamase (ESBL)-producing strains were used to develop and optimize the dHPLC genotyping assay. Further evaluation was carried out with a blinded panel of 62 clinical isolates. The results of blaCTX-M genotyping achieved by dHPLC were comparable to the typing results obtained by DNA sequencing. Applying the newly developed dHPLC-based genotyping method, we successfully genotyped all 73 blaCTX-M ESBL-producing strains from the 4-month survey study. Furthermore, we found the first reported cases in the United Kingdom of clinically significant disease caused by CTX-M-14- and CTX-M-1-producing Escherichia coli strains. We conclude that the novel dHPLC assay is highly accurate, rapid, and cost-effective for the genotyping of blaCTX-M-producing ESBLs and has great potential for determining the clinical relevance of different and new blaCTX-M genotypes, as well as for epidemiological studies and surveillance programs.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolated in Jiangsu Province, China, with a focus on fluoroquinolone resistance.

In this study, the phenotypic and genotypic resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Jiangsu Province, China, was analysed. In vitro susceptibility testing of eight antimicrobial agents, including ciprofloxacin and levofloxacin, against 95 clinical isolates was carried out. Detection of mutations in the gyrA and parC genes was performed by sequence analysis. The clinical isolates demonstrated 100% resistance to ciprofloxacin and 98.9% non-susceptibility to levofloxacin. All of the isolates were susceptible to cefotaxime and ceftriaxone. For cefepime, spectinomycin and tetracycline, 98.9, 94.7 and 1.1% of the isolates were susceptible, respectively. None of the isolates was susceptible to penicillin. Five types based on gyrA mutations could be categorized among 54 isolates with seven different mutation sites found on their parC gene. Analysis of sequence results showed that the gyrA mutation Asp-95-->Ala and the parC mutations Ser-87-->Arg and Ser-87-->Asn made a significant contribution to the resistance to fluoroquinolones, in addition to double mutations found in each gene. Therefore, the use of fluoroquinolones in the treatment of N. gonorrhoeae infections in Jiangsu Province is not recommended, while the use of third- and fourth-generation cephalosporins and spectinomycin is recommended.

Multicenter antimicrobial susceptibility survey of gram-negative bacteria isolated from patients with community-acquired infections in the People's Republic of China.

A survey of 2,099 gram-negative bacilli from community infections at seven centers in the People's Republic of China is reported. The rates of resistance of 1,615 isolates of the family Enterobacteriaceae were as follows: 40.8% for ciprofloxacin, 32.2% for gentamicin, 0% for imipenem or ertapenem, and 14.7% for cefotaxime. The rates of extended-spectrum beta-lactamase production were 16% for Escherichia coli and 17% for Klebsiella.

Comparison of antimicrobial resistance of Acinetobacter baumannii clinical isolates from Shanghai and Hong Kong.

OBJECTIVE: To compare the antimicrobial resistance patterns of Acinetobacter baumannii isolates from Shanghai and Hong Kong. MATERIALS AND METHODS: A total of 212 A. baumannii strains of one isolate per patient were collected from Shanghai and Hong Kong from August 2002 to August 2003 that were tested against 15 commonly used antimicrobial agents by the agar dilution method according to the NCCLS guidelines. RESULTS: Most beta-lactams showed no significant increase in activity after adding beta-lactamase inhibitors. The resistance rates of the isolates against ticarcillin-clavulanate, piperacillin-tazobactam and ampicillin-sulbactam were for Shanghai 74.9, 70.9, 69.1% and Hong Kong 24.3, 18.9, 13.5%, respectively. Only cefoperazone-sulbactam showed a significant increase in activity against both Shanghai and Hong Kong strains, as the resistance rates dropped from 93.7 to 8.6% and 83.8 to 5.4%, respectively. The resistance rates of ceftazidime, cefepime, and gentamicin against Shanghai strains were 69.7, 72.0, 73.7% and Hong Kong strains 69.7, 29.7, 18.9%, respectively. About 65% of Shanghai strains were found to be amikacin-resistant, however, all Hong Kong strains were sensitive. Fluoroquinolones including ciprofloxacin and levofloxacin had resistance rates over 60% against Shanghai strains, but only 13.5% against Hong Kong strains. Shanghai strains had imipenem and meropenem resistance rate of 6.3%. Though 10.8% Hong Kong strains were resistant to meropenem, only 2.7% of them were resistant to imipenem. CONCLUSION: A. baumannii isolated from Shanghai were more resistant to all drugs except meropenem than Hong Kong isolates. The results indicate a need for measures to control the abuse of antibiotic usage in order to prevent the emergence of more multidrug-resistant isolates in both cities.

In vitro activity of telithromycin against respiratory tract pathogens in comparison with other antimicrobial agents.

This study was done to evaluate the in vitro activity of a new ketolide telithromycin in comparison with clarithromycin, erythromycin, moxifloxacin and levofloxacin against Streptococcus pneumoniae (n = 67), Haemophilus influenzae (n = 139), and Moraxella catarrhalis (n = 46)collected between January and June 2003 in Hong Kong. Among the H. influenzae isolates, 25.2% produced beta-lactamase, while 97.8% of M. catarrhalis isolates produced beta-lactamase. Half of the S. pneumoniae isolates were nonsusceptible to penicillin, and 90.9% of these strains were resistant to clarithromycin and erythromycin. One (1.5%) S. pneumoniae strain was resistant to levofloxacin (MIC = 8 mg/l) and all isolates were sensitive to moxifloxacin and telithromycin with MIC <1 mg/l. H. influenzae isolates were sensitive to all fluoroquinolones tested and 2.2% of H. influenzae were resistant to clarithromycin. M. catarrhalis isolates were sensitive except 1 strain which was resistant to levofloxacin (MIC = 4 mg/l) and moxifloxacin (8 mg/l). All M. catarrhalis strains were sensitive to telithromycin with MIC90 = 0.5 mg/l. Telithromycin demonstrated high activity and no resistance was found in all these major respiratory tract pathogens.