Yoichi Hirakata

Pseudomonas (P.) aeruginosa is a major opportunistic pathogen especially in immunocompromised patients. To evaluate the invasiveness of respiratory pathogens, we developed monolayer culture systems and examined the degree of invasion by P. aeruginosa and invasive Salmonella (S.) typhimurium strains using human respiratory cell lines: A549 (derived from lung cancer), BEAS-2B (normal bronchial epithelium), and Calu-3 (pleural effusion of a patient with adenocarcinoma of the lung). Cells were seeded into filter units containing 0.33 cm2 filter membranes with 3.0 μm pores, and were incubated at 37°C under 5% CO2 for 4-10 days. By monitoring the trans-monolayer electrical resistance (TER), we judged that BEAS-2B cells (TER values: 436.2 ± 16.8 to 628.8 ± 66.3 Ω cm2) and Calu-3 cells (TER values: 490.5 ± 25.2 to 547.8 ± 21.6 Ω cm2) formed monolayers with tight junctions, but not A549 cells. On day 8 of culture, monolayer cultures were infected with bacteria, and the number of microorganisms penetrating into the basolateral medium was counted. Wild-type P. aeruginosa PAO1 (PAO1 WT) and S. typhimurium SL1344 were detected in the basolateral medium of BEAS-2B monolayer system by 3 h after inoculation, while only P. aeruginosa PAO1 WT was detected in the basolateral medium of Calu-3 monolayer, indicating poor invasiveness of S. typhimurium SL1344 in the Calu-3 system. These findings suggest that BEAS-2B or Calu-3 monolayer system could be useful for evaluating the invasiveness of respiratory pathogens. Because of the difference in bacterial invasiveness, we may need to choose a suitable cell system for each target pathogen.

β-Lactamase-negative ampicillin-resistant (BLNAR) isolates of Haemophilus influenza e have been emerging in some countries, including Japan. The Clinical and Laboratory Standards Institute has only a susceptible MIC breakpoint (≤1 μg/ml) for piperacillin-tazobactam and a disclaimer comment that BLNAR H. influenzae should be considered resistant, which was adapted without presentation of data. In addition, fluoroquinolone-resistant H. influenzae isolates have recently been occasionally reported worldwide. To address these problems, we examined susceptibilities to β-lactams, including piperacillin-tazobactam, and ciprofloxacin by microdilution and disk diffusion (only for piperacillin-tazobactam) methods, against a total of 400 recent H. influenzae clinical isolates, including 100 β-lactamase-negative ampicillin-susceptible, β-lactamase-positive ampicillin-resistant, BLNAR, and β-lactamase-positive amoxicillin-clavulanate-resistant (BLPACR) isolates each. BLNAR and BLPACR isolates were tested by PCR using primers that amplify specific regions of the ftsI gene. We also detected mutations in quinolone resistance-determining regions (QRDRs) by direct sequencing of the PCR products of DNA fragments. Among β-lactams, piperacillin-tazobactam exhibited potent activity against all isolates of H. influenzae , with all MICs at ≤0.5 μg/ml (susceptible). A disk diffusion breakpoint for piperacillin-tazobactam of ≥21 mm is proposed. We confirmed that all BLNAR and BLPACR isolates had amino acid substitutions in the ftsI gene and that the major pattern was group III-like (87.5%). One ciprofloxacin-resistant isolate (MIC, 16 μg/ml) and 31 ciprofloxacin-susceptible isolates (MICs, 0.06 to 0.5 μg/ml) had amino acid changes in their QRDRs. Piperacillin-tazobactam was the most potent β-lactam tested against all classes of H. influenzae isolates. It is possible that fluoroquinolone-resistant H. influenzae will emerge since several clinical isolates carried mutations in their QRDRs.

Four major causative bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes) of pediatric respiratory infections, 295 clinical isolates in total, were isolated at general practitioners' clinics in Sendai city, and evaluated the drug susceptibility to seven antibiotics for pediatric. Penicillin-resistant S. pneumoniae and penicillin-intermediate S. pneumoniae (PRSP-PISP) were 55.8% of all S. pneumoniae isolates. The MIC90 of penicillin and cephalosporin antibiotics in S. pneumoniae were good, 0.5-1 microg/mL, however, macrolide-resistant strains remarkably increased. As for H. influenzae, 50.0% of all isolates were ampicillin-intermediate and -resistant (MIC: > or = 2 microg/mL), the MIC90s of cephalosporin antibiotics had large differences between 0.5 to 8 microg/mL, and generally less susceptibility was shown to other antibiotics. M. catarrhalis showed less susceptibility to amoxicillin which behaved unstably to penicillinase, on the other hand, the MIC90s of other antibiotics were relatively good, 0.25-1 microg/mL. S. pyogenes remarkably tend to be resistant to macrolide antibiotics, however, the MIC90s of penicillin and cephalosporin antibiotics were very good, 0.03-0.06 microg/mL. Pediatric respiratory infections are required a treatment which results in inhibition of drug-resistant bacteria. Based on the results of drug susceptibility testing, we should make a proper selection of antibiotics by reference to disposition such as drug concentration in serum and transfer into cells.

From October 2005 to September 2006, we collected the specimen from 366 patients with lower respiratory tract infections in 12 institutions in Japan, and investigated the susceptibilities of isolated bacteria to various antibacterial agents and patients' characteristics. Of 411 strains that were isolated from specimen (mainly from sputum) and assumed to be bacteria causing in infection, 406 strains were examined. The isolated bacteria were: Staphylococcus aureus 70, Streptococcus pneumoniae 85, Haemophilus influenzae 78, Pseudomonas aeruginosa (non-mucoid) 46, P. aeruginosa (mucoid) 14, Klebsiella pneumoniae 21, and Moraxella subgenus Branhamella catarrhalis 40. Of 70 S. aureus strains, those with 2 microg/ml or less of MIC of oxacillin (methicillin-susceptible S. aureus: MSSA) and those with 4 microg/ml or more of MIC of oxacillin (methicillin-resistant S. aureus: MRSA) were 38 (54.3%) and 32 (45.7%) strains, respectively. Against MSSA, imipenem had the most potent antibacterial activity and inhibited the growth of 37 strains (97.4%) at 0.063 microg/ml or less. Against MRSA, arbekacin and vancomycin showed the most potent activity and inhibited the growth of all the strains at 1 microg/ml. Carbapenems showed the most potent activities against S. pneumoniae and in particular, panipenem inhibited the growth of all the strains at 0.063 microg/ml or less. Faropenem also had a preferable activity and inhibited the growth of all the strains at 0.25 microg/ml. In contrast, there were high-resistant strains (MIC: over 128 microg/ml) for erythromycin (38.1%) and clindamycin (22.6%). Against H. influenzae, levofloxacin showed the most potent activity and its MIC90 was 0.063 microg/ml or less. Meropenem showed the most potent activity against P. aeruginosa (mucoid) and its MIC90 was 0.5 microg/ml. Against P. aeruginosa (non-mucoid), arbekacin had the most potent activity and its MIC90 was 8 microg/ml. Against K. pneumoniae, cefozopran was the most potent activity and inhibited the growth of all the strains at 0.063 microg/ml or less. Also, all the antibacterial agents except ampicillin generally showed a potent activity against M. (B.) catarrhalis and the MIC90 of them were 2 microg/ml or less. The approximately half the number (53.6%) of the patients with respiratory infection were aged 70 years or older. Bacterial pneumonia and chronic bronchitis accounted for 44.3% and 29.8% of all the respiratory infection, respectively. The bacteria frequently isolated from the patients with bacterial pneumonia were S. aureus (15.4%), S. pneumoniae (23.4%), and H. influenzae (21.3%). S. aureus (25.4%) and S. pneumoniae (18.0%) also were frequently isolated from the patients with chronic bronchitis. Before the drug administration, the bacteria frequently isolated from the patients were S. pneumoniae (22.0%) and H. influenzae (21.4%). The bacteria frequently isolated from the patients treated with macrolides were S. pneumoniae and P. aeruginosa, and their isolation frequencies were each 35.3%.

We conducted 3 nationwide surveillance studies between 2001 and 2005 at 39 participating institutions throughout Japan according to the special survey plan to investigate susceptibility to ciprofloxacin (CPFX) and various parenteral antimicrobials using clinical isolates from patients with severe infection during the reexamination period of parenteral CPFX. Results of the first special survey (2001) were already reported in this journal. The current third special survey (2005) was conducted at 34 participating institutions throughout Japan to determine susceptibility to CPFX and 22 various parenteral antimicrobials with the use of the microdilution method with respect to 1696 strains isolated and identified from various clinical specimens between January and June 2005. The results of CPFX in this survey were compared with those in the first and second special surveys. The minimum inhibitory concentration of CPFX at which 90% of isolates were susceptible (MIC90) ranged from < or =0.063 to 2 microg/mL for methicillin-susceptible Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella spp., Citrobacter freundii, Enterobacter spp., Proteus spp., Serratia marcescens, and Acinetobacter baumannii, revealing no marked change from results of the first and second surveys. However, the CPFX-susceptibility rate of Escherichia coli decreased in the second and third surveys compared to that in the first survey. For Morganella morganii and Pseudomonas aeruginosa, the MIC90 of CPFX tended to increase with time. The CPFX-susceptibility rates calculated from the pneumonia breakpoint were 85.2% for P. aeruginosa and 67.9% for Stenotrophomonas maltophilia. With the exception of these 2 species, major causative organisms of respiratory tract infection had susceptibility rates as high as 90% or more for CPFX, which were similar to results of the first and second special surveys. These susceptibility rates for CPFX were similar to the rates for cefozopran and imipenem. These values generally indicated favorable CPFX susceptibility testing results of major bacteria and the potent antimicrobial activity of CPFX particularly against Gram-negative bacteria. Further surveillance is required regarding the trend in susceptibility of E. coli, M. morganii, and P. aeruginosa, which tended to become less susceptible with time.

Recently, proper use of antimicrobial agents for treatment of infections based on pharmacokinetics (PK)/ pharmacodynamics (PD) theory has been recommended to many clinical doctors. To consider the optimized administration method based on PK/PD theory for the elderly patients of gatifloxacin (GFLX), which was one of the oral respiratory quinolone antibacterial, Monte Carlo Simulation was conducted with community-acquired pneumonia and susceptibility data for Streptococcus pneumoniae (345 strains) collected by the second gatifloxacin surveillance study. From this results, the probabilities of achieving AUC/MIC 30 against pneumococcus for the elderly patients was 96.3% at twice-daily dosing of GFLX 100 mg, 97.2% at twice-daily dosing of GFLX 200 mg, therefore, it was suggested that the clinical effectiveness of the regimen of GFLX 100 mg was as well as the regimen of GFLX 200 mg.

A total of 18,639 clinical isolates in 19 species collected from 77 centers during 2004 in Japan were tested for their susceptibility to fluoroquinolones (FQs) and other selected antibiotics. The common respiratory pathogens, Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae showed a high susceptible rate against FQs. The isolation rate of beta lactamase non-producing ampicillin-resistant H. influenzae was approximately three times as large as those of western countries. Most strains of Enterobacteriaceae were also susceptible to FQs. The resistance rate of Escherichia coli against FQs has however been rapidly increasing so far as we surveyed since 1994. The FQs-resistant rate in methicillin-resistant Staphylococcus aureus (MRSA) showed approximately 90% except for 36%. of sitafloxacin while FQs-resistant rate in methicillin-susceptible S. aureus (MSSA) was around 5%. The FQs-resistant rate of methicillin-resistant coagulase negative Staphylococci (MRCNS) was also higher than that of methicillin-susceptible coagulase negative Staphylococci (MSCNS), however, it was lower than that of MRSA. In Pseudomonas aeruginosa clinical isolates, 32-34% from UTI and 15-19% of from RTI was resistant to FQs. Acinetobacter spp. showed a high susceptibility to FQs. Although FQs-resistant Neisseria gonorrhoeae have not been increased in western countries, it is remarkably high in Japan. In this survey, isolates of approximately 85% was resistant to FQs.

From October 2004 to September 2005, we collected the specimen from 319 patients with lower respiratory tract infections in 12 institutions in Japan, and investigated the susceptibilities of isolated bacteria to various antibacterial agents and patients' characteristics. Of 383 strains that were isolated from specimen (mainly from sputum) and assumed to be bacteria causing in inflammation, 381 strains were examined. The breakdown of the isolated bacteria were: Staphylococcus aureus 87, Streptococcus pneumoniae 80, Haemophilus influenzae 78, Pseudomonas aeruginosa (non-mucoid) 35, P. aeruginosa (mucoid) 9, Klebsiella pneumoniae 15, Moraxella subgenus Branhamella catarrhalis 30, etc. Of 87 S. aureus strains, those with 2 microg/mL or less of MIC of oxacillin (methicillin-sensitive S. aureus: MSSA) and those with 4 microg/mL or more of MIC of oxacillin (methicillin-resistant S. aureus: MRSA) were 40 (46.0%) and 47 (54.0%) strains, respectively. Against MSSA, imipenem had the most potent antibacterial activity and inhibited the growth of all the strains at 0.063 microg/mL. Against MRSA, vancomycin showed the most potent activity and inhibited the growth of all the strains at 1 microg/mL. Arbekacin (ABK) also showed the potent activity and its MIC90 was 2 microg/mL. Carbapenems showed the most potent activities against S. pneumoniae and inhibited the growth of all the strains at 0.25-0.5 microg/mL. Cefozopran (CZOP) also had a preferable activity (MIC90: 1 microg/mL) and inhibited the growth of all the strains at 2 microg/mL. In contrast, there were high-resistant strains (MIC: 128 microg/mL or more) for ABK (2.5%), erythromycin (37.5%), and clindamycin (38.8%). Against H. influenzae, levofloxacin showed the most potent activity and inhibited the growth of all the strains at 0.125 microg/mL. Meropenem showed the most potent activity against P. aeruginosa (mucoid) and inhibited the growth of all the strains at 2 microg/mL. Against P. aeruginosa (non-mucoid), amikacin (AMK) had the most potent activity and its MIC90 was 4 microg/mL. The activity of CZOP against the non-mucoid type also was preferable and its MIC90 was 8 microg/mL. Against K. pneumoniae, CZOP, cefmenoxime, cefpirome, flomoxef were the most potent activity and inhibited the growth of all the strains at 0.063 microg/mL. Also, all the agents generally showed a potent activity against M. (B.) catarrhalis and the MIC90 of them were 4 microg/mL or less. The approximately half the number (57.0%) of the patients with respiratory infection were aged 70 years or older. Bacterial pneumonia and chronic bronchitis accounted for 50.8% and 23.8% of all the respiratory infection, respectively. The bacteria frequently isolated from the patients with bacterial pneumonia were S. aureus (21.6%), S. pneumoniae (24.7%) and H. influenzae (20.1%). S. aureus (20.9%), S. pneumoniae (16.1%), and H. influenzae (16.1%) also were relatively frequently isolated from the patients with chronic bronchitis. Before the drug administration, the bacteria frequently isolated from the patients were S. pneumoniae (22.3%) and H. influenzae (25.1%). The bacteria relatively frequently isolated from the patients treated with macrolides were P. aeruginosa and the isolation frequency was 43.5%.