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In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-β-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6')-Iag, blaIMP-1, a truncated form of blaIMP-1, and a truncated form of aac(6')-Iag. The aac(6')-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6')-Iz. Recombinant AAC(6')-Iag protein showed aminoglycoside 6'-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6')-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for blaIMP-1 and aac(6')-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 2655 strains including 810 strains of Gram-positive bacteria, 1635 strains of Gram-negative bacteria, and 210 strains of anaerobic bacteria obtained from 30 medical institutions during 2009 was examined. The results were as follows; (1) MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multidrug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). (2) MEPM maintained potent and stable antibacterial activity against Pseudomonas aeruginosa. The proportion of MEPM-resistant strains to ciprofloxacin-resistant strains or imipenem-resistant strains were 53.1% and 58.0% respectively. (3) The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (26 strains) in enterobacteriaceae. And the proportion of metallo-beta-lactamase strains was 2.0% (6 strains) in P. aeruginosa. (4) Of all species tested, there were no species except for Bacteroides fragilis group, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 14 years passed after available for commercial use in Japan.

Objectives: The aim of this study was to evaluate the dissemination of metallo-beta-lactamase (MBL)-encoding genes among multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from major hospitals in the Hiroshima region., Methods: During July to December from 2004 to 2006, a surveillance of eight major hospitals in the Hiroshima region identified 387 non-duplicate isolates resistant to imipenem (MIC >or= 16 mg/L). They were screened for resistance to amikacin (MIC >or= 64 mg/L) and ciprofloxacin (MIC >or= 4 mg/L) and MBL-encoding genes. The structure of the variable regions of the integrons was determined using PCR mapping. Clonality was assessed using PFGE and multilocus sequence typing (MLST)., Results: The frequency of MBL-positive isolates in MDR P. aeruginosa isolates significantly increased from 42.3% in 2004 to 81.4% in 2006. Most of the MBL-positive isolates produced IMP-1 followed by VIM-2. The bla(IMP-1) and bla(VIM-2) genes were present in class 1 integrons. Characterization of the variable regions of the integron showed the presence of six different gene cassette arrays in bla(IMP-1) cassettes and a single array in bla(VIM-2) cassettes. The IMP-1 producers belonged to two clonal lineages using PFGE and MLST analyses and the integron variations correlated well with the clonal complexes. Among them, strains positive for a newly identified In113-derived bla(IMP-1) gene cassette array were most widely distributed in Hiroshima., Conclusions: This study shows a dramatic increase in MBL genes, primarily bla(IMP-1), in MDR P. aeruginosa isolates in Hiroshima during these 3 years. In addition, MDR P. aeruginosa with the newly discovered In113-derived bla(IMP-1) gene cassette array appears to be clonally expanding.

Susceptibility to a range of antimicrobial agents was determined among isolates of Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae collected in 12 centers throughout Japan during years 1-5 (the respiratory seasons of 1999-2004) of the longitudinal Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin study. The most frequent source of isolates of S. pneumoniae was from patients with community-acquired pneumonia (CAP) (25.3%). Reduced susceptibility to penicillin or erythromycin resistance was common among S. pneumoniae isolates (30.9-44.5% and 77.2-81.9%, respectively). The macrolide MIC(50) for S. pneumoniae was >or=128 microg/ml (azithromycin and erythromycin) and >or=64 microg/ml (clarithromycin). The erm(B) genotype accounted for the most erythromycin-resistant isolates in each study year. H. influenzae isolates were most commonly derived from patients with CAP (26.2%). The proportion of H. influenzae isolates that were beta-lactamase positive ranged between 4.3% and 9.7%. The prevalence of beta-lactamase-negative ampicillin-resistant isolates increased from 0.4% to 2.6% between years 1 and 4 then to 19.7% in year 5. S. pyogenes isolates were highly susceptible to most antimicrobial agents except macrolides and tetracycline. Telithromycin was highly active against all three pathogens examined throughout the study.

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 876 strains of Gram-positive bacteria, 1764 strains of Gram-negative bacteria, and 198 strains of anaerobic bacteria obtained from 30 medical institutions during 2006 was measured. The results were as follows; 1. MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus. 2. As for Pseudomonas aeruginosa, all of the MEPM-resistant strains were resistant to imipenem (IPM). MEPM showed low cross-resistant rate both againt IPM-resistant P. aeruginosa (41.8%) and ciprofloxacin-resistant P. aeruginosa (33.3%). 3. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 4.3% (6 strains) in Escherichia coli, 1.1% (1 strain) in Citrobacter freundii, 21.7% (5 strains) in Citrobacter koseri, 3.1% (4 strains) in Klebsiella pneumoniae, 3.3% (3 strains) in Enterobacter cloacae, 0.8% (1 strain) in Serratia marcescens, and 4.9% (2 strains) in Providencia spp. The proportion of metallo-beta-lactamase strains was 3.1% (10 strains) in P. aeruginosa. 4. Of all species tested, there were no species, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem at present, 11 years after available for commercial use.

Pseudomonas aeruginosa showing resistance to imipenem were found in 100 of 1,058 strains (9.5%) from six hospitals (a-f) in Hiroshima City, Japan. Of the 100 strains, 14 (14%) were double disk synergy test positive using sodium mercaptoacetic acid disks, and 18 (18%) were bla(IMP-1) or bla(VIM-2) allele positive by polymerase chain reaction (PCR). Among 100 imipenem-resistant strains, 32 were categorized into multi-drug resistant strains, in which 13 were positive for the metallo-beta-lactamase gene. Fifty-one strains (51%) among the 100 imipenem-resistant strains had elevated RND efflux pump activity against levofloxacin. But only 6 of 51 strains were classified as multi-drug resistant strains. The pulsed field gel electrophoresis analysis of the Spe I-digested DNA from the 100 isolates suggested not only clonal spread but spread of heterogeneous clones started to contribute to the prevalence of metallo-beta-lactamase producing P. aeruginosa strains in Japanese hospitals.

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 907 strains of Gram-positive bacteria, 1790 strains of Gram-negative bacteria, and 192 strains of anaerobic bacteria obtained from 30 medical institutions during 2004 was measured. The results were as follows; 1. MIC90 of MEPM for almost all of enterobacteriaceae and Haemophilus influenzae were 4-fold to 32-fold lower than those of other carbapenems. MEPM was more active than other carbapenem antibiotics against Gram-negative bacteria, especially against enterobacteriaceae and H. influenzae. MEPM were active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus. 2. As for Pseudomonas aeruginosa, imipenem (IPM) showed high cross-resistant rate againt meropenem-resistant P. aeruginosa (87.9%). MEPM showed low cross-resistant rate both againt IPM-resistant P. aeruginosa (49.2%) and ciprofloxacin-resistant P. aeruginosa (38.0%). 3. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (4 strains) in Escherichia coli, 8.0% (2 strains) in Citrobacter koseri, 2.5% (3 strains) in Klebsiella pneumoniae, 2.5% (2 strains) in Enterobacter cloacae, 0.9% (1 strains) in Serratia marcescens, and 2.2% (2 strains) in Proteus mirabilis. The proportion of metallo-beta-lactamase strains was 1.6% (5 strains) in P. aeruginosa. 4. Of all species tested, Peptostreptococcus spp. was the only species, which MIC90 of MEPM was more than 4-fold higher than that in our previous study using clinical isolates during 2002 (0.25 microg/ml --> 1 microg/ml). Therefore, there is almost no siginificant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem at present, 9 years after available for commercial use.

Burow's solution, or aluminum acetate solution, is effective in inhibiting various microorganisms including methicillin-resistant Staphylococcus aureus (MRSA) that are commonly observed in chronic suppurative otitis media. It takes several days to prepare Burow's solution using aluminum sulfate, and the pharmaceutical properties of the solution are not fully understood. In this study, the effect of storage (5 months) of Burow's solution prepared according to the Teine-Keijin Hospital manual on its pharmaceutical properties and antibacterial activities was examined. We also attempted to develop a rapid preparation method of aluminum acetate (or 1.7% aluminum) solution using two commercially available compounds of aluminum acetate basic (Al(2)O(CH(3)CO(2))(4), Al(OH)(CH(3)CO(2))(2)). The properties of Burow's solution, pH, osmolarity and antibacterial activity, were the same among different preparations and its storage for 5 months at 4 degrees C had no effect on these properties. The antibacterial potency of Burow's solution was dependent on aluminum concentration and its antibacterial potency against S. aureus and several MRSA strains was of the same magnitude. In a rapid preparation, aluminum acetate basic was mixed with appropriate amounts of tartaric acid and acetic acid, and the suspension was boiled for 2-2.5 hr until dissolved. The rapidly prepared aluminum acetate solution showed the same pharmaceutical properties and antibacterial activities as those of Burow's solution. The newly developed preparation method for aluminum acetate solution is expected to be convenient and feasible for hospital treatment of chronic suppurative otitis media.

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 899 strains of Gram-positive bacteria, 1500 strains of Gram-negative bacteria, and 158 strains of anaerobic bacteria obtained from 28 medical institutions during 2002 was measured. The results were as follows; 1. MEPM was more active than other carbapenem antibiotics against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MIC90 of MEPM against Pseudomonas aeruginosa was the lowest of the drugs tested. MEPM showed low cross-resistant rate against both imipenem-resistant P. aeruginosa and ciprofloxacin-resistant P. aeruginosa. MEPM was active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE). 2. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (4 strains) in Escherichia coli and 1.9% (2 strains) in Klebsiella pneumoniae. Carbapenems including MEPM were active against these ESBL strains. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem; at present, 7 years after available for commercial use.

This report discusses about the probability of the standardization of external quality control (control survey) in local medical area such as prefecture size. For example, our control survey by Hiroshima medical association in Hiroshima prefecture is selected and shown on their effort to the standardization of the control survey. This Hiroshima control survey is continuing for 27 years and its purpose is the improvement of the differences between laboratory facilities. For the all standardization of control survey system, tests, devices and reagents, the recent reports of Hiroshima control survey shows that the reference methods by Japan Society of Clinical Chemistry (JSCC) and the enzyme reference materials by Japan Committee of Clinical Laboratory Standardization (JCCLS) are very useful for the improvement of the differences between laboratory facilities in Hiroshima Prefecture.

To evaluate the resistance for major oral antimicrobial agents, mainly new quinolones, we carried out a drug susceptibility surveillance of 3,050 strains of 11 microbial species clinically isolated at 8 institutions such as general hospitals and examination centers in Hiroshima city. 10 antimicrobial agents were used: 3 new quinolone drugs, 5 beta-lactam drugs, minocycline and clarithromycin. Among Gram-positive bacteria, methicillin resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis showed low susceptibility to the new quinolone drugs, while methicillin susceptible Staphylococcus aureus (MSSA) and Streptococcus pneumoniae were highly sensitive to these drugs. Among Gram-negative bacteria, Pseudomonas aeruginosa showed high resistance for the new quinolone drugs, but enteric bacteria and Haemophilus influenzae did not show marked resistance, maintaining almost good sensitivity to these drugs. To reduce the appearance of resistant bacteria, appropriate antimicrobial agents should be selected. Drug susceptibility surveillance in the community will be also important in the future.

We carried out a prospective clinical trial of colon preparation with a regimen of oral antibiotics starting on the day before surgery. The patients were assigned to one of two groups consisting of either a mechanical preparation alone group (group 1, 45 cases) or a mechanical bowel preparation with oral antibiotics group (group 2, 38 cases). Group 2 received kanamycin and metronidazole three times on the day before surgery. Cefmetazole was administered for 3 consecutive days as prophylaxis in both groups. In a study using intraoperative mucosal swabs, the rates of group 2 patients with cultures yielding anaerobes or Gram-negative bacteria were significantly lower than those of group 1. There were no significant differences in the rates of patients with cultures yielding fungi or Gram-positive organisms. The positive culture rate in the peritoneal fluid of group 1 was also higher than that of group 2 (40%, 16%, P < 0.05). The surgical site infection rate was 18% in group 1 and 13% in group 2. Organisms isolated from the sites of postoperative infections were not identical with those from the peritoneal fluid. This relatively brief course preparation minimized the emergence of resistant strains. However, in spite of the colonic bacterial burden and the intraoperative inoculation in the patients with mechanical cleansing alone, their incidence of subsequent infections was comparable to that of patients who were administered oral antibiotics provided that the prophylactic antibiotic was administered for 3 days after surgery.

To determine the effect of antibiotics used for the treatment of postoperative infections on the normal intestinal flora in patients who have undergone gastroenterological surgery, we performed a prospective randomized study in patients who had undergone distal gastrectomy. Cefazoline was administered up to postoperative day 3 for prophylaxis. For the treatment of postoperative infections, either panipenem/betamipron (PAPM/BP; n = 5) or sulbactam/cefoperazone (SBT/CPZ; n = 5) was adminstered to ten patients with signs of postoperative infections. Feces were examined for bacteria before and 4 and 7 days after the commencement of administration of these therapeutic antibiotics. The anaerobic bacterial count decreased significantly in four of five patients treated with SBT/CPZ, from 1010.2+/-0.36/g before treatment to 105.22+/-3.51/g. In the PAPM/BP-treated group, the anaerobic bacterial count did not change after antibiotic therapy. We also investigated the major genera of colonic anaerobes, and found that the numbers of bifidobacteria were maintained during PAPM/BP administration. However, in the SBT/CPZ group, the number of bifidobacteria decreased to below the detection limit in four of five patients. Little change was observed in the aerobic bacterial count in either group. However, Enterobacteriaceae were replaced by enterococcus or candida in all patients from the SBT/CPZ group and in one of five patients from the PAPM/BP group. Physicians should be alert for the possible clinical consequences of changes in intestinal microflora after antibiotic treatment of postoperative infections.

We examined the pancreatic tissue concentrations of cefazolin in ten patients undergoing pancreatectomy, and determined the optimal intraoperative time to deliver a repeat dose of cefazolin. An intravenous bolus dose of 1 g cefazolin was administered at the time of skin incision. Peripheral blood, subcutaneous adipose tissue, and peritoneal samples were obtained intraoperatively every hour for 4 h after the antibiotic was first administered, and pancreatic tissue samples were obtained at the time of pancreatectomy. To determine adequate tissue levels of cefazolin, minimum inhibitory concentrations (MIC) were measured for four bacterial species, namely 360 isolates of methicillin-sensitive Staphylococcus aureus (MSSA), 204 isolates of Klebsiella pneumoniae, 314 isolates of Escherichia coli, and 30 isolates of Streptococcus spp. The antibiotic concentrations in adipose tissue and peritoneum 3 h after the administration of cefazolin were lower than the MIC80 for K. pneumoniae, E. coli, and Streptococcus spp. Most pancreatic tissue samples showed antibiotic concentrations greater than the MIC80 for these bacterial species; however, those from four patients complicated by severe chronic pancreatitis, massive intraoperative bleeding, or obesity showed concentrations lower than the MIC80. Thus, we recommend that a second dose of cefazolin be given 3 h after the first administration to maintain adequate levels of antibiotic activity.