Zenzo Nagasawa

Nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy in 2007: general view of the pathogens' antibacterial susceptibility.

For the purpose of nationwide surveillance of the antimicrobial susceptibility of bacterial respiratory pathogens collected from patients in Japan, the Japanese Society of Chemotherapy conducted a third year of nationwide surveillance during the period from January to April 2008. A total of 1,097 strains were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections. Susceptibility testing was evaluable with 987 strains (189 Staphylococcus aureus, 211 Streptococcus pneumoniae, 6 Streptococcus pyogenes, 187 Haemophilus influenzae, 106 Moraxella catarrhalis, 126 Klebsiella pneumoniae, and 162 Pseudomonas aeruginosa). A total of 44 antibacterial agents, including 26 β-lactams (four penicillins, three penicillins in combination with β-lactamase inhibitors, four oral cephems, eight parenteral cephems, one monobactam, five carbapenems, and one penem), three aminoglycosides, four macrolides (including a ketolide), one lincosamide, one tetracycline, two glycopeptides, six fluoroquinolones, and one oxazolidinone were used for the study. Analysis was conducted at the central reference laboratory according to the method recommended by the Clinical and Laboratory Standard Institute (CLSI). The incidence of methicillin-resistant S. aureus (MRSA) was as high as 59.8%, and those of penicillin-intermediate and penicillin-resistant S. pneumoniae (PISP and PRSP) were 35.5 and 11.8%, respectively. Among H. influenzae, 13.9% of them were found to be β-lactamase-non-producing ampicillin (ABPC)-intermediately resistant (BLNAI), 26.7% to be β-lactamase-non-producing ABPC-resistant (BLNAR), and 5.3% to be β-lactamase-producing ABPC-resistant (BLPAR) strains. A high frequency (76.5%) of β-lactamase-producing strains was suspected in Moraxella catarrhalis isolates. Four (3.2%) extended-spectrum β-lactamase-producing K. pneumoniae were found among 126 strains. Four isolates (2.5%) of P.aeruginosa were found to be metallo β-lactamase-producing strains, including three (1.9%) suspected multidrug-resistant strains showing resistance to imipenem, amikacin, and ciprofloxacin. Continual national surveillance of the antimicrobial susceptibility of respiratory pathogens is crucial in order to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis.

The first nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy. Part 1: a general view of antibacterial susceptibility

The Japanese Society of Chemotherapy (JSC) conducted the first nationwide surveillance of bacterial respiratory pathogens during the period from January to August 2006. With the cooperation of 32 medical institutions throughout Japan, a total of 924 strains belonging to seven clinically relevant bacterial species were collected from adult patients with well-diagnosed respiratory tract infections (RTIs). Antimicrobial susceptibility testing of the 887 evaluable strains (205 Staphylococcus aureus, 200 Streptococcus pneumoniae, 9 Streptococcus pyogenes, 165 Haemophilus influenzae, 91 Moraxella catarrhalis, 74 Klebsiella pneumoniae, and 143 Pseudomonas aeruginosa) to 42 antibacterial agents was conducted at the Central Laboratory of the Research Center for Anti-infective Drugs of the Kitasato Institute, according to recommendations issued by the Clinical and Laboratory Standards Institute (CLSI). The antibacterial agents employed were 25 β-lactams, three aminoglycosides, four macrolides (including one azalide and one ketolide), one lincosamide, one tetracycline, two glycopeptides, five fluoroquinolones, and one oxazolidinone. The incidence of methicillin-resistant S. aureus (MRSA) was 63.4%, and the incidences of penicillin-intermediately resistant S. pneumoniae (PISP) and penicillin-resistant S. pneumoniae (PRSP) were 35.0% and 4.0%, respectively. Among H. influenzae, 21.2% of the strains were found to be β-lactamase-nonproducing ampicillin (ABPC)-intermediately resistant (BLNAI), 29.1% to be β-lactamase-nonproducing ABPC-resistant (BLNAR), and 4.8% to be β-lactamaseproducing ABPC-resistant (BLPAR) strains. The incidence of extended-spectrum β-lactamase-producing K. pneumoniae was 2.7% (2 of 74 strains). Three (2.1%) of the 143 P. aeruginosa strains were found to be metallo-β-lactamaseproducing, including 1 (0.7%) multidrug-resistant strain. Through the nationwide surveillance, we obtained fundamental antimicrobial susceptibility data of clinically relevant bacterial pathogens in adult RTI to various antibacterial agents. These data will be a useful reference for future periodic surveillance studies, as well as for investigations to control antimicrobial-resistant pathogens.

Final report from the Committee on Antimicrobial Susceptibility Testing, Japanese Society of Chemotherapy, on the agar dilution method (2007)

In 1968, the agar dilution method was developed as an independent Japanese method for measuring the minimal inhibitory concentration (MIC) of antimicrobial agents. As this method differed in a few respects from the MIC measurement methods used in other countries, it was revised in 1981, by a committee headed by Susumu Mitsuhashi, and the revised method (Chemotherapy 29:76–79, 1981) has been used since then.In 1979, an agar dilution method for measuring the MIC of anaerobes was developed by a committee chaired by Nozomu Kosakai (Chemotherapy 27:559–561, 1979). In 1990, a committee headed by Sachiko Goto approved a broth microdilution method for nonfastidious bacteria (Chemotherapy 38:102–105, 1990). Later, a committee headed by Atsushi Saito examined media that would be suitable for nonfastidious bacteria and fastidious bacteria, and they endeavored to prepare a broth microdilution method for anaerobic bacteria. In this context, a new broth microdilution method was proposed at the 40th Annual Meeting of the Japanese Society of Chemotherapy (JSC) in Nagoya in 1992, and the proposal was adopted as the standard JSC method after some modification (Chemotherapy 41: 183–189, 1993).The agar dilution method has remained unrevised for approximately 20 years. A proposal to review this method was recently made, and the 2007 Committee on Antimicrobial Susceptibility Testing was formed, comprising the JSC members listed below. Under the auspices of this committee, the method revised in 1981 was reviewed in comparison to the international standard method (Clinical and Laboratory Standards Institute [CLSI] method).

The antagonistic effects of a combination of vancomycin and minocycline in Staphylococcus aureus with heterogeneous resistance to vancomycin

Some methicillin-resistant Staphylococcus aureus (MRSA) strains in which combinations of vancomycin (VCM) and β-lactam antibiotics show antagonism have recently emerged, and these strains are called β-lactaminduced VCM-resistant MRSA (BIVR). We examined whether various antibiotics exhibited an antagonistic effect with VCM when used against Mu3 and Fu10 (representative BIVR strains), using a simple agar disc method. Chloramphenicol, tetracyclines, macrolides, and lincosamides showed an antagonistic effect with VCM. We attempted to elucidate the antagonistic mechanism of a combination of VCM and minocycline (MINO) in BIVR strains. We determined the rates of autolysis, autolytic activities, and the change in morphology of Mu3 treated with a combination of VCM and MINO. We observed that Mu3 grown in a combination of VCM and MINO showed increasing rates of autolysis, and lower minimal bacteriolytic enzyme dose (MBD) values compared with Mu3 grown in VCM alone, but no cell wall thickening was observed. Taken together, these results suggest that cell wall thickening may not be essential in the increased resistance of BIVR strains. Our present data therefore suggest that these combination therapies of VCM with tetracyclines should be adopted with great care in order to prevent VCM treatment failure.

Pulmonary Colonization by Chrysosporium zonatum Associated with Allergic Inflammation in an Immunocompetent Subject

ABSTRACT We report a case of noninvasive pulmonary disease due to Chrysosporium zonatum in an immunocompetent male. The fungus colonized an existing tuberculous cavity and was isolated from transbronchial lavage fluid and from a percutaneous aspiration specimen. The disease was accompanied by the unusual feature of an allergic reaction. The fungus ball was successfully treated by intracavitary administration of amphotericin B. C. zonatum is the anamorph of the heterothallic ascomycete Uncinocarpus orissi, and the identity of the case isolate was verified by formation of ascospores in mating tests with reference isolates.

A fatal case of necrotizing fasciitis due to bacterial translocation of Klebsiella oxytoca

We report a 73-year-old man with hepatocellular cell carcinoma who had eruptions on and severe pain in the lower leg. Within several hours, the patient’s skin lesions had progressed markedly. Magnetic resonance imaging findings were consistent with necrotizing fasciitis. Klebsiella oxytoca was isolated from cultures of biopsy samples taken from the leg. The resulting DNA fingerprint pattern revealed that the enteric bacterium was the same as that obtained from the biopsy samples taken from the leg. Furthermore, a dendrogram showed that genetic proximity between samples was extremely high. These results confirmed that translocation of Klebsiella oxytoca as an enteric pathogen caused the necrotizing fasciitis in this patient.

Susceptibility of clinical isolates of Pseudomonas aeruginosa in the Northern Kyushu district of Japan to carbapenem antibiotics, determined by an integrated concentration method: evaluation of the method based on Monte Carlo simulation

In empirical antibacterial therapy, regional surveillance is expected to yield important information for the determination of the class and dosage regimen of antibacterial agents to be used when dealing with infections with organisms such as Pseudomonas aeruginosa, in which strains resistant to antibacterial agents have been increasing. The minimal inhibitory concentrations (MICs) of five carbapenem antibiotics against P. aeruginosa strains isolated in the Northern Kyushu district of Japan between 2005 and 2006 were measured, and 100 strains for which carbapenem MICs were ≤0.5–32 μg/ml were selected. In this study, MIC was measured by two methods, i.e., the common serial twofold dilution method and an integrated concentration method, in which the concentration was changed, in increments of 2 μg/ml, from 2 to 16 μg/ml. The MIC50/MIC90 values for imipenem, meropenem, biapenem, doripenem, and panipenem, respectively, with the former method were 8/16, 4/16, 4/16, 2/8, and 16/16 μg/ml; and the values were 6/10, 4/12, 4/10, 2/6, and 10/16 μg/ml with the latter method. The MIC data obtained with both methods were subjected to pharmacokinetic/pharmacodynamic (PK/PD) analysis with Monte Carlo simulation to calculate the probability of achieving the target of time above MIC (T>MIC) with each carbapenem. The probability of achieving 25% time above the MIC (T>MIC; % of T>MIC for dosing intervals) and 40% T>MIC against P. aeruginosa with any dosage regimen was higher with doripenem than with any other carbapenem tested. When the two sets of MIC data were subjected to PK/PD analysis, the difference between the two methods in the probability of achieving each % T>MIC was small, thus endorsing the validity of the serial twofold dilution method.

Evaluation of APTIMA Combo 2 for cross-reactivity with oropharyngeal Neisseria species and other microorganisms

Chlamydia trachomatis (CT) andNeisseria gonorrhoeae (GC) are two of the most common pathogens of sexually transmitted infections (STIs), and the estimated prevalence of CT and GC infections varies substantially in accordancewith populations. Recently, several reports have indicated that oral–genital sexual contact is relatively common and on the increase among adolescents and young adults [1,2], which may in part explain the widespread distribution of these pathogens. Among patients with gonorrhea at any site, positive rates of oropharyngeal infection have been documented in 3% to 7% of heterosexual men, 5% to 20% of heterosexual women, and 10% to 25% of homosexual men, and 39% to 96% of pregnant women [3]. More importantly, the rate of asymptomatic cases in oropharyngeal infection is higher than that in endocervical infection, and therefore, early diagnosis and treatment are vital for patients with symptomatic, asymptomatic and/or persistent infections to prevent complications and to control the spread of the infection. Several commercially available nucleic acid amplification tests (NAATs) are used for the detection of CT and/or GC [4–7]. The diagnosis of CT and GC infections typically requires a swab specimen collected fromtheposterior oropharynxaswell asfirst catchurine specimens and/ or swabs from the genital tract to check for oropharyngeal infection. However, several reports indicate that Neisseria species other than GC can be isolated from oropharyngeal samples and genital sites [1,8], and that the specificity of some commercially available assay kits is low for the detection of GC at the oropharyngeal site, especially in the polymerase chain reaction based assay kit by cross-reactionwith commensal nongonococcalNeisseria species such asN. subflavaandN. cinerea [9–11]. APTIMA Combo 2 (Gen-Probe, Incorporated, San Diego, CA) is a second generation NAAT based on the target capture and transcription-mediated amplification methods for the simultaneous detection of CT and GC in endocervical swab, urine specimens [5,12] as well as oropharyngeal swab [10]. This assay amplifies target ribosomal RNA, which is present at high copy number in all infected cells and bacteria, to induce a higher sensitivity [13]. Previous report [13] and the package insert of APTIMA Combo 2 suggested no cross-reaction with a total of 158 culture isolates, however, cross-reactivity data was not supplied about Actinomyces naeslundii, Actinomyces viscosus, Haemophilus parainfluenzae, Porphyromonas gingivalis, Prevotella intermedia and Streptococcus oralis. Therefore, we selected these habitual microorganisms in the oropharynx and the nasal cavity in this study. We also confirmed the cross-reactivity of Neisseria cinerea,Neisseria lactamica andNeisseria subflavawith APTIMA Combo 2 because the cross-reaction of these microorganisms with BD

Loop-mediated isothermal amplification (LAMP) 法を用いた Vibrio vulnificus の迅速検出

Vibrio vulnificus is found in marine waters near the coast around the world. Infection with this gram-negative rod, via ingestion of raw seafood or via a skin wound following contact with contaminated estuarine or marine water, can cause necrotizing fasciitis and sepsis. Most of patients with Vibrio vulnificus infection have underlying liver dysfunction or diabetes mellitus. Due to the high mortality and short latent periods, control of this infection depends on early identification of the bacterial species and prompt initiation of intensive care. Accordingly, the development of a technique that can identify this microbe quickly and accurately is of great importance. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method to detect specific genes with rapidity and high sensitivity. In this study, we developed LAMP for the detection of Vibrio vulnificus. Using 28 Vibrio vulnificus strains and 53 other bacterial strains, we confirmed the high specificity of this method. Moreover, our LAMP method also showed high sensitivity, with a minimum detection level of one colony-forming unit per test. Furthermore, we developed simplified and conventional pretreatments for the method using experimental animal models. All of these attempts have lod to our non being able to detect Vibrio vulnificus within 1 hour.

Application of Beta-lactam Therapeutic Drug Monitoring in Clinical Practice Using HPLC

In beta-lactam therapy, investigation of the pharmacokinetic-pharmacodynamic (PK-PD) relationship has provided surrogate makers to predict clinical outcome. This study was designed to verify the therapeutic efficacy and clinical utility of beta-lactam therapeutic drug monitoring (TDM) in critically ill patients using high-performance liquid chromatography (HPLC). This cohort study included 13 patients who were intravenously administered ceftazidime (n = 6), cefepime (n = 1), imipenem (n = 1), meropenem (n = 1), or piperacillin (n = 4). Blood samples were collected at 3 time points fitted to a 1-compartment model, and concentrations were determined using HPLC. The PK-PD target was the percentage of the dosing interval during which the antibiotic concentration exceeded the minimum inhibitory concentration for the pathogens (%T > MIC), which is 50% for penicillins (piperacillin), 60% for cephalosporins (ceftazidime and cefepime), and 40% for carbapenems (imipenem and meropenem). Our process using HPLC enables the analytical results of TDM to be available within half a day. The results revealed significant inter-patient pharmacokinetic variability. During the initial regimen, beta-lactam concentrations reached the target %T > MIC in all patients, and dosage reduction was required for 5 patients (38%). Of the 13 evaluable patients, clinical improvement was observed in 11 (85%), and microbiological success was observed in 10 (77%). In summary, beta-lactam TDM achieved the treatment goals and might also allow for the personalization to prevent overdosing for variable pharmacokinetic changes in critically ill patients. These findings indicate that beta-lactam TDM using HPLC can be used in the standard pharmacy clinical practice for critically ill patients.