Masatoshi Konno

Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae

ABSTRACT The affinity of [3H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of β-lactamase-negative ampicillin (AMP)-resistant (BLNAR)Haemophilus influenzae for which the AMP MIC was ≥1.0 μg/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of thedacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-Thr-Gly (KTG) motif, was substituted for Arg-517 in group I strains (n = 9), and Lys-526 was substituted for Asn-526 in group II strains (n = 12). In group III strains (n = 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of β-lactams forH. influenzae transformants into which the ftsIgene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for β-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in β-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to β-lactams in BLNAR strains.

Homology of mecA gene in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans to that of Staphylococcus aureus.

A penicillin-binding protein of molecular weight 76,000 inducible by beta-lactams was detected in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans. DNA from these strains hybridized to the mecA gene from Staphylococcus aureus; however, the chromosomal HindIII fragments containing the mecA genes were 3.4 kilobases in S. haemolyticus and 4.3 kilobases in S. simulans. Images

Identification of Penicillin and other Beta-Lactam Resistance in Streptococcus Pneumoniae by Polymerase Chain Reaction

To identify penicillin (Pc) and other β-lactam resistance in 310 clinical isolates ofStreptococcus pneumoniae by polymerase chain reaction (PCR), 3 sets of primers were designed to amplify Pc-binding protein (PBP) genes previously detected in Pc-susceptible strains: 1) a 430-bp fragment of thepbp1a gene, 2) a 292-bp fragment of thepbp2x gene, and 3) a 77-bp fragment of thepbp2b gene. The amplified regions of each PBP gene were positioned in highly divergent sequences of Pc-resistantS. pneumoniae. In other words, isolates for which these DNA fragments were detected were regarded as possessing sequences almost the same as that of the susceptible R6 strain and those for which these DNA fragments were not detected were assumed to have mutations. A set of primers that amplify 273 bp of the autolysin (lytA) gene to identifyS. pneumoniae was applied as well. Of 166 isolates for which the minimum inhibitory concentration (MIC) of Pc were ≤0.06 μg/mL, 83 (50.0%) were confirmed to be true susceptible strains with no PBP gene mutation and most of the remaining strains were found to possesspbp2x mutation. In contrast, most of 109 isolates for which the MIC of Pc were ≥0.5 μg/mL were confirmed to possess mutations in all three PBP genes. Thirty-five strain for which the MIC of Pc ranged from 0.125 to 0.25 μg/mL possessed various PBP gene mutations. The relationships between susceptibilities to 9 β-lactams ofS. pneumoniae and PBP gene mutation were analyzed by multiple regression analysis. Antibiotics were classified into 4 types according to the differences in PBP gene mutation affecting their MIC levels, 1) the MIC of Pc and ampicillin were affected bypbp1a andpbp2b mutations; 2) those of cefotaxime, cefpodoxime, and cefditoren were affected clearly bypbp2x mutation; 3) those of cefaclor and cefdinir were affected more strongly bypbp1a mutation than thepbp2x; and 4) the MIC of faropenem and imipenem were affected strongly bypbp2b mutation. These findings suggest that it may be possible to easily determine whether aS. pneumoniae isolate is susceptible or resistant to Pc, cefotaxime, and other β-lactams by applying PCR using a combination of primers.

Incidence of penicillin-resistant streptococcus pneumoniae in Japan, 1993–1995

The Working Group for PRSP was organized through the participation of 40 institutions to investigate the incidence of penicillin (Pc)-resistantStreptococcus pneumoniae (PRSP) in Japan. We collected 2410S. pneumoniae clinical isolates between October 1994 and March 1995. The susceptibility to Pc, erythromycin, and minocycline was determined by an agar dilution method using Mueller-Hinton agar supplemented with 5% sheep blood. Pc-susceptibleS. pneumoniae (PSSP) were defined as bacteria for which the minimum inhibitory concentration (MIC) was ≤0.06 μg/mL; Pc-intermediateS. pneumoniae (PISP) as those for which the MIC ranged from 0.125 to 0.25 μg/mL; PRSP, as those with a MIC≥0.5μg/mL. The incidence of resistant strains including PISP and PRSP was 41.8% in 1994 and 40.8% in 1995. Logistic regression analysis showed that PRSP was significantly more frequent in infants aged 0 to 2 years old than in the general population and PSSP was significantly more frequent in elderly patients aged 60 or older. The rate of PRSP was significantly higher in the throat than in the sputum. Among 10 regions studied nationwide, PRSP was detected less frequently in the areas of Hokkaido and Hokuriku and more frequently in the areas of Chugoku, Shikoku, and Kyushu. Most PRSP were resistant to erythromycin and minocycline. PSSP serotyping using the capsule-quellung reaction indicated a number of types. In contrast, most PISP and PRSP were serotyped to types 19, 23, and 6.

Nosocomial Infections Caused by Methicillin-Resistant Staphylococcus aureus in Japan

ConclusionIt is very important to develop better antibacterial agents and employ them properly in the treatment of large numbers of patients. This is also crucial for any future improvement of the health care system. However, if antibacterial agents are used too freely, problems will arise in the future. The problem of MRSA in Japan is a typical example.It is necessary to reconsider urgently the Japanese health care system to overcome this problem. Re-education of medical professionals, especially instruction on the proper use of antibiotics, is also needed to cope with the increasing number of patients who are placed at risk for developing antibiotic-resistant nosocomial infections because of their act of seeking medical treatment. However, the most important point after developing an excellent system is to have the wisdom not to be overly constrained by it, I believe that the only way to achieve this is through education.In this article, I have described the current status of nosocomial MRSA infections in Japan. I hope that this article will help people all over the world obtain greater benefits from antibiotics in the future.

A prospective clinical trial on prevention of catheter contamination using the hub protection cap for needleless injection device

BACKGROUND Catheter hub contamination has been recognized as a source of catheter-related bloodstream infections. We have investigated the efficacy of a protection cap for a needleless injection device in preventing intraluminal catheter contamination, compared with a conventional 3-way stopcock. METHODS Adult patients requiring an intravascular catheter placement for at least 48 hours in an intensive care unit were randomly assigned to receive either the needleless injection device with protection cap (test group, n = 31, number of devices = 151) or with a conventional 3-way stopcock (comparator group, n = 33, number of devices = 179). To evaluate intraluminal contamination, we examined the bacteria isolated in the inline bacterial filters, which were attached downstream of the injection ports. RESULTS The incidence of bacterial contamination was significantly different between the groups (test group 2/151 (1.3%) vs comparator group 11/179 (6.2%), P = .04). There was no correlation between the microbial contamination rate and the in situ time of catheter or numbers of injections. CONCLUSION The protection cap for needleless injection devices decreased microbial transfer from the injection port to the intraluminal fluid pathway and lowered the risk of catheter-related bloodstream infections.

In Vitro Evaluation of the Activity of β-Lactams, New Quinolones, and Other Antimicrobial Agents Against Streptococcus Pneumoniae

The in vitro activities of the β-lactam and quinolone antibiotics panipenem, cefpodoxime, cefdinir, cefditoren, faropenem, tosufloxacin, levofloxacin and grepafloxacin were compared with similar conventional antibiotics against penicillin-resistantStreptococcus pneumoniae (PRSP). Pneumococcal isolates collected from October 1994 to March 1995 (n=1283) consisted of penicillin-susceptibleS. pneumoniae (PSSP; 59.2%), penicillin-intermediately-resistantS. pneumoniae (PISP;11.2%), and PRSP (29.6%). The isolates were highly susceptible to panipenem, faropenem and cefditoren with MIC90 values of 0.125 μg/mL, 0.5 μg/mL and 0.5 μg/mL, respectively. Correlation coefficients for the relationships between the MICs of these β-lactam agents and that of penicillin G ranged from γ=0.7652 to γ=0.8022. These new β-lactam agents produced excellent bactericidal responses at concentrations greater than their MICs for PSSP concomitant with appropriate cellular morphologic changes. However, the bactericidal action of these antibiotics against PRSP was less pronounced and fewer instances of cell lysis were observed. The MIC90 of cefpodoxime was similar to that of cefaclor, whereas that of cefdinir was between those of faropenem and cefpodoxime. The MIC distribution of the new quinolone agents showed 1 peak, but the MIC90 values of tosufloxacin and grepafloxacin were both 0.5 μg/mL and that of levofloxacin was 2.0 μg/mL. Only 1% of all isolates demonstrated cross-resistance to all quinolone agents.

New Plasmid (pTU512), Mediating Resistance to Penicillin, Erythromycin, and Kanamycin, from Clinical Isolates of Staphylococcus aureus

Multiply drug-resistant strains of Staphylococcus aureus were isolated from pediatric patients with severe staphylococcal infections in 1974 through 1976. Resistance to benzylpenicillin, erythromycin, and kanamycin was jointly eliminated without exception from these multiply drug-resistant strains by treatment with ethidium bromide. It was also found that the triple drug resistance in a representative strain, TK512-200, was always transduced to a susceptible strain simultaneously. Moreover, a single class of plasmid deoxyribonucleic acid was isolated from a transductant and found to be 14.4 ± 0.6 μm in length, with a molecular weight corresponding to 29.8 × 106. From these results, it is concluded that the plasmid (pTU512) is a new one, mediating resistance to penicillin, erythromycin, and kanamycin. Images

The Administration Regimen of Isepamicin in Patients with Chronic Respiratory Tract Infection

A total of 34 patients with intractable chronic respiratory tract infections were treated with isepamicin and/or piperacillin in different dosage regimens. A comparison of the bacteriological effect using a cross over method showed a reduction in the count of Pseudomonas aeruginosa in sputum in the group receiving once-a-day isepamicin combined with piperacillin, compared with the twice-a-day combined administration. A comparison of the clinical and bacteriological efficacy between the different regimen groups revealed no noticeable difference. The clinical effect of this regimen is comparable to the conventional regimen, but has the advantages of a safer dosage and ease of administration.

Genotypic Identification of Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin 1 by the Enzymatic Detection of Polymerase Chain Reaction-Amplified DNA

The enzymatic detection of a polymerase chain reaction product (ED-PCR), a new detection method of PCR-amplified DNA, was evaluated for the identification of staphylococcal enterotoxin (SE) and toxic shock syndrome toxin 1 (TSST-1) genes. A total of 61Staphylococcus aureus strains, including reference strains and strains isolated from clinical specimens and food poisoning outbreaks, were examined by ED-PCR and by reverse passive latex agglutination (RPLA) phenotypic identification. There was 100% agreement between the genotypic and phenotypic identification of SEA, SEB, SEC, SEE strains and TSST-1. In the case of SED, however, 4 strains were positive by ED-PCR and negative by RPLA. ED-PCR offers an accurate alternative to traditional immunoassays or conventional PCR using electrophoresis for the detection of SE and TSST-1 production yielding results that are more precise than with older techniques.