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1.  Immunochromatographic Detection of the Group B Streptococcus Antigen from Enrichment Cultures 
Group B Streptococcus (GBS; Streptococcus agalactiae) is a leading cause of serious neonatal infections. The Centers for Disease Control and Prevention recommends GBS screening for all pregnant women during the 35th to 37th weeks of gestation. Although GBS screening has been performed mainly by the culture-based method, it takes several days to obtain a reliable result. In this study, we developed a rapid immunochromatographic test (ICT) for the detection of GBS-specific surface immunogenic protein in 15 min using an overnight enrichment culture. The ICT was prepared using two anti-Sip monoclonal antibodies. This ICT was able to detect recombinant Sip levels of 0.5 ng/ml, or about 106 CFU/ml of GBS cells, in tests with 9 GBS strains of different serotypes. The cross-reactivity test using 26 species of microorganism showed no detectable false-positive result. Reactivity of the ICT with 229 GBS strains showed one false-negative result that was attributable to the production of truncated Sip. Among 260 enrichment cultures of vaginal swabs, 17 produced red to orange pigments in Granada medium, and they were all GBS and Sip positive. Among 219 pigment-negative cultures, 12 were GBS positive and 10 were Sip positive. Two Sip-negative cultures contained GBS cells below the limit of detection by the ICT. Among 207 GBS-negative cultures, only one was Sip positive, which was attributable to GBS cell debris. Thus, the sensitivity and specificity of the ICT appeared to be 93.1% and 99.6%, respectively. The newly developed ICT is readily applicable to clinical use in the detection of GBS.
PMCID: PMC3889584  PMID: 23825191
2.  Nationwide survey of the development of drug resistance in the pediatric field in 2007 and 2010: drug sensitivity of Haemophilus influenzae in Japan (second report) 
The Drug-Resistant Pathogen Surveillance Group in Pediatric Infectious Disease conducted national surveillance for Haemophilus influenzae in 2007 (phase 3) and 2010 (phase 4), following the previous surveillance conducted from 2000 to 2001 (phase 1) and in 2004 (phase 2). We examined the antimicrobial susceptibility for H. influenzae derived from clinical specimens of pediatric patients collected nationwide from 27 institutions during phases 3 (386 strains) and 4 (484 strains). The frequency of β-lactamase-nonproducing ampicillin (ABPC)-resistant (BLNAR) strains, which rapidly increased from 11.4 % in phase 1 to 43.4 % in phase 2, has gradually decreased from 38.3 % in phase 3 to 37.8 % in phase 4. In contrast, On the other hand, the frequency of β-lactamase-producing strains, which continuously decreased from 8.3 % in phase 1 to 4.4 % in phase 3, has increased to 8.7 % in phase 4. Prevalence of β-lactamase-producing clavulanic acid/amoxicillin-resistant (BLPACR) strains, especially, has increased from 1.6 % in phase 3 to 4.8 % in phase 4. The oral antimicrobial agents with the lowest MIC90 were levofloxacin in both phases, and tosufloxacin in phase 4 (≤0.063 μg/ml), whereas for intravenous use the corresponding agent was tazobactam/piperacillin in both phases (0.125 μg/ml). There was no increase in the MIC90 of most β-lactams between phase 3 and phase 4. In relationship to sex, age, presence of siblings, attendance at a daycare center, siblings’ attendance at a daycare center, and prior administration of antimicrobial agents within 1 month, the frequency of β-lactamase-nonproducing ABPC-intermediately resistant (BLNAI) strains + BLNAR strains was high (P = 0.005) in cases with prior administration of antimicrobial agents in phase 3.
PMCID: PMC3682095  PMID: 23564330
Pediatric infectious disease; Surveillance; Haemophilus influenzae sensitivity; Drug resistance
3.  Nationwide survey of the development of drug-resistant pathogens in the pediatric field in 2007 and 2010: drug sensitivity of Streptococcus pneumoniae in Japan (second report) 
We previously conducted nationwide surveillance of Streptococcus pneumoniae in 2000–2001 (period 1) and 2004 (period 2) and reported the findings. Subsequent surveillance surveys conducted in 2007 (period 3) and 2010 (period 4) are now reported. Bacterial strains were clinically isolated from children with meningitis, sepsis, and respiratory tract infections at 27 hospitals participating in the Drug-Resistant Pathogen Surveillance Group in Pediatric Infectious Disease. Twenty-one drugs were investigated for 283 isolated strains in period 3, and 24 drugs were investigated for 459 strains in period 4. In period 3, 43.8 % of strains were penicillin-susceptible S. pneumoniae (PSSP), 52.3 % were penicillin-intermediate S. pneumoniae (PISP), and 3.9 % were penicillin-resistant S. pneumoniae (PRSP). In period 4, the percentages were PSSP 23.1 %, PISP 49.9 %, and PRSP 27.0 %. The resistance rates were 56.2 % and 76.9 %, respectively. Drug sensitivity was best with panipenem, at a minimum inhibitory concentration (MIC)90 ≤0.063 μg/ml in period 3, and with tebipenem (MIC90 ≤ 0.063 μg/ml) in period 4. Patients’ background factors related to increased bacterial resistance were investigated, and significant differences were found depending on whether a child had siblings (P = 0.0056) or was a daycare center attendee (P = 0.0195) in period 3, and age category (P = 0.0256) in period 4. No factors were common to both periods 3 and 4. Pneumococcus is a major causative organism of pediatric infectious disease, and we plan to continue conducting surveillance and providing information in the future.
PMCID: PMC3682105  PMID: 23564331
Drug resistance; Pediatric infectious disease; Sensitivity; Streptococcus pneumoniae; Surveillance
4.  Low level ß-lactamase production in methicillin-resistant staphylococcus aureus strains with ß-lactam antibiotics-induced vancomycin resistance 
BMC Microbiology  2012;12:69.
A class of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin only in the presence of ß-lactam antibiotics (BIVR). This type of vancomycin resistance is mainly attributable to the rapid depletion of free vancomycin in the presence of ß-lactam antibiotics. This means that ß-lactam antibiotics remain active or intact in BIVR culture, although most MRSA cells are assumed to produce ß-lactamase. We hypothesised that the BIVR cells either did not harbour the ß-lactamase gene, blaZ, or the gene was quiescent. We tested this hypothesis by determining ß-lactamase activity and conducting PCR amplification of blaZ.
Five randomly selected laboratory stock BIVR strains showed an undetectable level of ß-lactamase activity and were blaZ-negative. Five non-BIVR stock strains showed an average ß-lactamase activity of 2.59 ± 0.35 U. To test freshly isolated MRSA, 353 clinical isolates were collected from 11 regionally distant hospitals. Among 25 BIVR strains, only 16% and 8% were blaZ positive and ß-lactamase-positive, respectively. In contrast, 95% and 61% of 328 non-BIVR strains had the blaZ gene and produced active ß-lactamase, respectively. To know the mechanism of low ß-lactamase activity in the BIVR cells, they were transformed with the plasmid carrying the blaZ gene. The transformants still showed a low level of ß-lactamase activity that was several orders of magnitude lower than that of blaZ-positive non-BIVR cells. Presence of the ß-lactamase gene in the transformants was tested by PCR amplification of blaZ using 11 pairs of primers covering the entire blaZ sequence. Yield of the PCR products was consistently low compared with that using blaZ-positive non-BIVR cells. Nucleotide sequencing of blaZ in one of the BIVR transformants revealed 10 amino acid substitutions. Thus, it is likely that the ß-lactamase gene was modified in the BIVR cells to downregulate active ß-lactamase production.
We concluded that BIVR cells gain vancomycin resistance by the elimination or inactivation of ß-lactamase production, thereby preserving ß-lactam antibiotics in milieu, stimulating peptidoglycan metabolism, and depleting free vancomycin to a level below the minimum inhibitory concentration of vancomycin.
PMCID: PMC3424166  PMID: 22568976
Antibiotic resistance; ß-lactam; Vancomycin; Staphylococcus aureus; ß-lactamase; MRSA
5.  Development of an Immunochromatographic Strip for Simple Detection of Penicillin-Binding Protein 2′▿  
Infections with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MR-CNS) are a serious problem in hospitals because these bacteria produce penicillin-binding protein 2′ (PBP2′ or PBP2a), which shows low affinity to β-lactam antibiotics. Furthermore, the bacteria show resistance to a variety of antibiotics. Identification of these pathogens has been carried out mainly by the oxacillin susceptibility test, which takes several days to produce a reliable result. We developed a simple immunochromatographic test that enabled the detection of PBP2′ within about 20 min. Anti-PBP2′ monoclonal antibodies were produced by a hybridoma of recombinant PBP2′ (rPBP2′)-immunized mouse spleen cells and myeloma cells. The monoclonal antibodies reacted only with PBP2′ of whole-cell extracts and showed no detectable cross-reactivity with extracts from other bacterial species tested so far. One of the monoclonal antibodies was conjugated with gold colloid particles, which react with PBP2′, and another antibody was immobilized on a nitrocellulose membrane, which captures the PBP2′-gold colloid particle complex on a nitrocellulose strip. This strip was able to detect 1.0 ng of rPBP2′ or 2.8 × 105 to 1.7 × 107 CFU of MRSA cells. The cross-reactivity test using 15 bacterial species and a Candida albicans strain showed no detectable false-positive results. The accuracy of this method in the detection of MRSA and MR-CNS appeared to be 100%, compared with the results obtained by PCR amplification of the PBP2′ gene, mecA. This newly developed immunochromatographic test can be used for simple and accurate detection of PBP2′-producing cells in clinical laboratories.
PMCID: PMC3067364  PMID: 21177917
6.  A 25-year trace of methicillin-resistant Staphylococcus aureus dissemination in a geriatric hospital in Japan 
We analyzed 218 strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from the septicemia patients in a geriatric hospital for 25 years. These strains were classified into 11 major DNA types, A through K, and 27 minor types. The strains belonging to group A and B isolated before 1990 were susceptible to imipenem (IPM), fluoroquinolone, and most other antibiotics tested, except that they were markedly resistant to gentamicin. Strains mostly isolated in 1985 and thereafter were classified into group C through K, and they were mainly resistant to IPM, fluoroquinolones, and clindamycin. Analysis of the MRSA marker gene, staphylococcal cassette chromosome mec (SCCmec), of these strains revealed that the strains in groups A and B had mainly type IV and type I, respectively, and that strains in groups C through J had mainly type II. These results suggested that the strains holding type II SCCmec were resistant to IPM, fluoroquinolone, and clindamycin and they were dominant-resistant type after late 1980s. The antibiotic resistance profiles of MRSA dramatically changed during late 1980s, and these were correlated with the SCCmec types. The lesson from this study would be that consistent execution of surveillance study is needed to update the resistant profiles.
PMCID: PMC3008295  PMID: 21189839
methicillin-resistant Staphylococcus aureus; DNA typing; antibiotic resistance; survey; geriatric hospital
7.  Improved Selective Isolation of Bordetella pertussis by Use of Modified Cyclodextrin Solid Medium ▿  
Journal of Clinical Microbiology  2009;47(12):4164-4167.
We have developed a modified cyclodextrin solid (MCS) medium using the selective antibiotic cefdinir. MCS medium exhibited higher sensitivity (95.6%; any culture-positive sample as reference) and greater inhibition of nasopharyngeal flora than did Bordet-Gengou agar (65.2%, P = 0.009) or cyclodextrin solid medium (39.1%, P < 0.001).
PMCID: PMC2786639  PMID: 19846652
8.  CD46 Transgenic Mouse Model of Necrotizing Fasciitis Caused by Streptococcus pyogenes Infection▿  
Infection and Immunity  2009;77(11):4806-4814.
We developed a human CD46-expressing transgenic (Tg) mouse model of subcutaneous (s.c.) infection into both hind footpads with clinically isolated 11 group A streptococcus (GAS) serotype M1 strains. When the severity levels of foot lesions at 72 h and the mortality rates by 336 h were compared after s.c. infection with 1 × 107 CFU of each GAS strain, the GAS472 strain, isolated from the blood of a patient suffering from streptococcal toxic shock syndrome (STSS), induced the highest severity levels and mortality rates. GAS472 led to a 100% mortality rate in CD46 Tg mice after only 168 h postinfection through the supervention of severe necrotizing fasciitis (NF) of the feet. In contrast, GAS472 led to a 10% mortality rate in non-Tg mice through the supervention of partial necrotizing cutaneous lesions of the feet. The footpad skin sections of CD46 Tg mice showed hemorrhaging and necrotic striated muscle layers in the dermis, along with the exfoliation of epidermis with intracellular edema until 48 h after s.c. infection with GAS472. Thereafter, the bacteria proliferated, reaching a 90-fold or 7-fold increase in the livers of CD46 Tg mice or non-Tg mice, respectively, for 24 h between 48 and 72 h after s.c. infection with GAS472. As a result, the infected CD46 Tg mice appeared to suffer severe liver injuries. These findings suggest that human CD46 enhanced the progression of NF in the feet and the exponential growth of bacteria in deep tissues, leading to death.
PMCID: PMC2772545  PMID: 19737905
9.  Rapid Detection of Vaginal Candida Species by Newly Developed Immunochromatography▿ †  
For the diagnosis of vulvovaginal candidiasis, we developed a simple immunochromatographic method that enables the detection of vaginal Candida spp. within about 30 min. Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of this method appeared to be 80.3, 99.3, 98.0, and 92.0%, respectively.
PMCID: PMC2745012  PMID: 19656990
10.  Capsular Type and Antibiotic Resistance in Streptococcus agalactiae Isolates from Patients, Ranging from Newborns to the Elderly, with Invasive Infections▿  
Streptococcus agalactiae isolates (n = 189) from patients with invasive infections were analyzed for capsular type by PCR, for antimicrobial susceptibility, and for the presence of resistance genes. In contrast to the predominance of capsular type III in children, types Ib and V were most common among adults. All 45 levofloxacin-resistant strains had two amino acid substitutions, Ser81Leu in the gyrA gene and Ser79Phe in the parC gene, and showed similar pulsed-field gel electrophoresis patterns.
PMCID: PMC2687235  PMID: 19332682
11.  Rapid Depletion of Free Vancomycin in Medium in the Presence of β-Lactam Antibiotics and Growth Restoration in Staphylococcus aureus Strains with β-Lactam-Induced Vancomycin Resistance ▿  
A class of methicillin-resistant Staphylococcus aureus strains shows vancomycin resistance in the presence of β-lactam antibiotics (β-lactam-induced VAN-resistant methicillin-resistant S. aureus [BIVR]). Two possible explanations may be offered: (i) vancomycin in culture medium is depleted, and (ii) the d-Ala-d-Ala terminal of the peptidoglycan network is replaced with d-Ala-d-lactate. We tested these hypotheses by quantifying free vancomycin in the medium through the course of cell growth and by PCR amplification of the van genes. Growth of the BIVR cells to an absorption level of ∼0.3 at 578 nm required about 24 h in the presence of vancomycin alone at the MIC (4.0 μg/ml). However, growth was achieved in only about 10 h when 1/1,000 to 1/2,000 the MIC of β-lactam antibiotic was added 2 h prior to the addition of vancomycin, suggesting that the β-lactams shortened the time to recovery from vancomycin-mediated growth inhibition. Free vancomycin in the culture medium decreased to 2.3 μg/ml in the first 8 h in the culture containing vancomycin alone, yet cell growth was undetectable. When the vancomycin concentration dropped below ∼1.5 μg/ml at 24 h, the cells began to grow. In the culture supplemented with the β-lactam 2 h prior to the addition of vancomycin, the drug concentration continuously dropped from 4 to 0.5 μg/ml in the first 8 h, and the cells began to grow at a vancomycin concentration of ∼1.7 μg/ml or at 4 h of incubation. The gene encoding the enzyme involved in d-Ala-d-lactate synthesis was undetectable. Based on these results, we concluded that BIVR is attributable mainly to a rapid depletion of vancomycin in the medium triggered or promoted by β-lactam antibiotics.
PMCID: PMC2612165  PMID: 18936187
12.  Increased Macrolide Resistance of Mycoplasma pneumoniae in Pediatric Patients with Community-Acquired Pneumonia▿  
Among 380 Mycoplasma pneumoniae isolates from 3,678 pediatric patients with community-acquired pneumonia, 50 macrolide-resistant strains had an A2063G transition in domain V of the 23S rRNA, whereas 5 had an A2064G transition. These resistant strains increased rapidly from April 2002 to December 2006.
PMCID: PMC2223908  PMID: 17954691
13.  Mechanisms, molecular and sero-epidemiology of antimicrobial resistance in bacterial respiratory pathogens isolated from Japanese children 
The clinical management of community-acquired respiratory tract infections (RTIs) is complicated by the increasing worldwide prevalence of antibacterial resistance, in particular, β-lactam and macrolide resistance, among the most common causative bacterial pathogens. This study aimed to determine the mechanisms and molecular- and sero-epidemiology of antibacterial resistance among the key paediatric respiratory pathogens in Japan.
Isolates were collected at 18 centres in Japan during 2002 and 2003 from children with RTIs as part of the PROTEKT surveillance programme. A proportion of Haemophilus influenzae isolates was subjected to sequencing analysis of the ftsI gene; phylogenetic relatedness was assessed using multilocus sequence typing. Streptococcus pneumoniae isolates were screened for macrolide-resistance genotype by polymerase chain reaction and serotyped using the capsular swelling method. Susceptibility of isolates to selected antibacterials was performed using CLSI methodology.
Results and Discussion
Of the 557 H. influenzae isolates collected, 30 (5.4%) were β-lactamase-positive [BL+], 115 (20.6%) were BL-nonproducing ampicillin-resistant (BLNAR; MIC ≥ 4 mg/L) and 79 (14.2%) were BL-nonproducing ampicillin-intermediate (BLNAI; MIC 2 mg/L). Dabernat Group III penicillin binding protein 3 (PBP3) amino acid substitutions in the ftsI gene were closely correlated with BLNAR status but phylogenetic analysis indicated marked clonal diversity. PBP mutations were also found among BL+ and BL-nonproducing ampicillin-sensitive isolates. Of the antibacterials tested, azithromycin and telithromycin were the most active against H. influenzae (100% and 99.3% susceptibility, respectively). A large proportion (75.2%) of the 468 S. pneumoniae isolates exhibited macrolide resistance (erythromycin MIC ≥ 1 mg/L); erm(B) was the most common macrolide resistance genotype (58.8%), followed by mef(A) (37.2%). The most common pneumococcal serotypes were 6B (19.7%), 19F (13.7%), 23F (13.5%) and 6A (12.8%). Telithromycin and amoxicillin-clavulanate were the most active antibacterials against S. pneumoniae (99.8% and 99.6% susceptibility, respectively).
Approximately one-third of H. influenzae isolates from paediatric patients in Japan are BLNAI/BLNAR, mainly as a result of clonally diverse PBP3 mutations. Together with the continued high prevalence of pneumococcal macrolide resistance, these results may have implications for the clinical management of paediatric RTIs in Japan.
PMCID: PMC2020463  PMID: 17697316
14.  In Vitro Activity of Tebipenem, a New Oral Carbapenem Antibiotic, against Penicillin-Nonsusceptible Streptococcus pneumoniae 
The in vitro activity of tebipenem (TBM), a new oral carbapenem antibiotic, against Streptococcus pneumoniae clinical isolates (n = 202) was compared with those of 15 reference agents. The isolates were classified into five genotypic classes after PCR identification of abnormal pbp1a, pbp2x, and pbp2b genes: (i) penicillin-susceptible S. pneumoniae (PSSP) isolates with no abnormal pbp genes (n = 34; 16.8%), (ii) genotypic penicillin-intermediate S. pneumoniae (gPISP) isolates with only an abnormal pbp2x gene [gPISP (2x)] (n = 48; 23.8%), (iii) gPISP isolates with abnormal pbp1a and pbp2x genes (n = 32; 15.8%), (iv) gPISP isolates with abnormal pbp2x and pbp2b genes (n = 16; 7.9%), and (v) genotypic penicillin-resistant S. pneumoniae (gPRSP) isolates with three abnormal pbp genes (n = 72; 35.6%). The majority of the strains tested had mefA (n = 59; 29.2%) or ermB (n = 91; 45%) gene-mediating macrolide resistance. For these isolates the MIC at which 90% of isolates are inhibited was significantly lower for TBM than for the reference oral antibiotics, as follows: 0.002 μg/ml for PSSP, 0.004 μg/ml for gPISP (2x), 0.016 μg/ml for gPISP (isolates with abnormal pbp1a and pbp2x genes and isolates with abnormal pbp2x and pbp2b genes), and 0.063 μg/ml for gPRSP. In addition, TBM showed excellent bactericidal activity against gPRSP isolates, which exhibited a 3-log10 decrease within 2 h when they were incubated with a concentration greater than or equal to the MIC. Inhibition of cell wall synthesis toward the long axis and subsequent cell lysis were observed by scanning electron microscopy after a short-term exposure to TBM, unlike the effects seen with cephalosporins. These data suggest that TBM has potent activity against multidrug-resistant S. pneumoniae, the causative pathogen of community-acquired respiratory tract infections.
PMCID: PMC549228  PMID: 15728880
15.  Antibiotic Susceptibility in Relation to Penicillin-Binding Protein Genes and Serotype Distribution of Streptococcus pneumoniae Strains Responsible for Meningitis in Japan, 1999 to 2002 
The antibiotic susceptibilities, genotypes of penicillin (PEN)-binding protein genes (pbp), and serotype distributions of Streptococcus pneumoniae isolates from meningitis patients were investigated by a nationwide surveillance group in Japan between 1999 and 2002. We analyzed 146 isolates from children (≤17 years old) and 73 from adults (≥18 years old). Isolates with or without abnormal pbp1a, pbp2x, or pbp2b genes identified by PCR were classified into six genotype patterns and 90% MIC (MIC90) values for PEN: (i) strains with three normal genes (17.2% of isolates; MIC90, 0.031 μg/ml); (ii) strains with abnormal pbp2x (22.1%, 0.063 μg/ml); (iii) strains with abnormal pbp2b (1.0%, 0.125 μg/ml); (iv) strains with abnormal pbp2x and pbp2b (7.4%, 0.25 μg/ml); (v) strains with abnormal pbp1a and pbp2x (12.7%, 0.25 μg/ml); and (vi) strains with three abnormal PBP genes (39.7%, 4 μg/ml), which are termed genotypic PEN-resistant S. pneumoniae (gPRSP). Panipenem, a carbapenem, showed an excellent MIC90 (0.125 μg/ml) against gPRSP, followed by meropenem and vancomycin (0.5 μg/ml), cefotaxime and ceftriaxone (1 μg/ml), and ampicillin (4 μg/ml). Strains of gPRSP were significantly more prevalent in children (45.2%) than in adults (27.4%). The most frequent serotypes were 6B, 19F, 23F, 6A, and 14 in children and 23F, 22, 3, 10, 6B, and 19F in adults. Serotypes 6B, 6A, 19F, 23F, and 14 predominated among gPRSP. In children, 7- and 11-valent pneumococcal conjugate vaccines would cover 76.2 and 81.3% of isolates, respectively, although coverage would be lower in adults (43.9 and 56.0%, respectively). These findings suggest the need for early introduction of pneumococcal conjugate vaccines and continuous bacteriological surveillance for meningitis.
PMCID: PMC400554  PMID: 15105095
16.  Rapidly Increasing Prevalence of β-Lactamase-Nonproducing, Ampicillin-Resistant Haemophilus influenzae Type b in Patients with Meningitis 
A total of 395 Haemophilus influenzae strains from 226 Japanese institutions participating in the Nationwide Surveillance Study Group for Bacterial Meningitis were received from 1999 to 2002. All strains were analyzed by PCR to identify the resistance genes, and their susceptibilities to β-lactam agents were determined. Of these strains, 29.1% were β-lactamase nonproducing and ampicillin (AMP) susceptible (BLNAS) and lacked all resistance genes; 15.4% were β-lactamase producing and AMP resistant and had the blaTEM-1 gene; 30.6% were β-lactamase nonproducing and AMP resistant (low-BLNAR) and had a Lys-526 or His-517 amino acid substitution in ftsI encoding PBP 3; 13.9% were β-lactamase nonproducing and AMP resistant (BLNAR) and had an additional substitution of Thr-385 in ftsI; 9.1% were amoxicillin-clavulanic acid resistant (BLPACR I) and had the blaTEM-1 gene and a Lys-526 or His-517 amino acid substitution in ftsI; and 1.8% showed resistance similar to that of the BLPACR I group (BLPACR II) but had blaTEM-1 gene and ftsI substitutions, as was the case for the BLNAR strains. All but three strains were serotype b. The prevalence of BLNAR strains has increased rapidly: 0% in 1999, 5.8% in 2000, 14.1% in 2001, and 21.3% in 2002. The MICs at which 90% of BLNAR isolates were inhibited were as follows: AMP, 16 μg/ml; cefotaxime, 1 μg/ml; ceftriaxone, 0.25 μg/ml; and meropenem, 0.5 μg/ml. All of these values were higher than those for the BLNAS counterpart strains. The relatively wide distributions of the β-lactam MICs for BLNAR strains presumably reflect variations in ftsI gene mutations. Pulsed-field gel electrophoresis suggested the rapid spread of specific H. influenzae type b strains throughout Japan. Expedited vaccination, rapid identification, and judicious antibiotic use could slow their spread.
PMCID: PMC400528  PMID: 15105098
17.  Population Pharmacokinetics of Arbekacin, Vancomycin, and Panipenem in Neonates 
Immature renal function in neonates requires antibiotic dosage adjustment. Population pharmacokinetic studies were performed to determine the optimal dosage regimens for three types of antibiotics: an aminoglycoside, arbekacin; a glycopeptide, vancomycin; and a carbapenem, panipenem. Eighty-three neonates received arbekacin (n = 41), vancomycin (n = 19), or panipenem (n = 23). The postconceptional ages (PCAs) were 24.1 to 48.4 weeks, and the body weights (BWs) ranged from 458 to 5,200 g. A one-compartment open model with first-order elimination was applied and evaluated with a nonlinear mixed-effect model for population pharmacokinetic analysis. In the fitting process, the fixed effects significantly related to clearance (CL) were PCA, postnatal age, gestational age, BW, and serum creatinine level; and the fixed effect significantly related to the volume of distribution (V) was BW. The final formulas for the population pharmacokinetic parameters are as follows: CLarbekacin = 0.0238 × BW/serum creatinine level for PCAs of <33 weeks and CLarbekacin = 0.0367 × BW/serum creatinine level for PCAs of ≥33 weeks, Varbekacin = 0.54 liters/kg, CLvancomycin = 0.0250 × BW/serum creatinine level for PCAs of <34 weeks and CLvancomycin = 0.0323 × BW/serum creatinine level for PCAs of ≥34 weeks, Vvancomycin = 0.66 liters/kg, CLpanipenem = 0.0832 for PCAs of <33 weeks and CLpanipenem = 0.179 × BW for PCAs of ≥33 weeks, and Vpanipenem = 0.53 liters/kg. When the CL of each drug was evaluated by the nonlinear mixed-effect model, we found that the mean CL for subjects with PCAs of <33 to 34 weeks was significantly smaller than those with PCAs of ≥33 to 34 weeks, and CL showed an exponential increase with PCA. Many antibiotics are excreted by glomerular filtration, and maturation of glomerular filtration is the most important factor for estimation of antibiotic clearance. Clinicians should consider PCA, serum creatinine level, BW, and chemical features when determining the initial antibiotic dosing regimen for neonates.
PMCID: PMC375245  PMID: 15047516
18.  Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae 
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 the dacB 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 for H. influenzae transformants into which the ftsI gene 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.
PMCID: PMC90533  PMID: 11353613

Results 1-18 (18)