Noriyuki Nagano

First Molecular Characterization of Group B Streptococci with Reduced Penicillin Susceptibility

ABSTRACT Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 μg/ml, 2 to 8 μg/ml, and 4 to 128 μg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of β-lactam-insusceptible GBS from a phenotypic standpoint.

Nosocomial Transmission of CTX-M-2 β-Lactamase-Producing Acinetobacter baumannii in a Neurosurgery Ward

ABSTRACT Three strains of cefotaxime (CTX)-resistant Acinetobacter baumannii, FM0209680, FM0300106, and FM0301433, were isolated from transtracheal aspirate cultures of three patients with probable nosocomial infections in a neurosurgery ward in Japan. The CTX MICs for these isolates were greater than 128 μg/ml but were drastically reduced in the presence of 4 μg of clavulanic acid per ml. These strains were also resistant to ceftriaxone, cefpodoxime, and aztreonam but were susceptible to ceftazidime and imipenem. The profile of resistance to various broad-spectrum β-lactams was transferred by conjugation. Strain FM0209680 was not eradicated from case patient 1 by administration of imipenem, ceftazidime, and levofloxacin, even after a 6-month hospitalization period. Strains FM0300106 and FM0301433 were isolated from case patients 2 and 3 during the sixth week following admission, respectively, and then each patient was colonized for 3 weeks. Eradication of FM0300106 was successfully obtained from case patient 2 by imipenem treatment, while administration of imipenem was continued to prevent pneumonia. Prophylactic antimicrobial therapy was discontinued in case patient 3 because of the lack of pneumonic symptoms, and FM0301433 disappeared after the discontinuation of antimicrobial chemotherapy. All three strains carried the blaCTX-M-2 gene, and the appearance of colonies in the growth-inhibitory zones around disks of CTX and aztreonam in double-disk synergy tests suggested inducible β-lactamase production in these A. baumannii strains. The ribotyping investigation suggested that all these strains belong to the same clonal lineage. The plasmids harbored by A. baumannii had the same restriction profile as those harbored by Proteus mirabilis strains previously isolated in a urology ward of the Funabashi Medical Center.

Identification of a Highly Encapsulated, Genetically Related Group of Invasive Type III Group B Streptococci

Type III group B streptococci (GBS) isolated from Tokyo and Salt Lake City were classified according to the similarity of HindIII and Sse83871 restriction digest patterns (RDPs) of bacterial DNA. The bacteria were clustered into three RDP types, with excellent correlation between subtyping based on the two enzymes. The majority (91%) of invasive isolates obtained from neonates were RDP type III-3. The mean sialic acid content of the III-3 strains was higher than that of other type III strains. Closely related isolates were concordant for expression of the bacterial enzyme C5a-ase, but invasive strains were no more likely to be C5a-ase positive than were strains isolated from the genitourinary tract of pregnant women. These data indicate that a group of genetically related organisms with increased capsule production causes the majority of invasive type III GBS disease.

Genetic Heterogeneity in pbp Genes among Clinically Isolated Group B Streptococci with Reduced Penicillin Susceptibility

ABSTRACT The recent emergence of group B streptococcal isolates exhibiting increased penicillin MICs at the Funabashi Municipal Medical Center and other hospitals in Japan prompted a comparative analysis of the penicillin-binding proteins (PBPs) from those strains with the PBPs from penicillin-susceptible strains comprising four neonatal invasive strains isolated from 1976 to 1988 and two recent isolates. The PBP sequences of the penicillin-susceptible strains were highly conserved, irrespective of their isolation date. Of six strains with reduced susceptibility to penicillin (penicillin MICs, 0.25 to 0.5 μg/ml), strains R1, R2, R5, and R6 shared a unique set of five amino acid substitutions, including V405A adjacent to the 402SSN404 motif in PBP 2X and one in PBP 2B. The remaining two strains, R3 and R4, shared several substitutions, including Q557E adjacent to the 552KSG554 motif in PBP 2X, in addition to the substitutions in PBP 2B, which are commonly found among penicillin-insusceptible strains. Strains R7 and R8, which had a penicillin MIC of 1 μg/ml, shared a unique set of eight amino acid substitutions (two in PBP 2X; two in PBP 2B, including G613R adjacent to the 614KTG616 motif; three in PBP 1A; and one in PBP 2A), and the Q557E substitution in PBP 2X was common to R3 and R4. The binding of Bocillin FL was reduced or not detected in some PBPs, including PBP 2X of penicillin-insusceptible strains, but no significant reduction in the level of pbp2x transcription was found in such strains. The results of phylogenetic comparative analyses imply the absence of epidemic penicillin-insusceptible strains, and several genetic lineages of penicillin-insusceptible strains have been independently emerging through the accumulation of mutations in their pbp genes, especially in pbp2x.

Nosocomial Outbreak of Infections by Proteus mirabilis That Produces Extended-Spectrum CTX-M-2 Type β-Lactamase

ABSTRACT Nineteen multidrug-resistant Proteus mirabilis strains were isolated from 19 patients suffering from infections probably caused by P. mirabilis. These strains were recovered from urine or other urogenital specimens of 16 inpatients and three outpatients with a hospitalization history in a urology ward of Funabashi Medical Center, from July 2001 to August 2002. These strains demonstrated resistance to cefotaxime, ceftriaxone, cefpodoxime, and aztreonam, while they were highly susceptible to ceftazidime (MIC, ≤0.5 μg/ml). The resistance level of these strains to cefotaxime was decreased by the presence of clavulanic acid. Therefore, the strains were speculated to produce extended-spectrum class A β-lactamases. These strains were later found to carry blaCTX-M-2 genes by both PCR and sequencing analyses. The profiles of SmaI-digested genomic DNA of 19 isolates were distinguished into five different clusters by biased sinusoidal field gel electrophoresis. Four of them, consisting of 18 isolates, were suggested to be a clonal expansion. These findings suggested that a nosocomial outbreak of infections by CTX-M-2-producing P. mirabilis had occurred in our medical center. Most patients suffered from urogenital malignancies with long-term catheterization. Cefazolin, cefoperazone-sulbactam, and/or levofloxacin were mostly administered to the patients, but these agents seemed ineffective for eradication of CTX-M-2 producers. Early recognition and rapid identification of colonizing antimicrobial-resistant bacteria, including CTX-M-2-producing P. mirabilis, would be the most effective measures to cope with further spread of this kind of hazardous microorganism in clinical environments.

Nosocomial spread of multidrug-resistant group B streptococci with reduced penicillin susceptibility belonging to clonal complex 1

BACKGROUND Multiple group B Streptococcus (GBS) isolates with reduced penicillin susceptibility (PRGBS) were recovered from several patients, hence a probable nosocomial transmission of PRGBS in a hospital setting was suspected. METHODS Ten PRGBS recovered from eight patients in a general hospital were characterized. Sequence analysis of genes for penicillin-binding proteins (PBPs) and quinolone resistance-determining regions (QRDRs) of gyrA, gyrB and parC was performed, and the macrolide resistance genes were detected by PCR. Genetic relatedness among the isolates was examined by PFGE and multilocus sequence typing. RESULTS All the PRGBS had the key amino acid substitution V405A, together with F395L, R433H, H438Y and G648A in PBP 2X and T567I in PBP 2B. A 23S rRNA methylase gene, erm(B), was also found in all 10 PRGBS strains. PFGE analysis revealed considerable genetic relatedness among the isolates. Isolates of pulsotype I were obtained from four patients in ward A and one patient in ward B, while isolates of pulsotypes II and III were obtained from two patients in ward B and one patient in ward C, respectively. Isolates of pulsotype I were resistant to levofloxacin (MIC >8 mg/L) and had the following amino acid substitutions in the QRDRs: S81L in GyrA, E476K in GyrB and S79Y in ParC. However, pulsotype II strains resistant to levofloxacin (MIC 8 mg/L) had no change in GyrA, but changes in GyrB (E476K) and ParC (S79Y). All 10 PRGBS strains belonged to serotype VI and ST458 (where ST stands for sequence type). CONCLUSIONS This is the first description of the nosocomial spread of multidrug-resistant PRGBS strains belonging to the genetic lineage ST458.

Novel Chimeric β-Lactamase CTX-M-64, a Hybrid of CTX-M-15-Like and CTX-M-14 β-Lactamases, Found in a Shigella sonnei Strain Resistant to Various Oxyimino-Cephalosporins, Including Ceftazidime

ABSTRACT The plasmid-mediated novel β-lactamase CTX-M-64 was first identified in Shigella sonnei strain UIH-1, which exhibited resistance to cefotaxime (MIC, 1,024 μg/ml) and ceftazidime (MIC, 32 μg/ml). The amino acid sequence of CTX-M-64 showed a chimeric structure of a CTX-M-15-like β-lactamase (N- and C-terminal moieties) and a CTX-M-14-like β-lactamase (central portion, amino acids 63 to 226), suggesting that it originated by homologous recombination between the corresponding genes. The introduction of a recombinant plasmid carrying blaCTX-M-64 conferred resistance to cefotaxime in Escherichia coli, and the activities of cefotaxime and ceftazidime were restored in the presence of clavulanic acid. Of note, CTX-M-64 production could also confer consistent resistance to ceftazidime, which differs from the majority of CTX-M-type enzymes, which poorly hydrolyze ceftazidime. These results were consistent with the kinetic parameters determined with the purified CTX-M-64 enzyme. The blaCTX-M-64 gene was flanked upstream by an ISEcp1 sequence and downstream by an orf477 sequence. The sequence of the 45-bp spacer region between the right inverted repeat (IRR) of ISEcp1 and blaCTX-M-64 was exactly identical to that of ISEcp1-blaCTX-M-15-like. Moreover, the presence of a putative IRR of ISEcp1 at the right end of truncated orf477 is indicative of an ISEcp1-mediated transposition event in the blaCTX-M-64 gene. The emergence of CTX-M-64 by probable homologous recombination would suggest the natural potential of an alternative mechanism for the diversification of CTX-M-type β-lactamases.

Human Fulminant Gas Gangrene Caused by Clostridium chauvoei

ABSTRACT The first human case of fulminant gas gangrene caused by Clostridium chauvoei, a pathogen causing ruminant blackleg, was confirmed for a 58-year-old man suffering from diabetes mellitus. The patient developed conspicuous emphysematous gangrene in the right chest wall as well as intravascular gas entrapments and died 2 h after hospital arrival.

High frequency of fluoroquinolone- and macrolide-resistant streptococci among clinically isolated group B streptococci with reduced penicillin susceptibility

OBJECTIVES Recently several clinical isolates of Streptococcus agalactiae [also known as group B Streptococcus (GBS)] that have acquired reduced penicillin susceptibility (PRGBS) by amino acid substitutions in the penicillin-binding protein 2X have emerged. The frequency of fluoroquinolone (FQ)- and macrolide-resistant streptococci among PRGBS is not yet known. METHODS Fifty-seven GBS [19 PRGBS and 38 penicillin-susceptible GBS (PSGBS)], isolated from different medical institutions in Japan, were studied. For GBS, the MICs of penicillin G, levofloxacin and erythromycin were determined using the agar dilution method. Nineteen PRGBS were previously confirmed as genetically diverse streptococci by PFGE. Further, the mechanisms underlying penicillin, FQ and macrolide non-susceptibility/resistance were analysed. RESULTS The frequency of non-susceptibility to FQs among PSGBS was 18.4% (7/38), whereas that among PRGBS was 100% (19/19). The frequency of resistance to erythromycin among PSGBS was 7.9% (3/38), while that among PRGBS was 47.4% (9/19). Statistical significance was determined using Fishers exact test between reduced penicillin susceptibility and FQ non-susceptibility (P ≤ 0.0001) and macrolide resistance (P=0.0012). The resistance/non-susceptibility mechanisms among PRGBS were diverse, suggesting that the PRGBS examined were not clonal. CONCLUSIONS PRGBS isolates tend to show resistance to FQs and/or macrolides. Because the drug choice for treating these multidrug-resistant GBS is more limited than that for usual GBS, these strains may present future public health challenges.

Predominance of sequence type 1 group with serotype VI among group B streptococci with reduced penicillin susceptibility identified in Japan

BACKGROUND Although group B Streptococcus (GBS; i.e. Streptococcus agalactiae) has been considered to be uniformly susceptible to β-lactams, GBS isolates with reduced penicillin susceptibility (PRGBS) have been reported from Japan and North America. In this study, PRGBS from Japan were characterized by multilocus sequence typing (MLST) and the results compared with data on PRGBS reported from the USA. METHODS Twenty-eight clinical isolates of PRGBS recovered in Japan (including 22 isolates previously analysed by PFGE) were analysed by MLST and eBURST (http://eburst.mlst.net/). RESULTS Twenty-three isolates were found to belong to the sequence type 1 (ST1) group (11 ST458, 7 ST1, 3 ST297, 1 ST358 and 1 ST4), while the remaining 5 isolates formed the ST23 group. Among 11 ST458 and 7 ST1 isolates, 9 and 4 were serotype VI, respectively, indicating a probable correlation between the ST1 group and serotype VI for PRGBS in Japan. CONCLUSIONS PRGBS in Japan could be classified into at least two ST groups, ST1 and ST23, which are genetically different from the ST19 PRGBS isolated in the USA, though five allele variations were seen between ST1 and ST19, implying a slight genetic relatedness.