Identification of cfxA gene variants and susceptibility patterns in ß-lactamase-producing Prevotella strains.

OBJECTIVES: Antimicrobial-resistant isolates of Prevotella species, especially those resistant to ß-lactams, have become increasingly common. Here, we aimed to elucidate the underlying mechanisms contributing to the emergence and spread of antimicrobial resistance in Prevotella species. METHODS: Prevotella species were isolated from a variety of clinical specimens. ß-lactamase production was determined using nitrocefin discs, and the determination of minimum inhibitory concentration (MIC) to ten antimicrobials was done by the agar dilution method. Four resistance genes (cfxA, tetQ, ermF, and nim) and cfxA-flanking regions were detected using polymerase chain reaction. cfxA and the flanking regions were sequenced, and a phylogenetic tree was constructed based on CfxA amino acid sequences using the UPGMA method. RESULTS: Among the 45 Prevotella isolates identified, 35 (77.8%) produced ß-lactamases and had the cfxA genes. The tetQ, ermF, and nim genes were detected in 53.3%, 17.8%, and 0% of the 45 isolates, respectively. Among the 33 sequenced cfxA alleles, cfxA2 (45.5%) was the most frequent, followed by cfxA3 (42.4%) and a novel variant (cfxA7, 12.1%). The novel CfxA7 ß-lactamase had a novel L155F substitution not previously reported in CfxA variants. The MICs of all ß-lactam agents tested, excluding cefmetazole and meropenem, were lower among cfxA7-positive isolates than in cfxA2-and cfxA3-positive isolates. CONCLUSIONS: Differences in MICs of penicillins and cephalosporins may be due to amino acid substitutions in the CfxA variants, CfxA2, CfxA3, and CfxA7, among Prevotella isolates. Possession of cfxA-mobA, tetQ, and ermF may increase the risks of the emergence and spread of multidrug-resistant Prevotella species.

Complete Genome Sequence of Moraxella osloensis Strain KMC41, a Producer of 4-Methyl-3-Hexenoic Acid, a Major Malodor Compound in Laundry.

We report the complete genome sequence of Moraxella osloensis strain KMC41, isolated from laundry with malodor. The KMC41 genome comprises a 2,445,556-bp chromosome and three plasmids. A fatty acid desaturase and at least four ß-oxidation-related genes putatively associated with 4-methyl-3-hexenoic acid generation were detected in the KMC41 chromosome.

Draft Genome Sequence of Porphyromonas gingivalis Strain Ando Expressing a 53-Kilodalton-Type Fimbrilin Variant of Mfa1 Fimbriae.

Periodontopathic Porphyromonas gingivalis strain Ando abundantly expresses a 53-kDa-type Mfa1 fimbria. Here, we report the draft genome sequence of Ando, with a size of 2,229,994 bp, average G+C content of 48.4%, and 1,755 predicted protein-coding sequences.

Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia.

We report the complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia. The PAGU611 genome comprises a 2,078,348-bp chromosome and a 23,054-bp plasmid. The chromosome contains a unique genomic island, encoding a type VI secretion system and clustered regularly interspaced short palindromic repeat (CRISPR) loci.

In vitro antimicrobial activity of razupenem (SMP-601, PTZ601) against anaerobic bacteria.

We evaluated the in vitro antianaerobic activity of razupenem (SMP-601, PTZ601), a new parenterally administered carbapenem, against 70 reference strains and 323 clinical isolates. Razupenem exhibited broad-spectrum activity against anaerobes, inhibiting most of the reference strains when used at a concentration of ≤1 µg/ml. Furthermore, it exhibited strong activity, comparable to those of other carbapenems (meropenem and doripenem), against clinically isolated non-fragilis Bacteroides spp. (MIC90s of 2 µg/ml), with MIC90 values of 0.06, 0.03, and 0.5 µg/ml against Prevotella spp., Porphyromonas spp., and Fusobacterium spp., respectively. Clinical isolates of anaerobic Gram-positive cocci, Eggerthella spp., and Clostridium spp. were highly susceptible to razupenem (MIC90s, 0.03 to 1 µg/ml).

In vitro activity of tomopenem (CS-023/RO4908463) against anaerobic bacteria.

The antianaerobic activity of tomopenem, a new longer-half-life parenteral carbapenem, was compared with other carbapenems. Tomopenem showed broad activity against 63 reference species. The activity of tomopenem against 293 clinical isolates was potent (MIC(90), 0.06 to 4 microg/ml) and comparable to those of meropenem and doripenem and more potent than that of panipenem.

Essential involvement of CX3CR1-mediated signals in the bactericidal host defense during septic peritonitis.

Cecal ligation and puncture (CLP) caused septic peritonitis in wild-type (WT) mice, with approximately 33% mortality within 7 days after the procedure. Concomitantly, the protein level of intraperitoneal CX3CL1/fractalkine was increased, with infiltration by CX3CR1-expressing macrophages into the peritoneum. CLP induced 75% mortality in CX3CR1-deficient (CX3CR1(-/-)) mice, which, however, exhibited a similar degree of intraperitoneal leukocyte infiltration as WT mice. Despite this, CX3CR1(-/-) mice exhibited impairment in intraperitoneal bacterial clearance, together with a reduction in the expression of intraperitoneal inducible NO synthase (iNOS) and bactericidal proinflammatory cytokines, including IL-1beta, TNF-alpha, IFN-gamma, and IL-12, compared with WT mice. Bactericidal ability of peritoneal phagocytes such as neutrophils and macrophages was consistently attenuated in CX3CR1(-/-) mice compared with WT mice. Moreover, when WT macrophages were stimulated in vitro with CX3CL1, their bactericidal activity was augmented in a dose-dependent manner, with enhanced iNOS gene expression and subsequent NO generation. Furthermore, CX3CL1 enhanced the gene expression of IL-1beta, TNF-alpha, IFN-gamma, and IL-12 by WT macrophages with NF-kappaB activation. Thus, CX3CL1-CX3CR1 interaction is crucial for optimal host defense against bacterial infection by activating bacterial killing functions of phagocytes, and by augmenting iNOS-mediated NO generation and bactericidal proinflammatory cytokine production mainly through the NF-kappaB signal pathway, with few effects on macrophage infiltration.

Complete genome sequence of Finegoldia magna, an anaerobic opportunistic pathogen.

Finegoldia magna (formerly Peptostreptococcus magnus), a member of the Gram-positive anaerobic cocci (GPAC), is a commensal bacterium colonizing human skin and mucous membranes. Moreover, it is also recognized as an opportunistic pathogen responsible for various infectious diseases. Here, we report the complete genome sequence of F. magna ATCC 29328. The genome consists of a 1,797,577 bp circular chromosome and an 189,163 bp plasmid (pPEP1). The metabolic maps constructed based on the genome information confirmed that most F. magna strains cannot ferment most sugars, except fructose, and have various aminopeptidase activities. Three homologs of albumin-binding protein, a known virulence factor useful for antiphagocytosis, are encoded on the chromosome, and one albumin-binding protein homolog is encoded on the plasmid. A unique feature of the genome is that F. magna encodes many sortase genes, of which substrates may be involved in bacterial pathogenesis, such as antiphagocytosis and adherence to the host cell. The plasmid pPEP1 encodes seven sortase and seven substrate genes, whereas the chromosome encodes four sortase and 19 substrate genes. These plasmid-encoded sortases may play important roles in the pathogenesis of F. magna by enriching the variety of cell wall anchored surface proteins.

Comparative analysis of the four rRNA operons in Finegoldia magna ATCC29328.

There are four rRNA operons rrnA, rrnB, rrnC and rrnD on the genome of Finegoldia magna (formerly Peptostreptococcus magnus) ATCC29328, which, in contrast to those of Clostridia, are dispersed around the chromosome. Using a BAC library we determined the nucleotide sequences and structures of all four operons, including their flanking regions, and performed comparative analyses. We identified putative boxA sequences in the operons, which should be required for rRNA transcription antitermination, as well as their respective tandem promoters, AT-rich UP elements in the upstream region and Rho-independent terminators in the downstream region. The mosaic features of the operons were revealed. Multiple tRNAs were identified in the downstream region of two operons, 18 in rrnC and 11 in rrnD. They were presumed to form transcription units together with rRNAs. rrnA and rrnB had repeat units with Rho-independent terminators instead of tRNAs in the downstream region. rrnB and rrnC were the most similar in rrn upstream promoter region. Focusing on the sequence variations of rRNA genes, rrnB alone was heterogeneous. In light of previous reports, we also assessed the correlation between intercistronic rRNA sequence differences and distances between the operons, but no positive correlation was seen in this strain.

Bacterial artificial chromosome library of Finegoldia magna ATCC 29328 for genetic mapping and comparative genomics.

We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328 DNA to facilitate further genome analysis of F. magna. The BAC library contained 385 clones with an average insert size of 55 kb, representing a 10.1-fold genomic coverage. Repeated DNA hybridization using primer sets designed on the basis of BAC-end sequences yielded nine contigs covering 95% of the chromosome and two contigs covering 98% of the plasmid. The contigs were localized on the physical map of F. magna ATCC 29328 DNA. A total of 121 BAC-end sequences revealed 103 unique genes, which had not been previously reported for F. magna. The homolog ORF of albumin-binding protein (urPAB), one of the known virulence factors from F. magna, was sequenced and localized on the physical map. Homology analysis of 121 BAC-end sequences revealed that F. magna is most closely related to clostridia, particularly Clostridium tetani. This close relationship is consistent with the recent classification of peptostreptococci based on 16S rRNA sequence analysis. The BAC library constructed here will be useful for the whole genome sequencing project and other postgenomic applications.

Physical and genetic map of the Finegoldia magna (formerly Peptostreptococcus magnus) ATCC 29328 genome.

A physical and genetic map of Finegoldia magna (formerly Peptostreptococcus magnus) ATCC 29328 chromosome was constructed. The order of rare cleavage restriction fragments was determined by double digestion with the restriction enzymes I-CeuI, SgrAI, ApaI and PmeI, cross-hybridization and ApaI-linking clones. The size of the circular chromosome of F. magna was estimated to be 1.9 Mb. This strain also had a 200-kb megaplasmid. The chromosome contained four rrn operons, and the orientation of two rrn operons was opposite to the others. Fragment analysis of Peptostreptococcus anaerobius ATCC 27337(T) chromosome suggested that its size was much smaller than that of F. magna ATCC 29328.

Rapid identification of Streptococcus intermedius by PCR with the ily gene as a species marker gene.

Streptococcus intermedius belongs to the anginosus group of streptococci (AGS) and is associated with endogenous infections leading to abscesses in the oral cavity and at deepseated sites, such as the brain and liver. Two other species, S. anginosus and S. constellatus, and some presently unnamed taxa, are also classified as AGS. Recently, S. constellatus subsp. pharyngis, a new subspecies with biochemical characteristics similar to S. intermedius, was described with the potential for causing confusion when trying to identify isolates of these two species routinely with commercial identification kits, such as Rapid ID32 Strep and Fluo-Card Milleri. To correctly identify S. intermedius, this study attempted to develop an accurate PCR identification system with the ily gene as a species marker. This approach relies on amplification of an 819-bp fragment of the ily gene and its 3'-flanking region and is shown here to be specific for S. intermedius strains among all other streptococcal species. Moreover, this PCR system was applicable in direct rapid PCR with whole bacterial cells and TaKaRa Z-Taq (TaKaRa), a highly efficient DNA polymerase, as the template and DNA amplification enzyme, respectively.