Population-based incidence of hospitalized Clostridioides difficile infection among older adults in Ota-ku, Japan: A prospective surveillance study.

OBJECTIVES: Clostridioides difficile infection (CDI) burden is not well-characterized in Japan. Therefore, we conducted a population-based, hospitalized CDI incidence study, compared the results with standard-of-care (SOC) CDI testing, and generalized the results for nationwide incidence estimates. METHODS: Surveillance identified inpatients ≥50 years-of-age with diarrhea in nine Tokyo hospitals from December 17, 2018-March 30, 2020. A CDI case was defined as a patient with a PCR-positive/cell cytotoxicity neutralization assay (CCNA)-positive stool or a PCR-positive stool and pseudomembranous colitis (PMC). Incidence estimates were adjusted for the hospitalization share of participating hospitals and, in the sensitivity analysis, for missing CDI test results. SOC specimen collection and CDI testing occurred independently. RESULTS: Surveillance during 318 840 patient-days identified 4633 inpatients with diarrhea. Sixty-three CDI cases were identified; 11 (17·5%) had PMC, eight (12·7%) recurrent CDI, and nine (14·3%) died. The hospitalized CDI incidence was 97/100 000 population per year (PPY) in persons ≥50 years-of-age and, in the sensitivity analysis, 324/100 000 PPY. The incidence was 170 and 481/100 000 PPY in persons ≥65 and ≥85 years-of-age, respectively; these estimates increased to 569 and 1609/100 000 PPY in the sensitivity analysis, respectively. There were 12 primary SOC CDI cases in persons ≥50 years-of-age (18/100 000 PPY). CONCLUSIONS: The CDI incidence was high in older adults, with severe clinical consequences. SOC specimen collection and testing under-estimated CDI burden. There are >57 000 hospitalized CDI cases per year in Japan in persons ≥50 years-of-age. Public health interventions are needed to reduce the CDI burden in Japan.

Genome-Wide Screening for Identification of Novel Toxin-Antitoxin Systems in Staphylococcus aureus.

Toxin-antitoxin (TA) systems consist of toxin-inhibiting diverse cellular functions (e.g., DNA replication, transcription, and translation) and a noncoding RNA or protein antitoxin. TA systems are associated with various cellular events, such as stress responses, programmed cell death, and bacterial pathogenicity. Recent advances in genome sequencing and bioinformatics research have demonstrated that most bacteria harbor various kinds of TA modules on their chromosomes; however, there is little understanding of chromosomally encoded TA systems in the Gram-positive pathogen Staphylococcus aureus Here, we report on newly discovered S. aureus TA systems, each of which is composed of two proteins. Manual search and gene operon prediction analysis identified eight 2-gene operons as potential candidates for TA systems. Subsequently, using an Escherichia coli host killing and rescue assay, we demonstrated that four of the eight candidates worked as TA systems, designated tsaAT, tsbAT, tscAT, and tsdAT Moreover, the TsaT, TsbT, TscT, and TsdT toxins inhibited S. aureus growth, and the toxicity of TsbT was neutralized by coexpressing the tsbA gene in the native host, S. aureus Further, the bioinformatics analysis of the gene clusters revealed that TsaAT, TsbAT, TscAT, and TsdAT did not exhibit sequence similarity to known bacterial TA systems, and their homologues were present only within Staphylococcus species and not among any other bacteria. Our results further advance not only the understanding of S. aureus TA systems but also the study of unannotated TA systems in various bacterial species.IMPORTANCE Recent advances in genome sequencing and bioinformatics research have demonstrated that most pathogenic bacteria harbor a large number of chromosomally encoded toxin-antitoxin (TA) modules. However, little is known about the TA systems in S. aureus Here, we newly identified four S. aureus TA systems using a combination of manual base-by-base screening and functional analysis in E. coli Moreover, all toxins of the identified TA systems caused growth inhibition in the native host S. aureus Although the newly identified TA systems did not exhibit sequence similarity with known bacterial TA systems, their orthologues were conserved only among other Staphylococcus species, indicating their uniqueness to staphylococci. Our approach opens the possibility for studying unannotated TA systems in various bacterial species.

ACA-specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase.

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF-ec) cleaves RNA at A^CA. To date, a large number of MazF homologs that cleave RNA at specific three- to seven-base sequences have been identified from bacteria to archaea. MazF-ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate-binding site. Here, we investigated the role of the two loops in MazF-ec, which are closely associated with the interface of the MazF-ec dimer. We examined whether exchanging the loop regions of MazF-ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF-mx) and MazF from Mycobacterium tuberculosis (MazF-mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF-ec with loop 2 regions from either MazF-mx or MazF-mt3 created a new cleavage sequence at (A/U)(A/U)AA^C in addition to the original cleavage site, A^CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA^C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications.

Staphylococcus aureus YoeB homologues inhibit translation initiation.

YoeB is a bacterial toxin encoded by the yefM-yoeB toxin-antitoxin system found in various bacterial genomes. Here, we show that Staphylococcus aureus contains two YoeB homologues, both of which function as ribosome-dependent mRNA interferases to inhibit translation initiation in a manner identical to that of YoeB-ec from Escherichia coli.

Staphylococcus aureus MazF specifically cleaves a pentad sequence, UACAU, which is unusually abundant in the mRNA for pathogenic adhesive factor SraP.

Escherichia coli mRNA interferases, such as MazF and ChpBK, are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems present in its genome. A MazF homologue in Staphylococcus aureus (MazF(Sa)) has been shown to inhibit cell growth when induced in E. coli. Here, we determined the cleavage site for MazF(Sa) with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. MazF(Sa) specifically cleaves the RNA at a pentad sequence, U downward arrow ACAU. Bioinformatics analysis revealed that this pentad sequence is significantly abundant in several genes, including the sraP gene in the S. aureus N315 strain. This gene encodes a serine-rich protein, which is known to play an important role in adhesion of the pathogen to human tissues and thus in endovascular infection. We demonstrated that the sraP mRNA became extremely unstable in comparison with the ompA mRNA only when MazF(Sa) was induced in E. coli. Further bioinformatics analysis indicated that the pentad sequence is also significantly abundant in the mRNAs for all the pathogenic factors in S. aureus. This observation suggests a possible regulatory relationship between the MazEF(Sa) TA module and the pathogenicity in S. aureus.

In vitro antibacterial activities of new fluoroquinolones against clinical isolates of haemophilus influenzae with ciprofloxacin-resistance-associated alterations in GyrA and ParC.

BACKGROUND: The in vitro antimicrobial activities of new fluoroquinolones were tested against quinolone-resistant Haemophilus influenzae of clinical isolates. METHODS: The nucleotide sequences of the gyrA and parC genes from three ciprofloxacin-resistant strains of Haemophilus influenzae (MIC, 1.56-6.25 microg/ml) were determined. The gyrase was purified from the clinical isolates, and the inhibitory activities of quinolones against the enzyme were tested. RESULTS: These strains possessed at least one amino acid substitution in each of the GyrA (asparagine at residue 88 (Asp-88) to Tyr, Ser-84 to Leu or Ser-84 to Leu and Asp-88 to Asn) and ParC (Glu-88 to Lys). The antibacterial activity of olamufloxacin against the resistant strains was most potent compared with other quinolones, and the inhibitory activities correlated with quinolone resistance of these strains. CONCLUSIONS: These results warrant the clinical effects of new types of fluoroquinolones, such as olamufloxacin, against respiratory tract and otolaryngology infections caused by ciprofloxacin-resistant H. influenzae.