Yusuke Sato'o

Identification and Characterization of a Novel Staphylococcal Emetic Toxin.

ABSTRACT Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.

A novel comprehensive analysis method for Staphylococcus aureus pathogenicity islands.

Staphylococcus aureus pathogenicity islands (SaPIs) form a growing family of mobile genetic elements (MGEs) in Staphylococci. Horizontal genetic transfer by MGEs plays an important role in the evolution of S. aureus. Several SaPIs carry staphylococcal enterotoxin and SE‐like toxin genes. To comprehensively investigate the diversity of SaPIs, a series of primers corresponding to sequences flanking six SaPI insertion sites in S. aureus genome were designed and a long and accurate (LA)‐PCR analysis method established. LA‐PCR products of 13–17 kbp were observed in strains with seb, selk or selq genes. Restriction fragment length polymorphism (RFLP) analysis showed that the products have different RFLP characteristics than do previously described SaPIs; they were therefore predicted to include new SaPIs. Nucleotide sequencing analysis revealed seven novel SaPIs: seb‐harboring SaPIivm10, SaPishikawa11, SaPIivm60, SaPIno10 and SaPIhirosaki4, selk and selq‐harboring SaPIj11 and non‐superantigen‐harboring SaPIhhms2. These SaPIs have mosaic structures containing components of known SaPIs and other unknown genes. Strains carrying different SaPIs were found to have significantly different production of superantigen toxins. The present results show that the LA‐PCR approach can comprehensively identify SaPI diversity and is useful for investigating the evolution of S. aureus pathogenicity.

Molecular Epidemiology and Identification of a Staphylococcus aureus Clone Causing Food Poisoning Outbreaks in Japan

ABSTRACT Molecular characterization of isolates from staphylococcal food poisoning (SFP) outbreaks in Japan showed that the dominant lineage causing SFP outbreaks is clonal complex 81 (CC81), a single-locus variant of sequence type 1, coagulase type VII, positive for sea and/or seb, and positive for seh. Among various CC lineages producing staphylococcal enterotoxin A, CC81 showed the highest toxin productivity.

Effects of the Staphylococcus aureus and Staphylococcus epidermidis Secretomes Isolated from the Skin Microbiota of Atopic Children on CD4+ T Cell Activation

Interactions between the immune system and skin bacteria are of major importance in the pathophysiology of atopic dermatitis (AD), yet our understanding of them is limited. From a cohort of very young AD children (1 to 3 years old), sensitized to Dermatophagoides pteronyssinus allergens (Der p), we conducted culturomic analysis of skin microbiota, cutaneous transcript profiling and quantification of anti-Der p CD4+ T cells. This showed that the presence of S. aureus in inflamed skin of AD patients was associated with a high IgE response, increased expression of inflammatory and Th2/Th22 transcripts and the prevalence of a peripheral Th2 anti-Der p response. Monocyte-derived dendritic cells (moDC) exposed to the S. aureus and S. epidermidis secretomes were found to release pro-inflammatory IFN-γ and anti-inflammatory IL-10, respectively. Allogeneic moDC exposed to the S. aureus secretome also induced the proliferation of CD4+ T cells and this effect was counteracted by concurrent exposure to the S. epidermidis secretome. In addition, whereas the S. epidermidis secretome promoted the activity of regulatory T cells (Treg) in suppressing the proliferation of conventional CD4+ T cells, the Treg lost this ability in the presence of the S. aureus secretome. We therefore conclude that S. aureus may cause and promote inflammation in the skin of AD children through concomitant Th2 activation and the silencing of resident Treg cells. Commensals such as S. epidermidis may counteract these effects by inducing the release of IL-10 by skin dendritic cells.

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan

Staphylococcal food poisoning (SFP), one of the commonest food‐borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin‐encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.

Identification and characterization of novel Staphylococcus aureus pathogenicity islands encoding staphylococcal enterotoxins originating from staphylococcal food poisoning isolates

Horizontal transfer of Staphylococcus aureus pathogenicity islands (SaPIs) plays an important role in acquiring pathogenicity. This study aimed to identify novel SaPIs encoding staphylococcal enterotoxins (SEs) and to characterize their SE productivity and replication process.

The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable causative agents of emetic food poisoning in humans. New types of SEs and SE‐like (SEl) toxins have been reported. Several epidemiological investigations have shown that the SEs and SEl genes, particularly, SEK, SEL, SEM, SEN and SEO genes, are frequently detected in strains isolated from patients with food poisoning. The purpose of the present study was to evaluate the emetic activity of recently identified SEs using a small emetic animal model, the house musk shrew. The emetic activity of these SEs in house musk shrews was evaluated by intraperitoneal administration and emetic responses, including the number of shrews that vomited, emetic frequency and latency of vomiting were documented. It was found that SEs induce emetic responses in these animals. This is the first time to demonstrate that SEK, SEL, SEM, SEN and SEO possess emetic activity in the house musk shrew.

Molecular typing of Salmonella enterica serovar 4,[5],12:i:- isolates from humans, animals and river water in Japan by multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis

Fifty-one Salmonella enterica serovar 4,[5],12:i:- (S. 4, [5],12:i:-) isolates (14 human strains, 34 animal strains and 3 river water strains) which are assumed to be monophasic variants of S. Typhimurium were analyzed using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) in order to investigate their genetic diversities and relationships. PFGE, MLVA and combination of them identified 28, 27 and 34 profiles (Simpson’s diversity indices [DI]=0.94, 0.96 and 0.97), respectively. No correlations were detected between MLVA clustering and PFGE clustering or phage typing. These results suggested that S. 4,[5],12:i:- originated from multiple S. Typhimurium ancestors. Two cattle and one pig isolates showing identical phage types as well as PFGE and MLVA profiles to human isolates S. 4,[5],12:i:- suggested the existence of the links between human infections and animal reservoirs.

Efficient transformation of Staphylococcus aureus using multi-pulse electroporation.

A new multi-pulse electroporation system was evaluated to transform Staphylococcus aureus. Compared to the conventional electroporation system, it yielded high transformation efficiency to obtain more than 3.9×105S. aureus RN4220 transformed cells/1μg plasmid DNA using a single electroporation by manipulating the poring pulse and transfer pulse.