Takayuki Yamaguchi

Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B

ABSTRACT We identified a novel pathogenicity island in Staphylococcus aureus which contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, and edin-B gene in tandem and the phage resistance gene, flanked by hsdM, hsdS (restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), and Staphylococcus hyicus ETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced by S. aureus and is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive for edin-B were suggested to be clonally associated, and all edin-B-positive strains tested were positive for etd. Among 18 etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.

Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome

Bullous impetigo due to Staphylococcus aureus is one of the most common bacterial infections of man, and its generalized form, staphylococcal scalded skin syndrome (SSSS), is a frequent manifestation of staphylococcal epidemics in neonatal nurseries. Both diseases are mediated by exfoliative toxins (ETs), which show exquisite pathologic specificity in blistering only the superficial epidermis. We show that these toxins act as serine proteases with extremely focused molecular specificity to cleave mouse and human desmoglein 1 (Dsg1) once after glutamic acid residue 381 between extracellular domains 3 and 4. Mutation of the predicted catalytically active serine to alanine completely inhibits cleavage. The mutated ETs bind specifically to Dsg1 by immunofluorescence colocalization and by coimmunoprecipitation. Thus, ETs, through specific recognition and proteolytic cleavage of one structurally critical peptide bond in an adhesion molecule, cause its dysfunction and allow S. aureus to spread under the stratum corneum, the main barrier of the skin, explaining how, although they circulate through the entire body in SSSS, they cause pathology only in the superficial epidermis.

Phage conversion of exfoliative toxin A production in Staphylococcus aureus

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (φETA) that encodes ETA and determined the complete nucleotide sequence of the φETA genome. φETA has a head with a hexagonal outline and a non‐contractile and flexible tail. The genome of φETA is a circularly permuted linear double‐stranded DNA, and the genome size is 43 081 bp. Sixty‐six open reading frames (ORFs) were identified on the φETA genome, including eta, which was found to be located very close to a putative attachment site (attP). φETA converted ETA non‐producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that φETA or related phages are responsible for the acquisition of eta genes in S. aureus.

Complete Nucleotide Sequence of a Staphylococcus aureus Exfoliative Toxin B Plasmid and Identification of a Novel ADP-Ribosyltransferase, EDIN-C

ABSTRACT The complete nucleotide sequence of pETB, a 38.2-kbStaphylococcus aureus plasmid encoding the exfoliative toxin B (ETB), was determined. A total of 50 open reading frames were identified on the plasmid genome and, among these, 32 showed sequence similarity to known proteins. pETB contains three copies of IS257, which divide the pETB genome into three regions: (i) a cadmium resistance operon-containing region, (ii) a lantibiotic production gene-containing region, and (iii) the remaining part where genes for plasmid replication and/or maintenance are dispersed. In the third region, genes of various kinds of functions are present among the replication- and maintenance-related genes. They include two virulence-related genes, the etb gene and a gene encoding a novel ADP-ribosyltransferase closely related to EDIN, which belongs to the C3 family of ADP-ribosyltransferases modifying Rho GTPases. They also include genes for a cell wall-anchoring surface protein and a phage resistance protein. Based on the determined sequence of pETB, the genome structures of etb-bearing plasmids (ETB plasmids) from various clinical isolates were analyzed by the PCR scanning method. The data indicate that, although the ETB plasmids are highly heterogeneous in genome size, the fundamental genome organization is well conserved. The size variation of the plasmid is mainly attributed to defined regions which may be hot spots for gene shuffling.

Clinical Manifestations of Staphylococcal Scalded-Skin Syndrome Depend on Serotypes of Exfoliative Toxins

ABSTRACT We sought a possible correlation between the clinical manifestations of staphylococcal scalded-skin syndrome (SSSS) and the serotype of exfoliative toxins (ET) by PCR screening of the eta and etb genes in Staphylococcus aureus strains isolated from 103 patients with generalized SSSS and 95 patients with bullous impetigo. The eta gene and the etb gene were detected in, respectively, 31 (30%) and 20 (19%) episodes of generalized SSSS and 57 (60%) and 5 (5%) episodes of bullous impetigo. Both genes were detected in 52 (50%) episodes of generalized SSS and 33 (35%) episodes of bullous impetigo. To explain this link between etb and generalized SSSS, we examined the distribution of ETA- and ETB-specific antibodies in the healthy population (n = 175) and found that the anti-ETB antibody titer was lower than the anti-ETA titer. Thus, ETA is associated with bullous impetigo and ETB is associated with generalized SSSS, possibly owing to a lower titer of anti-ETB neutralizing antibodies in the general population.

Clonal association of Staphylococcus aureus causing bullous impetigo and the emergence of new methicillin-resistant clonal groups in Kansai district in Japan

A molecular epidemiological analysis was performed to reveal the clonal association of Staphylococcus aureus strains isolated from patients with bullous impetigo. Pulsed-field gel electrophoresis with cluster analysis, genetic and phenotypic characterizations, and antimicrobial susceptibility profiling of 88 S. aureus strains isolated from outpatients at 4 hospitals in the Kansai district in Japan were undertaken. Three distinct clonal groups were identified: 2 of them carried the exfoliative toxin (ET) A gene (eta), and the other carried the ETB gene (etb). The former groups represent 2 eta-positive clonal groups that have not been described previously. All the strains in the more dominant eta-positive clonal group and some of the strains in the etb-positive clonal group were methicillin-resistant S. aureus (MRSA) showing borderline-to-moderate resistance to beta-lactams. These MRSA strains appear to be emerging clonal groups that have not been considered in previous epidemiological studies of ET-producing S. aureus in Japan and thus pose a significant threat for future treatment of patients with bullous impetigo and/or staphylococcal scalded-skin syndrome.

Prevalence of Rho-Inactivating Epidermal Cell Differentiation Inhibitor Toxins in Clinical Staphylococcus aureus Isolates

Staphylococcus aureus produces exotoxins of the epidermal cell differentiation inhibitor (EDIN) family that ADP-ribosylate and inactivate Rho GTPases. The prevalence of genes encoding EDIN in clinical and nasal isolates of S. aureus was investigated. Of the 196 clinical S. aureus isolates tested, 15 (7.8%) were positive for 1 edin gene, whereas of 81 nasal isolates tested, only 3 (3.7%) were edin positive. Of the total 18 edin-positive isolates, 16 (90%) carried edin-B and 2 (10%) carried edin-C, but none was positive for edin-A. All edin-positive strains could produce the respective EDIN protein. Pulsed-field gel electrophoresis analysis suggested that the edin-B-positive S. aureus isolates are derived from one clone, and the edin-C-positive isolates are derived from another clone. Given that toxins acting on Rho GTPases are considered to be important for bacterial virulence, the EDIN toxins of S. aureus should receive more attention in future studies.

Emergence of Staphylococcus aureus Carrying Multiple Drug Resistance Genes on a Plasmid Encoding Exfoliative Toxin B

ABSTRACT We report the complete nucleotide sequence and analysis of pETBTY825, a Staphylococcus aureus TY825 plasmid encoding exfoliative toxin B (ETB). S. aureus TY825 is a clinical isolate obtained from an impetigo patient in 2002. The size of pETBTY825, 60.6 kbp, was unexpectedly larger than that of the archetype pETBTY4 (∼30 kbp). Genomic comparison of the plasmids shows that pETBTY825 has the archetype pETBTY4 as the backbone and has a single large extra DNA region of 22.4 kbp. The extra DNA region contains genes for resistance to aminoglycoside [aac(6′)/aph(2″)], macrolide (msrA), and penicillin (blaZ). A plasmid deletion experiment indicated that these three resistance elements were functionally active. We retrospectively examined the resistance profile of the clinical ETB-producing S. aureus strains isolated in 1977 to 2007 using a MIC determination with gentamicin (GM), arbekacin (ABK), and erythromycin (EM) and by PCR analyses for aac(6′)/aph(2″) and msrA using purified plasmid preparations. The ETB-producing S. aureus strains began to display high resistance to GM, which was parallel with the detection of aac(6′)/aph(2″) and mecA, after 1990. Conversely, there was no significant change in the ABK MIC during the testing period, although it had a tendency to slightly increase. After 2001, isolates resistant to EM significantly increased; however, msrA was hardly detected in ETB-producing S. aureus strains, and only five isolates were positive for both aac(6′)/aph(2″) and msrA. In this study, we report the emergence of a fusion plasmid carrying the toxin gene etb and drug resistance genes. Prevalence of the pETBTY825 carrier may further increase the clinical threat, since ETB-producing S. aureus is closely related to more severe impetigo or staphylococcal scalded-skin syndrome (SSSS), which requires a general antimicrobial treatment.

Tracing depositional consequences of environmental radionuclides (137Cs and 210Pb) in Slovenian forest soils

Depth distribution profiles of environmental radionuclides (137Cs and 210Pb) have been investigated in soil to elucidate the underlying environment of semi-natural temperate deciduous and/or coniferous forest soils in Slovenia (Žirovski vrh, Idrija, Kočevski Rog, Pohorie, Gorišnica and Rakitna). Surface enrichment of both nuclides was observed at all the sites investigated in this study, suggesting that the soils had undergone little natural or anthropogenic disturbance for at least the last several decades. Apparent annual burial rates of 137Cs (0.1–0.2 cm y−−1) were estimated to be about 1.3 times higher than those of 210Pb at individual sites of different lithology, which suggests strong affinity of 210Pb to soil organic matter. Variability of the vertical distribution profiles of these nuclides depends not only on “in situ” pedology but also on geographical and meteorological conditions, especially precipitation and wind direction.

Transmission dynamics of cholera in Yemen, 2017: a real time forecasting

BackgroundA large epidemic of cholera, caused by Vibrio cholerae, serotype Ogawa, has been ongoing in Yemen, 2017. To improve the situation awareness, the present study aimed to forecast the cholera epidemic, explicitly addressing the reporting delay and ascertainment bias.MethodsUsing weekly incidence of suspected cases, updated as a revised epidemic curve every week, the reporting delay was explicitly incorporated into the estimation model. Using the weekly case fatality risk as calculated by the World Health Organization, ascertainment bias was adjusted, enabling us to parameterize the family of logistic curves (i.e., logistic and generalized logistic models) for describing the unbiased incidence in 2017.ResultsThe cumulative incidence at the end of the epidemic, was estimated at 790,778 (95% CI: 700,495, 914,442) cases and 767,029 (95% CI: 690,877, 871,671) cases, respectively, by using logistic and generalized logistic models. It was also estimated that we have just passed through the epidemic peak by week 26, 2017. From week 27 onwards, the weekly incidence was predicted to decrease.ConclusionsCholera epidemic in Yemen, 2017 was predicted to soon start to decrease. If the weekly incidence is reported in the up-to-the-minute manner and updated in later weeks, not a single data point but the entire epidemic curve must be precisely updated.