C55 bacteriocin produced by ETB-plasmid positive Staphylococcus aureus strains is a key factor for competition with S. aureus strains.

Exfoliative toxin (ET) produced by Staphylococcus aureus is closely associated with the onset of bullous impetigo. To date, three ETs (ETA, ETB and ETD) have been identified. The gene encoding ETB is located in a plasmid designated pETB. Bacteriocin synthesis genes are also located in this plasmid and pETB-positive strains reportedly produce the C55 bacteriocin. In this study, the antibacterial activity against S. aureus strains of the bacteriocin produced by the pETB-positive strain TY4 was investigated. This bacteriocin demonstrated antibacterial activity against all pETB-negative but not pETB-positive strains, including TY4. Additionally, a TY4- strain from which the pETB plasmid had been deleted exhibited susceptibility to the bacteriocin. Further experiments revealed that two immunity factors (orf 46-47 and orf 48) downstream of the bacteriocin synthesis genes in the pETB plasmid are associated with immunity against the bacteriocin produced by TY4. The TY4- with orf46-47 strain exhibited complete resistance to bacteriocin, whereas the TY4- with orf48 strain exhibited partial resistance. Whether bacteriocin affects the proportion of each strain when co-cultured with S. aureus strains was also investigated. When TY4 or TY4- was co-cultured with 209P strain, which is susceptible to the bacteriocin, the proportion of 209P co-cultured with TY4 was significantly less than when 209P was co-cultured with TY4-, whereas the proportion of TY4- with orf46-48 co-cultured with TY4 was greater than with TY4-. These results suggest that the C55 bacteriocin produced by pETB-positive strains affects the proportion of each strain when pETB-positive and -negative strains co-exist.

[Homologous Analysis Using Repetitive-sequence-based PCR Typing of Exfoliative Toxin-producing Staphylococcus aureus Isolated from Our Hospital].

We examined staphylococcal coagulase types and homologous analysis using the DiversiLab repetitive-sequence-based PCR system in exfoliative toxin (ET)-producing Staphylococcus aureus. Twenty-two isolates (17 methicillin-sensitive Staphylococcus aureus (MSSA) and 5 methicillin-resistant Staphylococcus aureus (MRSA) isolates) obtained in our hospital from January 2012 and December 2013 were used. Three groups were classified according to the coagulase types and serotypes of ET. The first group (4 MSSA) showed coagulase type I and ET-A, and the second group (3 MSSA and 2 MRSA) showed coagulase type I and ET-B. The third group (10 MSSA and 3 MRSA) showed coagulase type V and ET-B. An analysis by DiversiLab demonstrated that homology was high in both the first and second groups. The homogenousness was high among the third group isolates except for the ocular isolates. In our hospital, three important groups were present according to a coagulase type and an ET type, and the homology of ocular isolates could be different from other materials isolates.

Emergence of Staphylococcus aureus carrying multiple drug resistance genes on a plasmid encoding exfoliative toxin B.

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.

Regulatory mechanism for exfoliative toxin production in Staphylococcus aureus.

The exfoliative toxin (ET) is a major virulence factor of Staphylococcus aureus that causes bullous impetigo and its disseminated form, staphylococcal scalded-skin syndrome (SSSS). ET selectively digests one of the intracellular adhesion molecules, desmoglein 1, of epidermal keratinocytes and causes blisters due to intraepidermal cell-cell dissociation. Most S. aureus strains that cause blistering disease produce either ETA or ETB. They are serologically distinct molecules, where ETA is encoded on a phage genome and ETB is enocded on a large plasmid. ETA-producing S. aureus strains are frequently isolated from impetigo patients, and ETB-producing S. aureus strains are isolated from SSSS. ET-induced blister formation can be reproduced with the neonatal mouse. To determine the regulatory mechanism of ET production, we investigated the role of the two-component systems and global regulators for eta or etb expression in vitro and in vivo with the mouse model. Western blot and transcription analyses using a series of mutants demonstrate ETA production was downregulated by sigB, sarS, and sarA, while ETB production was downregulated by sigB and sarA but not by sarS. Production of both toxins is upregulated by saeRS, arlRS, and agrCA. Furthermore, by the in vivo neonatal mouse model, sigB and sarS but not sarA negatively regulate the exfoliation activity of the ETA-producing strain, while sarA negatively regulates the ETB-producing strain. In both strains, saeRS, arlRS, and agrCA positively regulate the exfoliation activity in vivo. The data illustrate similar but distinct regulatory mechanisms for ETA and ETB production in S. aureus in vitro as well as in vivo.

Bullous impetigo in children infected with methicillin-resistant Staphylococcus aureus alone or in combination with methicillin-susceptible S. aureus: analysis of genetic characteristics, including assessment of exfoliative toxin gene carriage.

Among bullous impetigo isolates, exfoliative toxin (ET) gene carriage was found in 61.5% of methicillin-resistant Staphylococcus aureus (MRSA) isolates versus 90.6% of methicillin-susceptible S. aureus (MSSA) isolates. MRSA-only cases were ETB or ETA positive, while MRSA/MSSA coinfection cases were ET negative for MRSA but ETA positive for MSSA. Collagen adhesin may facilitate some MRSA infections.

Characterization of toxin production of coagulase-negative staphylococci isolated from food and starter cultures.

In this study a comprehensive analysis of toxin production of food associated coagulase-negative staphylococci (CNS) was investigated. The strains belong to the following staphylococcal species, Staphylococcus carnosus, Staphylococcus condimenti, Staphylococcus equorum, Staphylococcus piscifermentans, Staphylococcus succinus, and Staphylococcus xylosus, which were isolated from fermented food and starter cultures. A collection of 330 strains were analyzed with respect to their hemolytic activity. 59% of the strains exhibited weak to moderate hemolytic activity with human blood and 34% with sheep blood after 48 h incubation. A selection of 35 strains were tested by immunoblot analysis for their ability to produce toxins, such as the most common staphylococcal enterotoxins (SEs), the toxic shock syndrome toxin 1 (TSST-1), and the exfoliative toxin A (ETA). 18 of the 35 strains produced at least one of the toxins with the SED and SEH being the most common. These indicate that the use of CNS in food production demands a safety evaluation.

Staphylococcus aureus virulence factors associated with infected skin lesions: influence on the local immune response.

OBJECTIVES: To evaluate Staphylococcus aureus isolates from infected skin lesions for their potential to produce immune system-modulating toxins and to correlate these with white blood cell (WBC) counts associated with these lesions. DESIGN: Specimens were obtained for bacterial culture and gram staining from 105 infected skin lesions, and the number of WBCs per low-power field (LPF) was determined. Chromosomal DNA was prepared from 84 bacterial isolates and subjected to real-time polymerase chain reaction analysis to determine the presence of genes encoding potential immunomodulating toxins. Bacterial populations were divided into 2 groups: those associated with low WBC counts (0-5 WBCs/LPF) and those with high WBC counts (> 5 WBCs/LPF). We applied chi(2) statistical analyses to compare the toxin gene profiles associated with WBC counts on initial swab for culture. PATIENTS: Samples were obtained from patients at a single geographic location. RESULTS: A higher than expected percentage of bacteria capable of producing the exfoliative toxins A and/or B (ETA and/or ETB) and Panton-Valentine leukocidin (PVL) was seen in all skin lesions infected with S aureus without regard to WBC count with initial cultures. Comparison of the toxins associated with the low WBC group vs the high WBC group showed that low WBC counts were associated with ETA and ETB, while high WBC counts were associated with PVL and toxic shock syndrome toxin. There were no differences in the clinical appearance of the lesions between groups. CONCLUSIONS: Staphylococcus aureus virulence factors ETA, ETB, and PVL are associated with WBC counts from infected skin lesions. The exact role they play in affecting the WBC counts remains to be determined.

Outbreak of staphylococcal bullous impetigo in a maternity ward linked to an asymptomatic healthcare worker.

An outbreak of staphylococcal bullous impetigo occurred over a period of five months in a maternity ward involving seven infected and two colonised neonates. The skin lesions were due to epidermolytic toxin A-producing Staphylococcus aureus. Infection control measures were implemented and a retrospective case-control study performed. Contact with an auxiliary nurse was the only risk factor for cases of bullous impetigo (P<0.01). The nurse cared for all seven cases and was an asymptomatic nasal carrier of the epidemic strain. Repeated courses of decontamination treatment failed to eradicate carriage. Nine months after the last case, another neonate developed a more severe form of bullous impetigo and the auxiliary nurse was reassigned to an adult ward.

Production of exfoliative toxin by isolates of Staphylococcus hyicus from different countries.

A total of 218 isolates of Staphylococcus hyicus from pigs in eight countries (Belgium, Croatia, Germany, Japan, Korea, Slovenia, the uk and the usa) and 44 isolates from other animals in Belgium, India, Japan and the usa were examined for the genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD by multiplex pcr. The expression of the toxins was confirmed by immunoblot analysis, using monoclonal or polyclonal antibodies specific for each of the toxins. The porcine isolates were from pigs with exudative epidermitis, pigs with other lesions and from healthy pigs, and one or more of the toxins could be found among the isolates from the pigs in all the countries. Toxigenic strains of S hyicus were isolated from both healthy and diseased pigs, but the chance of isolating toxigenic strains from pigs with exudative epidermitis was greater than from pigs with other lesions or healthy pigs. Of the 44 isolates from other animal species, only one isolate, from a hare from Belgium, produced ExhB, and one isolate, from a cow with mastitis from Japan, produced ExhA.

Structural similarities and differences in Staphylococcus aureus exfoliative toxins A and B as revealed by their crystal structures.

Staphylococcal aureus epidermolytic toxins (ETs) A and B are responsible for the induction of staphylococcal scalded skin syndrome, a disease of neonates and young children. The clinical features of this syndrome vary from localized blisters to severe exfoliation affecting most of the body surface. Comparison of the crystal structures of two subtypes of ETs-rETA (at 2.0 A resolution), rETB (at 2.8 A resolution), and an active site variant of rETA, Ser195Ala at 2.0 A resolution has demonstrated that their overall topology resembles that of a "trypsin-like" serine protease, but with significant differences at the N- and C-termini and loop regions. The details of the catalytic site in both ET structures are very similar to those in glutamate-specific serine proteases, suggesting a common catalytic mechanism. However, the "oxyanion hole," which is part of the catalytic sites of glutamate specific serine proteases, is in the closed or inactive conformation for rETA, yet in the open or active conformation for rETB. The ETs contain a unique amphipathic helix at the N-terminus, and it appears to be involved in optimizing the conformation of the catalytic site residues. Determination of the structure of the rETA catalytic site variant, Ser195Ala, showed no significant perturbation at the active site, establishing that the loss of biological and esterolytic activity can be attributed solely to disruption of the catalytic serine residue. Finally, the crystal structure of ETs, together with biochemical data and mutagenesis studies, strongly confirms the classification of these molecules as "serine proteases" rather than "superantigens."

Microbiologic characteristics of exfoliative toxin-producing Staphylococcus aureus.

Exfoliative toxin (ET) production, phage types and antibiotic susceptibilities of 74 strains of Staphylococcus aureus isolated from patients with generalized staphylococcal scalded skin syndrome or bullous impetigo were studied. Of 74 staphylococcal isolates, 61 strains were found to produce ET by the newborn mouse assay method and the latex agglutination method. Fifteen strains were positive for ET-A, 32 for ET-B and 14 for both ET-A and ET-B. Among 61 ET-producing strains 27 (44%) were classified as Phage Group II, 16 (26%) as Group III, and 14 (23%) as Groups I and III. Of 27 Phage Group II strains 14 produced ET-A and 13 produced both ET-A and ET-B, but no strain was positive solely for ET-B. On the other hand 15 of 16 Phage Group III strains and all 14 Phage Group I and III strains produced only ET-B. It was demonstrated that the phage types of staphylococci were closely related to ET types. Characteristically the minimal inhibitory concentrations of penicillin G against ET-producing strains were less than 2 micrograms/ml, in contrast to other pathogenic staphylococci, 60 to 70% of which are highly penicillin G-resistant.

Genotypic and phenotypic characterization of Staphylococcus aureus strains isolated in subjects with atopic dermatitis. Higher prevalence of exfoliative B toxin production in lesional strains and correlation between the markers of disease intensity and colonization density.

Staphylococcus aureus strains generally colonize eczematous lesions of subjects with atopic dermatitis much more frequently than in the skin of normal individuals. The aim of this study was to provide a detailed genotypic and phenotypic analysis of S. aureus strains colonizing four different sites (lesional and non-lesional skin areas, nasal and pharyngeal mucosas) of 49 patients with atopic dermatitis. The 88 isolates were analyzed in duplicate by pulsed field gel electrophoresis and in their exfoliative toxin A or B production by latex test. The patients were characterized by age, sex, severity scoring of atopic dermatitis and serum eosinophil cationic protein. Fourteen (28.6%) of the patients were completely negative for S. aureus while 35 (71.4%) were positive in at least one site. The severity scores and eosinophil cationic protein levels were significantly correlated variables (P<0.001), linked to the colonization intensity (P ranging between 0.05 and <0.001 depending on the site) and to the number of colonized sites (P at least <0.01). The genotypic patterns, widely heterogeneous, showed no restriction to peculiar patterns. Only eight strains produced exfoliative toxin B which was significantly restricted to the lesional isolates (P=0.012).

Nasal, axillary, and perineal carriage of Staphylococcus aureus among women: identification of strains producing epidermolytic toxin.

Following two outbreaks of staphylococcal scalded skin syndrome in a maternity unit, 500 pregnant women attending an antenatal clinic were screened for carriage of epidermolytic toxin producing Staphylococcus aureus. Nasal, axillary, and perineal swabs were collected from women whose gestational ages ranged from 12-40 weeks. Isolates of S aureus were purified, phage typed, and tested for methicillin sensitivity and production of epidermolytic toxin. The results showed that 164 (33%) women carried S aureus; of these, 100 (61%) were from the nose and three (2%) from axillae, but 41 (25%) strains were isolated from the perineum alone. Screening for nasal carriage alone will therefore miss 25% of carriers. More than one strain of S aureus was identified in seven of 20 women with multiple site carriage. Three (2%) methicillin resistant strains were isolated during the survey, and five (3%) isolates produced epidermolytic toxin. Phage typing identified 63 (34%) strains as non-typable, but 50% of isolates typed either groups I, II or III, and a further 10% represented varying combinations of these and other phage groups. These results provide baseline information on S aureus in the community, and identification of methicillin resistant and toxin producing strains shows a reservoir of outbreak potential which could become relevant on hospital admission of such a carrier.

A novel method for rapid production and purification of exfoliative toxin A of Staphylococcus aureus.

The exfoliative toxins of Staphylococcus aureus are the causative agents of the scalded-skin syndrome. Previously described methods of toxin production and purification require large quantities of culture medium, take a long time and often produce low yields of toxin. A novel method of toxin production and purification using a dialysis sac to separate the culture medium from the staphylococci is described. This method produces up to 12 mg of crude toxin per ml of bacterial cell culture bathing the surface of the dialysis sac within 36 h and almost 10 mg of purified toxin per ml of cell culture within 3 days, in contrast to previous procedures that took over a week to produce 0.1-1.0 mg ml(-1) crude toxin and less than 0.01 mg ml(-1) purified toxin. This rapid method of toxin production should speed up future research into the pathogenesis of the staphylococcal scalded-skin syndrome.

Superantigenic exotoxin production by isolates of Staphylococcus aureus from the Kawasaki syndrome patients and age-matched control children.

Nineteen strains of Staphylococcus aureus were isolated from the throat or the tooth surfaces of 19 cases amongst 127 patients with Kawasaki syndrome (KS) during the acute phases and 11 S. aureus isolates were obtained from five of 17 diseased controls and six healthy controls. The production of exotoxins, particularly superantigenic toxic shock syndrome toxin-1 (TSST-1), coagulase serotype, pigment production, haemolytic activity and tryptophan auxotrophy of these isolates were compared. Among 10 KS S. aureus strains isolated in 1990-1991, five (50%) secreted TSST-1, a higher frequency than two (18%) of 11 control isolates. In contrast, none of the nine KS strains collected in 1984 produced TSST-1. Four of five TSST-1-secreting KS strains produced white or white to golden pigmentation, whereas the two control strains capable of TSST-1 production formed golden colonies. There were no noticeable differences between S. aureus strains from KS patients and control children in the production of staphylococcal exotoxins A-E, coagulase serotype, haemolysis of sheep erythrocytes and tryptophan auxotrophy. The pathological or aetiological role of a new TSST-1-secreting S. aureus clone in patients with KS was not confirmed.

Staphylococcal scalded skin syndrome.

Staphylococcal scalded skin syndrome (SSSS) is a recognised clinical entity that affects primarily the very young and, in rare cases, the very old or the immunocompromised. Koch's postulates have been fulfilled in that: (i) Staphylococcus aureus is isolated from every case; (ii) S. aureus can reproduce the syndrome in an experimental animal model; (iii) a specific extracellular toxin can reproduce the syndrome; and (iv) antibody to the toxin can protect experimental animals. Although exfoliative toxin (ET) is responsible for the skin loosening seen in SSSS, it does not account for all the symptoms of the disease. Purified ET does not cause erythema in either neonatal mice or man, and the lesions are not painful unless the loosened epidermis is removed. This suggests that other factors, e.g., delta-haemolysin, are involved in the pathogenesis of this condition. Although much has been learned about the pathogenesis of the syndrome, we are still largely ignorant of the factors which govern host resistance to SSSS (i.e., intoxication by ET-producing strains of S. aureus). It is fortunate from the patient's point of view that the aetiological agent can be destroyed readily by the use of appropriate antibiotic therapy.

Beta-lactamase-negative, methicillin-resistant Staphylococcus aureus in a newborn nursery: report of an outbreak and laboratory investigations.

An outbreak of skin infection caused by a beta-lactamase-negative strain of methicillin-resistant Staphylococcus aureus (MRSA) occurred during a five-week period in a newborn nursery. Twelve babies, two mothers and two members of staff were involved. One baby had a diagnosis of staphylococcal scalded skin syndrome and two others required treatment with antibiotics. The infecting strain produced exfoliative toxin A. It was thought that it had been introduced from a different maternity unit by a nasal carrier. Laboratory investigations tended to support this hypothesis.

Outbreak of staphylococcal scalded skin syndrome among neonates.

Over a period of 2 months, 12 babies born in the maternity unit at Guy's Hospital developed staphylococcal scalded skin syndrome in two distinct outbreaks. Staphylococci isolated from the babies, together with those from the mothers and attending medical staff were phage-typed. All isolates from the babies were of type 3A/3C. During the first outbreak only one carrier of the epidemic strain (a paediatrician) was found but a further 12 persons were identified as possible carriers during the second outbreak. In order to confirm the link between outbreaks, all phage group II isolates were subjected to reverse phage-typing, testing for metal-ion resistance, plasmid profiling and in-vivo testing for production of epidermolytic toxin. It was shown that the same epidemic strain of toxin-producing Staphylococcus aureus was responsible for both outbreaks. The affected neonates responded rapidly to a short course of intravenous flucloxacillin. The outbreak ceased after appropriate treatment of all carriers and the implementation of an extensive disinfection policy within the maternity unit.

Cloning of the gene coding for Staphylococcus intermedius exfoliative toxin and its expression in Escherichia coli.

An exfoliative toxin (SIET)-producing strain (D-52) of Staphylococcus intermedius derived from canine pyoderma did not possess large plasmids. Therefore, the gene coding for SIET was considered to be located on the chromosomal DNA. The SIET gene was cloned from the chromosomal DNA of S. intermedius and was expressed in Escherichia coli. The nucleotide sequence of the SIET gene consists of a coding region of 990 bp specifying a polypeptide of 330 amino acid residues, which included a putative 42-residue signal sequence.

Phage conversion of exfoliative toxin A in Staphylococcus aureus isolated from cows with mastitis.

An exfoliative toxin produced by Staphylococcus aureus is the causative agent of staphylococcal scalded-skin syndrome (SSSS) in young children. Recently, we reported that only few isolates of S. aureus from bovine mastitis contained the eta gene encoding exfoliative toxin A (ETA) and produced ETA in vitro. In this study, we isolated temperate phages from two ETA-positive bovine isolates of S. aureus by treatment with mitomycin C. Polymerase chain reaction (PCR) assay of the phage genomes suggested that the temperate phages carried the structural gene for ETA. Moreover, the nucleotide sequence analysis of the PCR products revealed that the eta gene was located very close to an amidase gene on the phage genomes. The nucleotide sequence for the amidase gene of the bovine phage (bovine phi ETA) differed at nine positions from that of the amidase gene of phi ETA from a human isolate reported by Yamaguchi et al. [Mol. Microbiol. 38 (2000) 694], suggesting that eta-converting phages are heterogeneous. Bovine phi ETA had a head with a hexagonal outline and a non-contractile and flexible tail. Bovine phi ETA was able to lysogenize ETA-negative bovine isolates of S. aureus, and the lysogenized S. aureus isolates had the ability to produce ETA. These results suggest the possibility of horizontal transmission of the eta gene by temperate bacteriophages among bovine isolates of S. aureus.