Crystal structure of exfoliative toxin D from Staphylococcus aureus.

Staphylococcus aureus exfoliative toxins (ETs) are serine proteases responsible for staphylococcal scalded skin syndrome. Four ETs, ETA, ETB, ETD, and ETE, have been identified, all of which cleave desmoglein-1. This study presents the crystal structure of ETD at 1.75 Å resolution. The protein exhibits a structure composed of two ß-barrels and two α-helices as described in previous studies of ETs. A predicted model of ETD in complex with Ile380-Glu381-Gly382-Pro383 (IEGP), a segment of human desmoglein-1 (hDsg1), was constructed. Glu381 of hDsg1 was predicted to interact with as many as six amino acid residues in ETD, whereas two amino acid residues in ETD primarily constituted subsite S1', and a space near subsite S1' was noted. It is likely that polypeptide chains located near the IEGP segment in the predicted structure of hDsg1 bind to this space. The structure of loop D, which was predicted to participate in subsite S2', in ETD was markedly different from those in other ETs.

Virulence and host specificity of staphylococci from Staphylococcus intermedius group of pigeon origin with an emphasis on Staphylococcus intermedius.

The Staphylococcus intermedius group (SIG) includes coagulase-positive staphylococci commonly found in animals. The taxonomic classification within the SIG has evolved with molecular techniques distinguishing five species. Despite their similarities, these species exhibit varied host affinities, with unclear implications for virulence and host interaction. This study aimed to investigate the presence of coagulase-positive staphylococci in pigeons and to detect genes encoding for selected virulence factors in isolated strains. Another goal was to determine the adhesion capabilities of randomly selected pigeon S. intermedius, S. delphini, and canine S. pseudintermedius strains to canine and pigeon corneocytes and their adhesion and invasion abilities to canine keratinocytes in vitro. In total, 121 coagulase-positive strains were isolated from domestic and feral pigeons. The most prevalent species were S. delphini B and S. intermedius in domestic and feral pigeons, respectively. We proved that pigeon strains carried genes encoding for exfoliative toxin SIET and leukotoxin Luk-I. Moreover, we found that S. intermedius showed higher adherence to pigeon than to canine corneocytes, aligning with its presumed natural host. No difference in adherence abilities of S. pseudintermedius to canine and pigeon corneocytes was observed. In this study, we also observed that S. pseudintermedius could successfully invade the canine keratinocytes, in contrary to S. delphini and S. intermedius. Moreover, only S. intermedius was not able to invade canine keratinocytes at all. These findings highlight the complex interplay between SIG bacteria, and their hosts, underscoring the need for further research to understand the mechanisms of host adaptation and pathogenicity within this group.

Staphylococcus aureus Exfoliative Toxin E, Oligomeric State and Flip of P186: Implications for Its Action Mechanism.

Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.

Antibiotic susceptibility of human-associated Staphylococcus aureus and its relation to agr typing, virulence genes, and biofilm formation.

BACKGROUND AND OBJECTIVE: Carriage of virulence factors confers some evolutionary benefit to bacteria, which favors the resistant strains. We aimed to analyze whether antibiotic susceptibility of Staphylococcus aureus strains is affected by agr typing, biofilm formation ability, and virulence profiles. METHODS: A total of 123 S. aureus clinical isolates were subjected to antimicrobial susceptibility testing by disk diffusion method, biofilm formation by microtiter plate method, as well as polymerase chain reaction screening to identify virulence genes and the accessory gene regulator (agr) types I-IV. A P value < 0.05 was considered significant. RESULTS: The most prevalent virulence gene was staphyloxanthin crtN, followed by hemolysin genes, capsular cap8H, toxic shock toxin tst, and enterotoxin sea, respectively. Resistant isolates were more commonly found in the agr-negative group than in the agr-positive group. Isolates of agr type III were more virulent than agr I isolates. Strong biofilm producers showed more antibiotic susceptibility and carried more virulence genes than non-strong biofilm producers. Associations were found between the presence of virulence genes and susceptibility to antibiotics. Carriage of the virulence genes and agr was higher in the inpatients; while, resistance and strong biofilms were more prevalent in the outpatients. CONCLUSION: These findings indicated the presence of several virulence factors, biofilm production capacity, agr types and resistance to antibiotics in clinical S. aureus isolates. Considering the importance of S. aureus for human medicine, an understanding of virulence and resistance relationships would help to reduce the impact of S. aureus infections.

Antimicrobial Resistance and Molecular Analysis of Staphylococcus aureus in Staphylococcal Scalded Skin Syndrome among Children in Korea.

BACKGROUND: Staphylococcal scalded skin syndrome (SSSS) is a skin disease characterized by blistering and desquamation caused by exfoliative toxins (ETs) of Staphylococcus aureus (S. aureus). Although many countries show predominance of methicillin-susceptible S. aureus (MSSA), cases of methicillin-resistant S. aureus (MRSA) have been reported. METHODS: Twenty-six children aged <15 years diagnosed with SSSS from January 2010 to December 2017 from three hospitals were included. S. aureus isolates from cases were analyzed for multilocus sequence types and ETs. Medical records were reviewed for clinical characteristics, treatment, and antimicrobial susceptibility patterns of S. aureus. RESULTS: Among the 26 cases, mean age was 2.3 years. According to skin manifestations patients were classified as generalized (n = 10, 38.5%), intermediate (n = 11, 42.3%), and abortive (n = 5, 19.2%). Among all cases, 96.2% (25/26) were due to MRSA and the macrolide-resistance rate was 92.3% (24/26). ST89 (n = 21, 80.8%) was the most prevalent clone, followed by single clones of ST1, ST5, ST72, ST121, and ST1507. The eta gene was detected in one (3.8%) isolate which was MSSA. The etb gene was detected in 14 (53.8%) isolates, all of which were ST89. Nafcillin or first-generation cephalosporin was most commonly prescribed (n=20, 76.9%). Vancomycin was administered in four patients (15.4%) and clindamycin in nine patients (34.6%). Among MRSA cases, there was no difference in duration of treatment when comparing the use of antimicrobials to which the causative bacteria were susceptible or non-susceptible (9.75 vs. 8.07 days, P > 0.05). CONCLUSION: S. aureus isolated from children with SSSS in Korea demonstrated a high prevalence of methicillin-resistant ST89 clones that harbored the etb gene. The predominance of MRSA suggests that antibiotics to which MRSA are susceptible may be considered for empirical antibiotic treatment in children with SSSS in Korea. Further studies on the role and effectiveness of systemic antibiotics in SSSS are warranted.

Skin Infections Caused by Staphylococcus aureus.

Staphylococcus aureus is the most common pathogen involved in skin infections worldwide, regardless of the patient's age, the climate or geographical area. The main skin clinical manifestations can be linked to a few toxins produced by the bacteria, which give rise to a rich and varied clinical spectrum. Panton Valentine leucocidin, exfoliatins, enterotoxins and toxin shock syndrome toxin 1 are the main toxins involved in most dermatological manifestations associated with S. aureus. Other less frequent cutaneous manifestations can occur in endocarditis, bacteraemia. Currently, the most important event is worldwide emergence of community-acquired S. aureus resistant to methicillin (CA-MRSA), mainly causing skin infections.

Characterization of Staphylococcus aureus from Pasteur Institute in Côte d'Ivoire: Methicillin Resistance, Reduced Sensitivity to Vancomycin, Panton-Valentine Leucotoxin and Exfoliatins.

Staphylococcus aureus, which causes various infections, particularly suppurations, expresses many virulence factors. The resistance of S. aureus to methicillin (MRSA) which can spread to vancomycin constitutes a major challenge in infectiology. The search for virulence and resistance factors is therefore of interest to better understand the mechanisms of this pathogenicity. The objectives of this study were to determine the frequency of phenotypic and genotypic (mecA, vanB) resistances, the frequency of virulence genes (eta, etb, and lukS) and to investigate the resistant strains for the presence of virulence genes. On thirty-one strains isolated from infections at the Pasteur Institute of Côte d'Ivoire, the study of susceptibility to methicillin and vancomycin was carried out by phenotypic and molecular methods. We observed phenotypic and genotypic resistance to methicillin of 41.9% and 32.3% respectively. Despite a suspicion of very high vancomycin susceptibility reduced, 25.8% by phenotypic method, the vanB gene was only found in 3.2% of strains. The prevalence of virulence genes was high with the eta gene, 96.8%, and the lukS gene 45.2%. The mecA gene was present with an eta gene in 32.3% of strains and in 9.7% with the lukS gene, however the vanB gene was not present in any strain carrying virulence factors. These results should lead to the screening of other van genes for resistance to vancomycin.

The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function.

Bacterial superantigens (SAgs) cause Vß-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vß-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis.

Frequency of superantigen encoding genes of Staphylococcus aureus isolates collected from multiple sclerosis (MS) patients and nasal carriers.

BACKGROUND: Bacterial superantigens are potent T cell activators that can have acute or chronic effects on the central nervous system. OBJECTIVES: In this study, the role of enterotoxins, exfoliative toxins and toxic shock syndrome toxin of Staphylococcus aureus was investigated in MS patients and healthy nasal carriers. METHODS: Three-hundred fifty nasal swabs were collected from healthy nasal carriers (n = 210) and MS (n = 140) patients. Staphylococcus aureus superantigens were detected by multiplex PCR. Antimicrobial susceptibility pattern was performed using disk diffusion method. RESULTS: The highest rates of nasal colonization were seen in MS patients (46.42%). The rates of nasal colonization in the healthcare workers were 30.95%. The most commonly detected superantigens were SEA (31.5%), SEB (17.7%) and ETA (16.9%). The Staphylococcus aureus isolates had the highest levels of resistance against erythromycin (57.7%), clindamycin (55.4%) and co-trimoxazole (43.1%). All isolates were susceptible to vancomycin, linezolid, and mupirocin. CONCLUSION: Our results revealed that the frequency of superantigen producing Staphylococcus aureus isolates is high in the MS patients. As well as these isolates are sensitive to mupirocin. Thus it is better to use of mupirocin for nasal decolonization of Staphylococcus aureus in the MS patients.

An outbreak of skin infections in neonates due to a Staphylococcus aureus strain producing the exfoliative toxin A.

PURPOSE: Staphylococcus aureus is an important cause of infections in hospitalized neonates. Preterm or low birthweight infants are especially at risk to develop a S. aureus infection due to the immaturity of the immune system, length of hospital stay and invasive procedures. Exfoliative toxin (ET)-producing S. aureus is often responsible for neonatal infections, causing clinical manifestations such as staphylococcal scalded skin syndrome, characterized by both localized blisters or generalized exfoliation of the skin. METHODS: We describe an outbreak due to an S. aureus strain producing ETA occurring in a local hospital in Northern Italy. Molecular typing of the isolates included spa typing and multilocus sequence typing. DNA microarray hybridization was also performed on one representative strain. RESULTS: In the period from July 2013 to February 2014, 12 neonates presented with skin infections, mainly bullae or pustules. Cultures of skin swabs yielded methicillin-susceptible S. aureus (MSSA). By molecular typing, an epidemic strain (t1393/ST5) was identified in nine neonates; microarray analysis and PCR revealed that it contained the ETA encoding gene. Screening of staff, mothers and healthy neonates and environmental cultures did not reveal the presence of the epidemic strain. However, the father of an infected neonate was found to be a carrier of MSSA t1393 five months after the outbreak started. CONCLUSION: Implementation of hygiene procedures and sanitization of the ward twice terminated the outbreak. Timely surveillance of infections, supported by molecular typing, is fundamental to prevent similar episodes among neonates.

Two highly divergent lineages of exfoliative toxin B-encoding plasmids revealed in impetigo strains of Staphylococcus aureus.

Exfoliative toxin B (ETB) encoded by some large plasmids plays a crucial role in epidermolytic diseases caused by Staphylococcus aureus. We have found as yet unknown types of etb gene-positive plasmids isolated from a set of impetigo strains implicated in outbreaks of pemphigus neonatorum in Czech maternity hospitals. Plasmids from the strains of clonal complex CC121 were related to archetypal plasmid pETBTY4. Sharing a 33-kb core sequence including virulence genes for ETB, EDIN C, and lantibiotics, they were assigned to a stand-alone lineage, named pETBTY4-based plasmids. Differing from each other in the content of variable DNA regions, they formed four sequence types. In addition to them, a novel unique plasmid pETB608 isolated from a strain of ST130 was described. Carrying conjugative cluster genes, as well as new variants of etb and edinA genes, pETB608 could be regarded as a source of a new lineage of ETB plasmids. We have designed a helpful detection assay, which facilitates the precise identification of the all described types of ETB plasmids.

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.

Antibiotic susceptibility and genomic variations in Staphylococcus aureus associated with Skin and Soft Tissue Infection (SSTI) disease groups.

BACKGROUND: Staphylococcus aureus is associated with human skin and soft tissue infections (SSTIs); however, the involvement of virulence factors in different clinical presentations is unclear. METHODS: We analyzed methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains from Taiwan to determine correlations among the clinical characteristics of SSTIs, antimicrobial susceptibility and virulence factors of S. aureus with specific genetic backgrounds. RESULTS: We identified 177 MRSA isolates and 130 MSSA isolates among the 307 SSTI-associated S. aureus isolates. Hospital-acquired (HA)- and community-acquired (CA)-MRSA isolates accounted for 61.6 % and 38.4 % of the isolates, respectively. Clinical presentations in SSTI patients differed significantly for the disease groups. Deep-seated MRSA infections presented with higher amputation rate than MSSA infections. MRSA isolates were all susceptible to linezolid, teicoplanin, and vancomycin, and >94 % of isolates were erythromycin- and clindamycin-resistant. Staphylococcal cassette chromosome (SCCmec) types IV, V, and VII were the most frequent in the CA-MRSA group (n = 68); types III, IV and V were the most frequent in the HA-MRSA group (n = 109). Panton-Valentine leukocidin (PVL) genes were significantly more frequent in CA-MRSA strains (75.0 %) than in HA-MRSA (33.0 %) and MSSA (24.6 %) and were found in 66.7 % (74/111) strains isolated from the abscess group. Exfoliatin A genes were more common in catheter-related exit-site MSSA infections (37.5 %) compared with other MSSA disease groups (P < 0.05). Exfoliatin B and superantigen exotoxin genes were uncommon in all SSTI disease types. Pulsotypes A (ST239), C, and D (ST59) were the predominant MRSA genotypes in deep-seated infections. CONCLUSIONS: If not treated appropriately, deep-seated MRSA-associated infections present with higher amputation rates than deep-seated MSSA-associated infections. PVL-positive MRSA strains caused more frequently pus-forming lesions and less bacteremia and invasive diseases. Methods for discriminating CA-MRSA from HA-MRSA strains are now unreliable due to circulation of both ST 239 and ST 59 strains in the community and nosocomial settings. Initial antibiotic treatments should consider MRSA for patients with SSTIs in areas where MRSA is prevalent.

Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: Structural basis for the high specificity of exfoliative toxins.

Exfoliative toxins are serine proteases secreted by Staphylococcus aureus that are associated with toxin-mediated staphylococcal syndromes. To date, four different serotypes of exfoliative toxins have been identified and 3 of them (ETA, ETB, and ETD) are linked to human infection. Among these toxins, only the ETD structure remained unknown, limiting our understanding of the structural determinants for the functional differentiation between these toxins. We recently identified an ETD-like protein associated to S. aureus strains involved in mild mastitis in sheep. The crystal structure of this ETD-like protein was determined at 1.95 Å resolution and the structural analysis provide insights into the oligomerization, stability and specificity and enabled a comprehensive structural comparison with ETA and ETB. Despite the highly conserved molecular architecture, significant differences in the composition of the loops and in both the N- and C-terminal α-helices seem to define ETD-like specificity. Molecular dynamics simulations indicate that these regions defining ET specificity present different degrees of flexibility and may undergo conformational changes upon substrate recognition and binding. DLS and AUC experiments indicated that the ETD-like is monomeric in solution whereas it is present as a dimer in the asymmetric unit indicating that oligomerization is not related to functional differentiation among these toxins. Differential scanning calorimetry and circular dichroism assays demonstrated an endothermic transition centered at 52 °C, and an exothermic aggregation in temperatures up to 64 °C. All these together provide insights about the mode of action of a toxin often secreted in syndromes that are not associated with either ETA or ETB.

The distribution of pathogenic and toxigenic genes among MRSA and MSSA clinical isolates.

Staphylococcus aureus (S. aureus) is considered as a notorious nosocomial pathogen among hospitalized patients and community-dwelling subjects. Its increasing morbidity and mortality is believed to be due to antibiotic resistance. However, the data concerning molecular properties of infecting strains are few. In this study, a total of 192 S. aureus strains, including 88 (45.8%) meticillin-sensitive S. aureus (MSSA) and 104 (54.2%) meticillin-resistant S. aureus (MRSA) were recovered from clinical samples. The prevalence of subtypes containing staphylococcal cassette chromosome mec (SSCmec), staphylococcal enterotoxins (SEs), toxic shock syndrome toxin (TSST) and exfoliative toxin was assessed by PCR. Antibiotic susceptibility pattern and vancomycin resistance of each isolate were evaluated by disk diffusion method and micro-dilution method, respectively. 9 (2.3%) strains required MIC > 2 mg/l of vancomycin, which significantly increased among multi drug resistant (MDR), MRSA and SCCmec type III strains (p < 0.05). 171 (89%), 140 (72.91%), 7 (3.6), 78 (48.6%), 5 (2.6%), 151 (78.64%), 129 (67.18%), 178 (92.7%) and 15 (7.8%) of 192 isolates harbored mecA, entA, entB, entC, entD, entE, eta, etb and tsst-1 genes, respectively. 31 (16.14%), 5 (2.6%), 95 (49.48%) and 7 (3.64%) of 192 isolates carried SCCmec type I, II, III and IV, respectively. We found a significantly higher rate of MRSA and resistance to all tested antibiotics, except to penicillin G, kanamycin and linezolide among the SCCmec type III class (p < 0.05). According to our findings, MSSA isolates should be taken as seriously as MRSA strains due to the potential presence of broad spectrum virulence factor genes.

Complete genome analysis of two new bacteriophages isolated from impetigo strains of Staphylococcus aureus.

Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated ϕB166 and ϕB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

Exfoliative toxin A staphylococcal scalded skin syndrome in preterm infants.

UNLABELLED: Staphylococcal scalded skin syndrome (SSSS) demonstrates dermal symptoms due to exfoliative toxin (ET) A or ETB produced by Staphylococcus aureus. We examined the association between anti-ETA antibodies and SSSS onset in neonates. Three preterm infants carried an ETA-producing strain of S. aureus, manifesting as either SSSS or bullous impetigo; a full-term infant carrying the same strain was asymptomatic. The infants (n=106) were categorized into three groups according to their gestational age (GA) as follows: <30 weeks, 30-37 weeks, and >37 weeks. The measured levels of anti-ETA antibody in the three infants displaying SSSS were low before the onset of dermal symptoms; only the asymptomatic full-term infant displayed a high antibody level. Anti-ETA antibody levels in the preterm group with a GA of <30 weeks were statistically lower than those in the term infant group; the prevalences of anti-ETA antibodies above a cutoff value in the three groups of neonates were 55 % (18/33) among preterm infants with a GA <30 weeks, 73 % (25/34) among those with a GA of 30-37 weeks, and 90 % (35/39) among infants with a GA >37 weeks. CONCLUSION: The presence of anti-ETA antibodies below a particular cutoff level might be associated with SSSS onset in preterm infants.

[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.

Necrotic activity of ExhC from Mammaliicoccus sciuri is mediated by specific amino acid residues.

Mammaliicoccus sciuri, a commensal and pathogenic bacterium of significant clinical and veterinary relevance, expresses exfoliative toxin C (ExhC), a specific glutamyl endopeptidase belonging to the chymotrypsin family as the principal virulence factor. However, unlike most members of this family, ETs are inactive against a wide range of substrates and possess exquisite specificity for desmoglein-1 (Dsg1), a cadherin-like adhesion molecule that is crucial to maintain tissue integrity, thereby preventing the separation of skin cells and the entry of pathogens. ExhC is of clinical importance since in addition to causing exfoliation in pigs and mice, it induces necrosis in multiple mammalian cell lines, a property not observed for other ETs. Previous experiments have implicated the ExhC79-128 fragment in causing necrosis. Site-directed mutagenesis of specific residues within this fragment were studied and led to the design of an ExhC variant containing four-point mutations (ExhCmut4) lacking necrotic potential but retaining nearly wild-type (wt) levels of enzymatic activity. Moreover, the determination of the ExhCwt and ExhCmut4 crystal structures identified the conformation in the necrosis-linked region. These results constitute an important step toward the understanding of the mechanisms underlying the necrotic and epidermolytic activity of ExhC.

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.