Staphylococcus sciuri exfoliative toxin C is a dimer that modulates macrophage functions

Staphylococcus sciuri causes multiple infections in humans. Recently, a strain of S. sciuri (HBXX06) carrying exfoliative toxin C (ExhC) was reported to cause fatal exudative epidermal skin pathology in piglets and might be considered as a potential zoonotic agent. However, little is known about the pathogenicity of this bacterium. In this study, we examined the activity of recombinant ExhC-his (rExhC) protein using newborn mice as the model and investigated the effect of rExhC on macrophage functions. Interestingly, we found that both rExhC and S. sciuri ExhC existed as dimers and that rExhC inhibited the phagocytosis of RAW264.7 cell lines but enhanced the production of proinflammatory mediators, such as interleukin-6, interleukin-12, tumor necrosis factor α, and nitric oxide, by murine peritoneal macrophages and RAW264.7 cells. These results suggest that ExhC may play an important role in innate immune response against S. sciuri infection.     

A highly pathogenic strain of Staphylococcus sciuri caused fatal exudative epidermitis in piglets

Staphylococcus sciuri are important human pathogens responsible for endocarditis, peritonitis, septic shock, urinary tract infection, pelvic inflammatory disease and wound infections. However, little information is known regarding the pathogenicity of S. sciuri to animals. From the pericardial fluid of a diseased piglet with exudative epidermitis (EE), we isolated a strain of Staphylococcus in pure culture. Surprisingly, this isolate was a member of S. sciuri rather than S. hyicus as identified by its biochemical traits and also by analysis of 23S ribosomal DNA using Internal Transcribed Spacer PCR. In addition, inoculation of newborn piglets with 1x10(10) CFU of the isolate by oral feeding or intra-muscular injection successfully reproduced EE in piglets, which suggested that the oral intake of the pathogen by the animals is one of the major routes of exposure. These unexpected findings prioritized S. sciuri as important zoonotic agents, which may have ramifications for human medicine.     

Critical roles of amino acids Ser231, His107 and Asp156 of Staphylococcus sciuri exfoliative toxin C (ExhC) in the induction of skin exfoliations in neonate mice

Staphylococcus sciuri is a rare pathogen in humans, but it can cause a wide array of human infections. Recently a strain of S. sciuri (HBXX06) carrying exfoliative toxin C (ExhC) was reported to cause fatal exudative epidermitis in piglets and might be considered as a potential zoonotic agent. However, little is known regarding the pathogenicity of this bacterium. In this study, we predicted the three-dimensional structure of S. sciuri HBXX06 ExhC and replaced Ser231 or His107 or Asp156 in the active site of ExhC by site-directed mutagenesis, and examined the effects of mutant ExhC on BHK-21 cells and newborn mice as models. Interestingly, we found that mutant ExhC lost its exfoliative effects on newborn mice but could still induce necrosis in cultured cells if any one of the three amino acid residues in the active site was replaced. These results suggest that Ser231, His107 and Asp156 of ExhC play a critical role in the induction of skin exfoliation in neonate mice, which may help to further understand the mechanisms underlying the actions of exfoliative toxins.     

Panton-Valentine leukocidin does play a role in the early stage of Staphylococcus aureus skin infections: a rabbit model

Despite epidemiological data linking necrotizing skin infections with the production of Panton-Valentine leukocidin (PVL), the contribution of this toxin to the virulence of S. aureus has been highly discussed as a result of inconclusive results of in vivo studies. However, the majority of these results originate from experiments using mice, an animal species which neutrophils--the major target cells for PVL--are highly insensitive to the action of this leukocidin. In contrast, the rabbit neutrophils have been shown to be as sensitive to PVL action as human cells, making the rabbit a better experimental animal to explore the PVL role. In this study we examined whether PVL contributes to S. aureus pathogenicity by means of a rabbit skin infection model. The rabbits were injected intradermally with 10(8) cfu of either a PVL positive community-associated methicillin-resistant S. aureus isolate, its isogenic PVL knockout or a PVL complemented knockout strain, and the development of skin lesions was observed. While all strains induced skin infection, the wild type strain produced larger lesions and a higher degree of skin necrosis compared to the PVL knockout strain in the first week after the infection. The PVL expression in the rabbits was indirectly confirmed by a raise in the serum titer of anti-LukS-PV antibodies observed only in the rabbits infected with PVL positive strains. These results indicate that the rabbit model is more suitable for studying the role of PVL in staphylococcal diseases than other animal models. Further, they support the epidemiological link between PVL producing S. aureus strains and necrotizing skin infections.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.     

Local Inflammation Exacerbates the Severity of Staphylococcus aureus Skin Infection

Staphylococcus aureus is the leading cause of skin infections. In a mouse model of S. aureus skin infection, we found that lesion size did not correlate with bacterial burden. Athymic nude mice had smaller skin lesions that contained lower levels of myeloperoxidase, IL-17A, and CXCL1, compared with wild type mice, although there was no difference in bacterial burden. T cell deficiency did not explain the difference in lesion size, because TCR βδ (-/-) mice did not have smaller lesions, and adoptive transfer of congenic T cells into athymic nude mice prior to infection did not alter lesion size. The differences observed were specific to the skin, because mortality in a pneumonia model was not different between wild type and athymic nude mice. Thus, the clinical severity of S. aureus skin infection is driven by the inflammatory response to the bacteria, rather than bacterial burden, in a T cell independent manner.     

An outbreak of staphylococcal dermatitis in laboratory mice

An epizootic of dermatitis with erosion, ulcer and crust broke out in an experimental colony of JCL-ICR mouse over a period from December 1975 to June 1976. The disease was detected in 592 of a total of 1831 mice of 3-24 months old, especially in males of 7-24 months old (517/821). At the beginning of December 1975, only a few males of 12 months old were found to have the lesion on the back skin, and thereafter the dermatitis prevailed gradually among the mice. Histopathologic examinations showed the loss of the epidermis, necrosis and/or collapse of the corium, accumulation of serous exudate with neutrophilic cell infiltration and a few cocci scattered on the surface. In chronic cases, fibrous granulation tissues with neutrophilic cell infiltration were formed in the corium. Staphylococcus aureus was isolated in pure culture from the skin lesions in all of the mice examined. Skin disease similar to that of the field case was reproduced in mice inoculated subcutaneously with 10(7) viable organisms of the fresh isolate. By giving chlortetracycline in drinking water for 7 days, treatment of the affected mice was efficacious in mild cases, but not in severe cases.     

High-level (beta)-lactam resistance and cell wall synthesis catalyzed by the mecA homologue of Staphylococcus sciuri introduced into Staphylococcus aureus

A close homologue of mecA, the determinant of broad-spectrum beta-lactam resistance in Staphylococcus aureus was recently identified as a native gene in the animal commensal species Staphylococcus sciuri. Introduction of the mecA homologue from a methicillin-resistant strain of S. sciuri into a susceptible strain of S. aureus caused an increase in drug resistance and allowed continued growth and cell wall synthesis of the bacteria in the presence of high concentrations of antibiotic. We determined the muropeptide composition of the S. sciuri cell wall by using a combination of high-performance liquid chromatography, mass spectrometric analysis, and Edman degradation. Several major differences between the cell walls of S. aureus and S. sciuri were noted. The pentapeptide branches in S. sciuri were composed of one alanine and four glycine residues in contrast to the pentaglycine units in S. aureus. The S. sciuri wall but not the wall of S. aureus contained tri- and tetrapeptide units, suggesting the presence of dd- and ld-carboxypeptidase activity. Most interestingly, S. aureus carrying the S. sciuri mecA and growing in methicillin-containing medium produced a cell wall typical of S. aureus and not S. sciuri, in spite of the fact that wall synthesis under these conditions had an absolute dependence on the heterologous S. sciuri gene product. The protein product of the S. sciuri mecA can efficiently participate in cell wall biosynthesis and build a cell wall using the cell wall precursors characteristic of the S. aureus host.     

Intranasal vaccination with a double mutant of staphylococcal enterotoxin C provides protection against Staphylococcus aureus infection

Staphylococcus aureus expresses a repertoire of factors including staphylococcal exotoxins (SEs), exoenzymes, and numerous cell-associated components that contribute to the pathogenesis of disease. We constructed and expressed a nontoxic double mutant SEC (dmSEC), devoid of superantigenic activity, and investigated the ability of intranasal vaccination with dmSEC plus cholera toxin (CT) adjuvant to protect mice against S. aureus infection. Mice were vaccinated with dmSEC and inoculated with a viable S. aureus clinical isolate strain. The survival rate in the immunized mice was higher, and bacterial counts in the organs were significantly lower than those in the control group. Intranasal vaccination with dmSEC induced the production of SEC-specific antibodies such as IgG1, IgG2b and IgA. dmSEC-vaccinated mice elicited significantly higher titers of interleukin-4 (IL-4) and IL-10, and lower levels of interferon-gamma (IFN-gamma) after challenge with S. aureus compared with the control group. Furthermore, the sera from dmSEC-immunized mice significantly inhibited IFN-gamma and tumor necrosis factor-alpha production in vitro. These results indicate that intranasal vaccination with dmSEC devoid of superantigenic properties induces systemic immune responses and provides protection against S. aureus infection.     

Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein

The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing approximately 70% by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4-5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor(-/-) mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of "small proline rich proteins", and repetin, a member of the "fused gene" subgroup of the S100 gene family.     

A comparative evaluation of phenotypic and molecular methods in the identification of members of the Staphylococcus sciuri group

Members of the Staphylococcus sciuri group (S. sciuri, S. lentus, and S. vitulinus) are coagulase-negative, novobiocin-resistant staphylococci that could be distinguished from other staphylococci on the basis of positive oxidase activity. In the present study, a scheme based on conventional methods and utilization of various carbohydrates was evaluated for the identification of oxidase-positive staphylococci, and validated using two molecular techniques. Of the 173 oxidase-positive staphylococcal tested strains, 161 were identified as S. sciuri, 9 as S. lentus, 2 as S. vitulinus, and one as S. fleurettii by our scheme. The level of agreement with tRNA intergenic length polymorphism analysis (tDNA-PCR) was high (97.5-100% correlation). The accuracy and ease of use of this protocol suggest that it may be useful and valuable in microbiological laboratories for the identification of members of this group.     

Approximate dimensions of membrane lesions produced by streptolysin S and streptolysin O

Membrane lesions produced by the streptococcal membranolysins streptolysin S and streptolysin O were investigated. Escape of labeled marker molecules of various sizes from resealed sheep erythrocyte ghosts treated with the toxins for 30 min allowed estimation of the sizes of the primary channels formed. Streptolysin S formed lesions ranging in size up to 45 A in diameter, and even high toxin concentrations did not result in larger channels. The lesions produced by streptolysin O exceeded 128 A in diameter. Kinetics experiments demonstrated that the primary streptolysin O lesions were formed rapidly (1-2 min), but release of marker molecules from streptolysin S-treated vesicles began only after a 5-15-min lag period. Label release from large unilamellar liposomes treated with streptolysin S suggested that membrane fluidity does not affect the size of the streptolysin S lesions.     

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 °C, subsequently displayed a high thermotolerance, being able to grow at 42 °C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.     

Developmental cell death in the liver and newborn lethality of Ku86 deficient mice suppressed by antioxidant N-acetyl-cysteine

Repair of DNA double-strand breaks (DSBs) is essential for genome integrity and cell survival. Ku86 is involved in the repair of DNA DSBs by non-homologous end joining (NHEJ). Mice deficient in Ku86 show growth retardation, dwarfism, premature aging, and immunodeficiency. In this study, we observed severely compromised survival of Ku86(-/-) mice, such that most Ku86(-/-) mice died within the first postnatal weeks and only 1.5% of the expected 25% from heterozygous crosses survived for 1 month. Since post-mortem analysis was not possible due to parental cannibalism, histopathological examination was performed on Ku86(-/-) fetuses to assess possible causes of newborn death. Eighty percent and 75% of Ku86(-/-) fetuses exhibited apoptosis and necrosis in the liver, while only 20% and 10% of Ku86(+/+) littermates had apoptosis and necrosis, respectively. In addition, the severity of liver damage was significantly higher in Ku86(-/-) fetuses. Developmental liver damage may have led to postnatal lethality because the fetal liver with pre-existing injury may not be able to undergo transformation from a lymphohematopoietic to an indispensable metabolic organ. Free radicals can cause chromosomal breaks and lead to cell death. We postulated that endogenous oxidative stress might be involved in the resulting liver damage and animal lethality in Ku86(-/-) mice deficient in DNA DSB repair. This hypothesis was tested by treating Ku86(-/-) mice with the well known free radical scavenger, thiol antioxidant N-acetyl-cysteine (NAC), during embryonic development. We found that a significantly higher percentage, 7.7% of NAC treated Ku86(-/-) offspring versus 1.5% untreated Ku86(-/-) mice were alive at 1 month of age. In addition, the incidence of liver necrosis decreased by 21% and the severity of necrosis significantly reduced. Thus, Ku86 deficiency results in severe developmental liver damage and newborn lethality associated with oxidative stress.     

Production of a Host-Specific Toxin by Germinating Spores of Alternaria tenuissima Causing Leaf Spot of Pigeon Pea

Production of a host-specific toxin by Alternaria tenuissima , the cause of pigeon pea leaf spot, was investigated in spore-germination fluids (SGF). The SGF selectively induced necrosis on pigeon pea leaves in a deteched leaf assay. Necrotic lesions were observed when a toxin from SGF was applied onto detached young leaves of the pigeon pea cultivar Bahar at concentration as low as 5 ng/ml. The resistant line Tanzania and nonhosts tolerated at least 20,000 times higher concentration of the toxin. The differential activity of the toxin on hosts and nonhosts of the fungus, as well as on susceptible and resistant cultivars or lines, suggested host-specific property of the toxin. At a concentration of 10 ng/ml, the toxin induced susceptibility of pigeon pea leaves to a non-pathogenic isolate of Alternaria alternata. The toxin possibly plays a role as a disease determinant of A. tenuissima , because the toxin was released from germinating spores as early as 3 h of incubation andthe, amount detected within 9 h was about 6 times of the concentration required for necrotic toxicity.     

复合生物保鲜剂对松鼠葡萄球菌的抑菌性能及其作用机理研究

以冷藏带鱼中分离出的革兰氏阳性优势菌——松鼠葡萄球菌(Staphylococcus sciuri)为试验菌,研究复合生物保鲜剂(配比浓度为:壳聚糖10.0 g/L,溶菌酶0.3 g/L与茶多酚3.0 g/L)对松鼠葡萄球菌的抑菌效果与作用机理。通过牛津杯法确定复合保鲜剂对松鼠葡萄球菌的最小抑菌浓度(MIC)与最小杀菌浓度(MBC),结合抑菌活力、细菌生长曲线、碱性磷酸酶(AKP)活性、细胞膜完整性、膜通透性与细菌超微结构观察等,综合评价不同浓度复合保鲜剂在不同处理时间内对松鼠葡萄球菌的作用效果。结果表明,复合保鲜剂对松鼠葡萄球菌的MIC与MBC分别为0.8与1.6 mg/mL,随着处理时间的延长,复合生物保鲜剂明显抑制松鼠葡萄球菌的生长,使菌体细胞外的AKP量增多,造成细菌菌体细胞壁通透性增大,细胞结构的完整性受到破坏,菌液电导率值显著升高,菌体电解质等内容物外泄,影响细胞内环境和细胞膜的稳定性,菌体皱缩变形,表面粗糙,细胞壁塌陷,细胞质外泄渗出,导致菌体死亡。     

A comparison of biodegradation of phenol and homologous compounds by Pseudomonas vesicularis and Staphylococcus sciuri strains

Pseudomonas vesicularis and Staphylococcus sciuri were isolated as dominant strains from phenol-acclimated activated sludge. P. vesicularis was an efficient degrader of phenol, catechol, p-cresol, sodium benzoate and sodium salicylate in a single substrate system. Under similar conditions S. sciuri degraded only phenol and catechol from among aromatic compounds that were tested. Cell-free extracts of P. vesicularis grown on phenol (376 mg l(-1)), sodium benzoate (576 mg l(-1)) and sodium salicylate (640 mg l(-1)) showed catechol 2,3-dioxygenase activity initiating an extradiol (meta) splitting pathway. The degradative intradiol (ortho) pathway as a result of catechol 1,2-dioxygenase synthesis was induced in P. vesicularis cells grown on catechol (440 mg l(-1)) orp-cresol (432 mg l(-1)). Catechol 1,2-dioxygenase and the ortho-cleavage has been also reported in S. sciuri cells capable of degrading phenol (376 mg l(-1)) or catechol (440 mg l(-1)). In cell-free extracts of S. sciuri no meta-cleavage enzyme activity was detected. These results demonstrated that gram-positive S. sciuri strain was able to effectively metabolize some phenols as do many bacteria of the genus Pseudomonas but have a different capacity for degrading of these compounds.     

Heat-labile toxin-producing isolates of Pasteurella multocida from rabbits.

Five of one hundred forty seven isolates of Pasteurella multocida from rabbits were found to produce heat-labile toxin. Each isolate was assayed for the ability of potassium thiocyanate (KSCN) extracts to cause dermonecrosis in guinea pig skin, ability of bacteria or filtrates to cause cytotoxicity in cell cultures, and reactivity with monoclonal antibodies to heat-labile P. multocida toxin. Five capsular type D isolates produced dermonecrosis and reacted with monoclonal antibodies to toxin. Filtrates of all five of these isolates were cytotoxic for cell cultures. Potassium thiocyanate extracts of all five isolates caused pleuritis and pneumonia in rabbits after intranasal inoculation. Turbinate atrophy was seen in 5 of 19 rabbits inoculated intranasally with toxic extracts. Heat-labile toxin was not produced by 109 capsular type A isolates or 19 nontypable isolates.     

Coagulase-Negative,Novobiocin-Resistant Staphylococci on the Skin of Animals and Man,on Meat and in Milk

The occurrence of coagulase-negative, novobiocin-resistant staphylococci, i.e. Staphylococcus cohnii, Staphylococcus saprophyticus, Staphylococcus sciuri and Staphylococcus xylosus, on the skin of animals and man has been studied. On cultures from cats, cows, dogs, guinea pigs, mice, rabbits and sheep studied, such organisms were predominant among the coagulase-negative staphylococci. From the skin of the hands of 21 of 38 persons whose professions brought them into contact with animals, e.g. inséminât ors, slaughterhouse workers and veterinarians, coagulase-negative, novobiocin-resistant staphylococci were isolated. This finding contrasted with that regarding 50 persons lacking such contacts, of whom only 1 harboured such bacteria. S. saprophyticus was isolated only from those slaughterers presenting with wounds on their hands. Coagulase-negative, novobiocin-resistant staphylococci were also isolated from every second specimen collected from the surface of meat at a slaughterhouse. No difference in the culture results could be demonstrated from specimens collected before and after cutting-up of the carcasses. Of 26 strains of coagulase-negative, DNase-negative staphylococci isolated from milk with pathological CMT, all but 5 were novobiocin-resistant. Fifteen were classified as S. xylosus, 4 as S. sciuri and 1 as S. cohnii. Of another 15 DNase-positive strains, 3 were resistant to novobiocin. Finally, clinical infections with coagulase-negative, novobiocin-resistant staphylococci in man, e.g. urinary tract infections caused by S. saprophyticus, are considered in relation to possible contagious reservoirs and modes of spread.