Polymerase chain reaction compared to other laboratory findings and to clinical evaluation in the diagnosis of cutaneous tuberculosis and atypical mycobacteria skin infection

Cutaneous tuberculosis has re-emerged in the last 15 years together with the higher incidence of pulmonary tuberculosis and multidrug resistance. The choice for a single diagnostic tool among the many available today is a challenge. Our objective was to compare polymerase chain reaction (PCR) with other exams in the diagnosis of cutaneous tuberculosis and atypical mycobacteria skin infection. PCR and a set of five different exams were performed in 32 patients (34 samples of paraffin-embedded tissue) evaluated for 3 years in a university hospital, considering the response to mycobacterial infection treatment as a positive case. PCR was the most sensitive (88%) and specific (83%) exam. Culture, immunohistochemistry and acid-fast bacilli were not in agreement with clinical response to treatment. Although PCR is a useful tool, careful clinical exam is still the gold standard for the evaluation and treatment of cutaneous tuberculosis and mycobacteria skin infection.

Skin infection caused by Scedosporium apiospermum in immunocompromised patients. Report of two cases.

Scedosporium apiospermum is a filamentous fungus that can cause cutaneous or extracutaneous disease. A large number of cases have been published over the last decades, mainly in patients immunocompromised as a result of their disease or treatment. These kinds of infections can progress rapidly and become disseminated, leading to very serious or even fatal complications. We report two new cases of skin infection by Scedosporium apiospermum from our hospital.

Restoration of Pattern Recognition Receptor Costimulation to Treat Chromoblastomycosis, a Chronic Fungal Infection of the Skin

Chromoblastomycosis is a chronic skin infection caused by the fungus Fonsecaea pedrosoi. Exploring the reasons underlying the chronic nature of F. pedrosoi infection in a murine model of chromoblastomycosis, we find that chronicity develops due to a lack of pattern recognition receptor (PRR) costimulation. F. pedrosoi was recognized primarily by C-type lectin receptors (CLRs), but not by Toll-like receptors (TLRs), which resulted in the defective induction of proinflammatory cytokines. Inflammatory responses to F. pedrosoi could be reinstated by TLR costimulation, but also required the CLR Mincle and signaling via the Syk/CARD9 pathway. Importantly, exogenously administering TLR ligands helped clear F. pedrosoi infection in vivo. These results demonstrate how a failure in innate recognition can result in chronic infection, highlight the importance of coordinated PRR signaling, and provide proof of the principle that exogenously applied PRR agonists can be used therapeutically.

Schistosoma mansoni: comparative evaluation of different routes of experimental infection

Experiments were carried out with Sw albino mice and it was concluded that the percutaneous route via abdominal skin was significatively more efficient than tail immersion method and subcutaneous infection; the subcutaneous injection was significatively more efficient than the percutaneous infection through the tail; this latter and the intraperitoneal injection, resulted in similar infections, but were significatively less efficient than the others. Significative difference was also observed in the comparison between the subcutaneous route and percutaneous infection through ear pinna. The influence of the site of skin infection by percutaneous route was also discussed.

Rhodococcus equi infection after reduction mammaplasty in an immunocompetent patient

The majority of infections caused by R. equi occur in hosts with some degree of cell-mediated immunodeficiency. Immunocompetent individuals are infrequently affected and usually present with localized disease. Infections of the skin or related structures are uncommon and are usually related to environmental contamination. The microbiology laboratory plays a key role in the identification of the organism since it may be mistaken for common skin flora. We describe a 31 year-old woman without medical problems who presented nine weeks after breast reduction with right breast cellulitis and purulent drainage from the surgical wound. She underwent incision and drainage, and cultures of the wound yielded Rhodococcus equi. The patient completed six weeks of antimicrobial therapy with moxifloxacin and rifampin with complete resolution.

Low and high-dose intradermal infection with Leishmania majorand Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensiswith low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 10³ or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 10³ parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensisdisplayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensisin the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.

Evaluation of the immune response to infection with metastatic and non-metastatic "Leishmania guyanensis" parasites

ABSTRACT : Les infections par le parasite Leishmania guyanensis se caractérisent par une dissémination depuis le site initial d'infection jusqu'aux tissus naso-pharyngés, responsable de la Leishmaniose à lésions secondaires muco-cutanées (LMC). Les lésions des patients atteints de LMC montrent une massive infiltration de cellules immunitaires, une réponse immunitaire élevée et la présence de parasites (bien qu'en très faible quantité). La LMC engendre une augmentation de l'expression de TNFa ainsi qu'un défaut dans le contrôle de la réponse immunitaire caractérisé par une absence de réponse à l'IL 10. La réponse immunitaire de l'hôte ainsi que la virulence du parasite sont deux facteurs reconnus pour le contrôle de l'infection. Le mécanisme de la pathogenèse de la LMC restent grandement incompris, surtout le mécanisme de dissémination de l'infection du site d'inoculation jusqu'aux sites secondaires d'infection (métastases) ainsi que les détails de la réponse de l'hôte contre le pathogène. Dans un modèle d'infection d' hamsters avec des parasites du Nouveau Monde, la classification des parasites Leishmania se fait en fonction de leur capacité à développer des métastases. Ce modéle d'infection a permis de caractériser différentes souches de parasites selon la classification de l'Organisation Mondiale de la Sante (OMS) tel que la souche de référence W>É-II/BR/78/M5313 qui est reconnue comme hautement métastatique alors que ces clones dérivés de M5313 montrent de grandes variations quand a leur capacité à créer des métastases. Les clones 13 et 21 sont métastatiques (M+) alors que les clones 3 et 17 sont nonmétastatiques (NI-). Les objectifs de cette thèse ont été d'étudier le rôle de la réponse immunitaire innée des macrophages après infection in vitro avec différents clones métastatiques et non-métastatiques du parasite L. guyanensis, ainsi que d'étudier la réponse immunitaire générée suite à une infection in vivo par les clones M+ et M- de L. guyanensis dans un modèle marin. L'analyse de la .réponse immunitaire des macrophages in vitro montrent qu'il y aune augmentation significative de leur statut d'activation après infection par des parasites M+ indiquée par la modulation des marqueurs d'activation de surface CD80, CD86 et CD40, ainsi que une augmentation significative de CXCL 10, CCLS, IL6 et TNFa au niveau transcription de l'ARNm et au niveau de la protéine. Cette phénomène d'activation a été observée chez les deux souches de souris C57BL/6 et BALB/c. L'utilisation d'un inhibiteur d'entrée des parasites (Cytochalsin D) ou d'un inhibiteur des fonctions endosomales (Chloroquine) diminue de manière significative la réponse des macrophages aux parasites M+. L'utilisation de macrophages déficients en TLR, MyD88, et TRIF a démontré que la réponse générée après infection par les parasites M+ était dépendante de la voie de signalisation de TRIF et TLR3. Lors d'infection in vivo par des parasites M5313, au moins 50% des souris BALB/c présentent un phénotype sensible caractérisé par des lésions non-nécrotiques qui ne guérissent pas, persistent plus de 13 semaines après infection et contiennent un nombre considérable de parasites. Ces souris développent une réponse immunitaire de type T helper 2 (Th2) avec un niveau élevé d'IL-4 et d'IL-10. Les autres souris ont un phénotype non-sensible, les souris développant peu ou pas de lésion, avec peu de parasites et une réponse immunitaire diminuée, caractérisée par un niveau faible d'IFNy, d'IL4 et d'IL10. De plus, les souris BALB/c infectées par un parasite L. guyanensis isolé à partir des lésions muco-cutanées d'un patient humain atteint de LMC ont démontrés un phénotype similaire aux souris infectées par la souche M5313 avec 50% des souris développant des lésions persistantes, alors qu'un parasite dérivé des lésions cutanées humains n'a montré qu'une faible sensibilité avec une lésion transitoire qui finit par guérir. Nous avons montré que la sensibilité de ces souris BALB/c dépend de l'IL-4 et de l'IL-10 car les souris IL-10-/sur fond génétique BALB/c ainsi que les souris BALB/c traitée avec de l'anti-IL4 étaient capables de contrôler l'infection par M5313. Les souris C57BL/6 sont résistantes à l'infection par le parasite M5313. Elles développent une lésion transitoire qui guérit 9 semaines après infection. Ces souris résistantes ont un très faible taux de parasites au site d'infection et développent une réponse immunitaire de type Thl avec un niveau élevé d'IFNr et peu d'IL4 et d'IL10. Les infections in vivo de souris déficientes en MyD88, TRIF, TLR3 ou TLR9 (sur fond génétique C57BL/6) ont indiqué que MyD88 et TLR9 étaient impliqués dans la résistance à l'infection par L. guyanensi, et que TRIF et TLR3 avaient un rôle important dans la sensibilité. Ce travail met en évidence le fait que la réponse immunitaire de l'hôte est modulée par le parasite selon leur caractérisation d'être soit M+ ou M-. Nous avons démontré également que plusieurs gènes et voies de signalisations étaient impliqués dans cette réponse favorisant le développement d'une LMC. ABSTRACT : Leishmania guyanensis parasites are able to disseminate from the initial site of cutaneous skin infection to the nasopharyngeal tissues resulting in destructive secondary lesions and the disease Mucocutaneous Leishmaniasis (MCL). The secondary lesions in patients have intense immune cell infiltration, elevated immune responses and the presence (albeit at low levels) of parasites. More specifically, MCL patients produce higher levels of TNFa and display impairment in their ability to control the immune response due to a defect in their ability to respond to IL10. Little is known about the pathogenesis of MCL, especially about the dissemination of the infection from the site of inoculation to secondary sites (metastasis) and the response of the host to the pathogen. The hamster model of L. guyanensis infection has previously characterized the WHO reference strain, L. guyanensis WHI/BR/78/M5313, as being highly metastatic. Clones of parasites derived from this reference strain show a differential ability to metastasize. This thesis studied the differential immune response generated by macrophages in vitro, or by mice in vivo, following infection with L. guyanensis parasites. A significant increase in the activation status of macrophages derived from C57BL/6 or BALB/c mice was observed after in vitro infection with L. guyanensis parasites when compared to non-metastatic parasites. This change in status was evidenced by the increased expression of surface activation markers, together with the chemokines, CXCL 10, CCLS, and cytokines, IL6 and TNFa. Furthermore, in vitro infection of macrophages isolated from mice deficient in either a specific Toll Like Receptor (TLR) or the adaptor molecules MyD88 or TRIF, indicated that the immune response generated following L. guyanensis metastatic parasite infection was reliant on the TRIF dependent TLR3 signalling pathway. In vivo footpad infection of BALB/c mice with the L. guyanensis M5313 parasites showed a reproducible susceptible phenotype, whereby at least 50% of infected mice developed non-healing, nonnecrosing lesions with high parasitemia that persisted over 13 weeks post infection. This phenotype was characterized by a Th2 type cytokine immune response with increased levels of IL4 and IL10 detected in the draining lymph nodes. IL 10 deficient mice on a BALB/c background, or BALB/c mice treated with anti-IL4 were able to control infection with L. guyanensis M5313 parasites, thereby proving that these cytokines were indeed implicated in the susceptibility to infection. Moreover, infection of BALB/c mice with patient isolated L. guyanensis parasites confirmed that MCL derived parasites were able to induce a susceptibility phenotype similar to that of L. guyanensis M5313. C57BL/6 mice, on the other hand, were highly resistant to infection with L. guyanensis M5313 parasites and produced transient footpad swelling that healed by week 9 post infection, together with low degrees of footpad parasitemia and a Thl polarized immune response. Infection of mice deficient in MyD88, TRIF, TLR3, and TLR9 (on a C57BL/6 background), indicated that MyD88 and TLR9 were involved in the resistance of these mice to infection, and that TRIF and TLR3 were involved in the susceptibility. This study has shown that the host response can be differentially modulated depending on the infecting parasite with several genes and pathways being identified that could be involved in promoting the development of MCL.

Effects of the basic fibroblast growth factor and its anti-factor in the healing and collagen maturation of infected skin wound

PURPOSE: The infection is one of the main factors that affect the physiological evolution of the surgical wounds. The aim of this work is to evaluate the effects of fibroblast growth factor (FGFâ) and anti-FGFâ in the healing, synthesis and maturation of collagen when topically used on infected skin wounds of rats. METHODS: An experimental study was perfomed in 60 male Wistar rats. All animals were divided in two groups (A and B). Each group was divided in three subgroups A1, B1; A2, B2 and A3, B3. After anesthesia with pentobarbital, two open squared wounds (1cm2), 4cm distant to each other, were done in the dorsal skin of all the rats. In group A (n=30) the wounds were contaminated with multibacterial standard solution, and in group B(n=30) the wounds were maintained sterile. These wounds were named F1 (for inflammation analysis) and F2 (for collagen study). The open wounds of A1 and B1 rats were topically treated with saline solution, A2 and B2 were treated with FGFâ and subgroups A3 and B3 were treated with FGFâ and anti-FGFâ. The rats were observed until complete epitelization of F2 wounds for determination of healing time and the expression of types I and III collagen, using Picro Sirius Red staining. Inflammatory reaction in F1 wounds was studied using hematoxilineosin staining. The three variable was measured by the Image Pro-Plus Média Cybernetics software. The statistical analysis was performed by ANOVA and Tukey test, considering p<0.05 as significant. RESULTS: It was observed that infection retarded significantly (p<0.05) the time of wound scarring and the topical application of FCFb reverted the inhibition of healing caused by bacteria. The inflammatory reaction was greater in the subgroup B2 than in B1 and A3, and the difference was significant (p<0.05). It was observed greater expression of type I collagen in all the subgroups treated with FCFb, when compared with the untreated subgroups. Type III collagen was significantly decreased in wounds of B3 rats, comparing to the other subgroups. CONCLUSIONS: The FCFb accelerated the healing of open infected wounds and contributed with maturation of collagen, enhancing the type I collagen density. The anti-FCFb antibody was able to attenuate the production of both type I and III collagen

Nanotechnology-based drug delivery systems for treatment of hyperproliferative skin diseases - a review

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Studies of skin antisepsis and enhanced penetration of chlorhexidine

Current evidence-based guidelines recommend that 2% (w/v) chlorhexidine digluconate (CHG), preferentially in 70% (v/v) isopropyl alcohol (IIPA), is used for skin antisepsis prior to incision of the skin. In this current study, the antimicrobial efficacy of CHG, six essential oils [tea tree oil (TTO), thymol, eucalyptus oil (EO), juniper oil, lavender oil and citronella] and novel benzylidenecarboxamidrazone and thiosemicarbazone compounds were determined against a panel of microorganisms commonly associated with skin infection (Staphylococcus epidermidis, S. aureus, meticillin-resistant S. aureus, Propionibacterium acnes, Acinetobacter spp., Pseudomonas aeruginosa and Candida albicans) The results demonstrated synergistic activity of CHG in combination with EO against biofilm cultures of S. epidermidis, with significantly reduced concentrations of CHG and EO required to inhibit biofilm growth compared to CHG or EO alone. Skin permeation of CHG was subsequently investigated using an in vitro human skin model (Franz cell) and the penetration profile was determined by serial sectioning of the full thickness human skin. Two percent (w/v) CHG in aqueous solution and in 70% (v/v) IPA demonstrated poor skin permeation; however, the skin permeation was significantly enhanced in combination with 5% - 50% (v/v) EO. Detectable levels of CHG did not permeate through full thickness skin in 24 h. Skin permeation of 2% (w/v) CHG in 70% (v/v) IPA in the presence of 10% (v/v) EO was subsequently studied. The results demonstrated a significantly enhanced skin penetration of CHG after a 2 min application, with CHG detected at significant levels to a depth of 600 m with CHG in combination with EO and IPA compared to 100 m with IPA alone. Combination antisepsis comprising CHG and EO may be beneficial for skin antisepsis prior to invasive procedures to reduce the number of microorganisms on and within the skin due to enhanced skin penetration of CHG and improved efficacy against S. epidermidis in a biofilm mode of growth.

Effects of the basic fibroblast growth factor and its anti-factor in the healing and collagen maturation of infected skin wound

PURPOSE: The infection is one of the main factors that affect the physiological evolution of the surgical wounds. The aim of this work is to evaluate the effects of fibroblast growth factor (FGFâ) and anti-FGFâ in the healing, synthesis and maturation of collagen when topically used on infected skin wounds of rats. METHODS: An experimental study was perfomed in 60 male Wistar rats. All animals were divided in two groups (A and B). Each group was divided in three subgroups A1, B1; A2, B2 and A3, B3. After anesthesia with pentobarbital, two open squared wounds (1cm2), 4cm distant to each other, were done in the dorsal skin of all the rats. In group A (n=30) the wounds were contaminated with multibacterial standard solution, and in group B(n=30) the wounds were maintained sterile. These wounds were named F1 (for inflammation analysis) and F2 (for collagen study). The open wounds of A1 and B1 rats were topically treated with saline solution, A2 and B2 were treated with FGFâ and subgroups A3 and B3 were treated with FGFâ and anti-FGFâ. The rats were observed until complete epitelization of F2 wounds for determination of healing time and the expression of types I and III collagen, using Picro Sirius Red staining. Inflammatory reaction in F1 wounds was studied using hematoxilineosin staining. The three variable was measured by the Image Pro-Plus Média Cybernetics software. The statistical analysis was performed by ANOVA and Tukey test, considering p<0.05 as significant. RESULTS: It was observed that infection retarded significantly (p<0.05) the time of wound scarring and the topical application of FCFb reverted the inhibition of healing caused by bacteria. The inflammatory reaction was greater in the subgroup B2 than in B1 and A3, and the difference was significant (p<0.05). It was observed greater expression of type I collagen in all the subgroups treated with FCFb, when compared with the untreated subgroups. Type III collagen was significantly decreased in wounds of B3 rats, comparing to the other subgroups. CONCLUSIONS: The FCFb accelerated the healing of open infected wounds and contributed with maturation of collagen, enhancing the type I collagen density. The anti-FCFb antibody was able to attenuate the production of both type I and III collagen

Effects of the basic fibroblast growth factor and its anti-factor in the healing and collagen maturation of infected skin wound

PURPOSE: The infection is one of the main factors that affect the physiological evolution of the surgical wounds. The aim of this work is to evaluate the effects of fibroblast growth factor (FGFâ) and anti-FGFâ in the healing, synthesis and maturation of collagen when topically used on infected skin wounds of rats. METHODS: An experimental study was perfomed in 60 male Wistar rats. All animals were divided in two groups (A and B). Each group was divided in three subgroups A1, B1; A2, B2 and A3, B3. After anesthesia with pentobarbital, two open squared wounds (1cm2), 4cm distant to each other, were done in the dorsal skin of all the rats. In group A (n=30) the wounds were contaminated with multibacterial standard solution, and in group B(n=30) the wounds were maintained sterile. These wounds were named F1 (for inflammation analysis) and F2 (for collagen study). The open wounds of A1 and B1 rats were topically treated with saline solution, A2 and B2 were treated with FGFâ and subgroups A3 and B3 were treated with FGFâ and anti-FGFâ. The rats were observed until complete epitelization of F2 wounds for determination of healing time and the expression of types I and III collagen, using Picro Sirius Red staining. Inflammatory reaction in F1 wounds was studied using hematoxilineosin staining. The three variable was measured by the Image Pro-Plus Média Cybernetics software. The statistical analysis was performed by ANOVA and Tukey test, considering p<0.05 as significant. RESULTS: It was observed that infection retarded significantly (p<0.05) the time of wound scarring and the topical application of FCFb reverted the inhibition of healing caused by bacteria. The inflammatory reaction was greater in the subgroup B2 than in B1 and A3, and the difference was significant (p<0.05). It was observed greater expression of type I collagen in all the subgroups treated with FCFb, when compared with the untreated subgroups. Type III collagen was significantly decreased in wounds of B3 rats, comparing to the other subgroups. CONCLUSIONS: The FCFb accelerated the healing of open infected wounds and contributed with maturation of collagen, enhancing the type I collagen density. The anti-FCFb antibody was able to attenuate the production of both type I and III collagen

Phytosterols isolated from endophytic fungus Colletotrichum gloeosporioides(Melanconiaceae)

ABSTRACTEndophytic fungi are fungi that colonize internal tissues of plants. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, the aim this study was the isolation and structural identification of sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6) from fungus Colletotrichum gloeosporioidesisolated as endophytic from Virola michelli, a typical Amazon plant, used in folk medicine against skin infection. Compounds were isolated by chromatography column on silica and identified by 1H and 13C NMR and MS. The presence of phytosterols in fungi is rare and this is the first report of the isolation of the phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum.

VARIABILITY OF PHENOTYPIC, PROTEOMIC AND TRANSCRIPTOMIC EXPRESSION OF STAPHYLOCOCCUS AUREUS SURFACE ADHESINS

Staphylococcus aureus is a highly successful pathogen responsible of a wide variety of diseases, from minor skin infection to life-threatening sepsis or infective endocarditis, as well as food poisoning and toxic shock syndrome. This heterogeneity of infections and the ability of S. aureus to develop antibiotic-resistance to virtually any available drugs reflect its extraordinary capacity to adapt and survive in a great variety of environments. The pathogenesis of S. aureus infection involves a wide range of cell wall-associated adhesins and extracellular toxins that promote host colonization and invasion. In addition, S. aureus is extremely well equipped with regulatory systems that sense environmental conditions and respond by fine tuning the expression of metabolic and virulence determinants. Surface adhesins referred to MSCRAMMs - for Microbial Surface Component Recognizing Adherence Matrix Molecules - mediate binding to the host extracellular matrix or serum components, including fibrinogen, fibronectin, collagen and elastin, and promote tissue colonization and invasion. Major MSCRAMMs include a family of surface-attached proteins covalently bound to the cell wall peptidoglycan via a conserved LPXTG motif. Genomic analyses indicate that S. aureus contain up to 22 LPXTG surface proteins, which could potentially act individually or in synergy to promote infection. In the first part of this study we determined the range of adherence phenotypes to fibrinogen and fibronectin among 30 carriage isolates of S. aureus and compared it to the adherence phenotypes of 30 infective endocarditis and 30 blood culture isolates. Overall there were great variations in in vitro adherence, but no differences were observed between carriage and infection strains. We further determined the relation between in vitro adherence and in vivo infectivity in a rat model of experimental endocarditis, using 4 isolates that displayed either extremely low or high adherence phenotypes. Unexpectedly, no differences were observed between the in vivo infectivity of isolates that were poorly and highly adherent in vitro. We concluded that the natural variability of in vitro adherence to fibrinogen and fibronectin did not correlate with in vivo infectivity, and thus that pathogenic differences between various strains might only be expressed in in vivo conditions, but not in vitro. Therefore, considering the importance of adhesins expression for infection, direct measurement of those adhesins present on the bacterial surface were made by proteomic approach. 5 In the second series of experiments we assessed the physical presence of the LPXTG species at the staphylococcal surface, as measured at various time points during growth in different culture media. S. aureus Newman was grown in either tryptic soy broth (TSB) or in Roswell Park Memorial Institute (RPMI) culture medium, and samples were removed from early exponential growth phase to late stationary phase. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa) and clumping factor A (ClfA). Peptides of surface proteins were recovered by "trypsin-shaving" of live bacteria, and semi-quantitative proteomic analysis was performed by tandem liquid-chromatography and mass-spectrometry (LC-MS). We also determined in parallel the mRNA expression by microarrays analysis, as well as the phenotypic adherence of the bacteria to fibrinogen in vitro. The surface proteome was highly complex and contained numerous proteins theoretically not belonging to the bacterial envelope, including ribosomal proteins and metabolic enzymes. Sixteen of the 21 known LPXTG species were detected, but were differentially expressed. As expected, 9 known agr-regulated proteins (e.g. including Spa, FnBPA, ClfA, IsdA, IsdB, SasH, SasD, SasG and FmtB) increased up to the late exponential growth phase, and were abrogated in agr-negative mutants. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr negative mutant, while all other LPXTG proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in in vitro fibrinogen adherence tests during late growth (24h), whereas it remained poorly detected by proteomics. Differential expression was also detected in iron-rich TSB versus iron-poor RPMI. Proteins from the iron-regulated surface determinant (isd) system, including IsdA, IsdB and IsdH were barely expressed in iron-rich TSB, whereas they increased their expression by >10 time in iron-poor RPMI. We conclude that semi-quantitative proteomic analysis of specific protein species is feasible in S. aureus and that proteomic, transcriptomic and adherence phenotypes demonstrated differential profiles in S. aureus. Furthermore, peptide signatures released by trypsin shaving suggested differential protein domain exposures in various environments, which might be relevant for antiadhesins vaccines. A comprehensive understanding of the S. aureus physiology should integrate all these approaches.

Human microbiota in health and disease

Each human body plays host to a microbial population which is both numerically vast (at around 1014 microbial cells) and phenomenally diverse (over 1,000 species). The majority of the microbial species in the gut have not been cultured but the application of culture-independent approaches for high throughput diversity and functionality analysis has allowed characterisation of the diverse microbial phylotypes present in health and disease. Studies in monozygotic twins, showing that these retain highly similar microbiota decades after birth and initial colonisation, are strongly indicative that diversity of the microbiome is host-specific and affected by the genotype. Microbial diversity in the human body is reflected in both richness and evenness. Diversity increases steeply from birth reaching its highest point in early adulthood, before declining in older age. However, in healthy subjects there appears to be a core of microbial phylotypes which remains relatively stable over time. Studies of individuals from diverse geopraphies suggest that clusters of intestinal bacterial groups tend to occur together, constituting ‘enterotypes’. So variation in intestinal microbiota is stratified rather than continuous and there may be a limited number of host/microbial states which respond differently to environmental influences. Exploration of enterotypes and functional groups may provide biomarkers for disease and insights into the potential for new treatments based on manipulation of the microbiome. In health, the microbiota interact with host defences and exist in harmonious homeostasis which can then be disturbed by invading organisms or when ‘carpet bombing’ by antibiotics occurs. In a portion of individuals with infections, the disease will resolve itself without the need for antibiotics and microbial homeostasis with the host’s defences is restored. The administration of probiotics (live microorganisms which when administered in adequate amounts confer a health benefit on the host) represents an artificial way to enhance or stimulate these natural processes. The study of innate mechanisms of antimicrobial defence on the skin, including the production of numerous antimicrobial peptides (AMPs), has shown an important role for skin commensal organisms. These organisms may produce AMPs, and also amplify the innate immune responses to pathogens by activating signalling pathways and processing host produced AMPs. Research continues into how to enhance and manipulate the role of commensal organisms on the skin. The challenges of skin infection (including diseases caused by multiply resistant organisms) and infestations remain considerable. The potential to re-colonise the skin to replace or reduce pathogens, and exploring the relationship between microbiota elsewhere and skin diseases are among a growing list of research targets. Lactobacillus species are among the best known ‘beneficial’ bacterial members of the human microbiota. Of the approximately 120 species known, about 15 are known to occur in the human vagina. These organisms have multiple properties, including the production of lactic acid, hydrogen peroxide and bacteriocins, which render the vagina inhospitable to potential pathogens. Depletion of the of the normal Lactobacillus population and overgrowth of vaginal anaerobes, accompanied by the loss of normal vaginal acidity can lead to bacterial vaginosis – the commonest cause of abnormal vaginal discharge in women. Some vaginal anaerobes are associated with the formation of vaginal biofilms which serve to act as a reservoir of organisms which persists after standard antibiotic therapy of bacterial vaginosis and may help to account for the characteristically high relapse rate in the condition. Administration of Lactobacillus species both vaginally and orally have shown beneficial effects in the treatment of bacterial vaginosis and such treatments have an excellent overall safety record. Candida albicans is a frequent coloniser of human skin and mucosal membranes, and is a normal part of the microbiota in the mouth, gut and vagina. Nevertheless Candida albicans is the most common fungal pathogen worldwide and is a leading cause of serious and often fatal nosocomial infections. What turns this organism from a commensal to a pathogen is a combination of increasing virulence in the organism and predisposing host factors that compromise immunity. There has been considerable research into the use of probiotic Lactobacillus spp. in vaginal candidiasis. Studies in reconstituted human epithelium and monolayer cell cultures have shown that L. rhamnosus GG can protect mucosa from damage caused by Candida albicans, and enhance the immune responses of mucosal surfaces. Such findings offer the promise that the use of such probiotic bacteria could provide new options for antifungal therapy. Studies of changes of the human intestinal microbiota in health and disease are complicated by its size and diversity. The Alimentary Pharmabiotic Centre in Cork (Republic of Ireland) has the mission to ‘mine microbes for mankind’ and its work illustrates the potential benefits of understanding the gut microbiota. Work undertaken at the centre includes: mapping changes in the microbiota with age; studies of the interaction between the microbiota and the gut; potential interactions between the gut microbiota and the central nervous system; the potential for probiotics to act as anti-infectives including through the production of bacteriocins; and the characterisation of interactions between gut microbiota and bile acids which have important roles as signalling molecules and in immunity. The important disease entity where the role of the gut microbiota appears to be central is the Irritable Bowel Syndrome (IBS). IBS patients show evidence of immune activation, impaired gut barrier function and abnormal gut microbiota. Studies with probiotics have shown that these organisms can exert anti-inflammatory effects in inflammatory bowel disease and may strengthen the gut barrier in IBS of the diarrhoea-predominant type. Formal randomised trials of probiotics in IBS show mixed results with limited benefit for some but not all. Studies confirm that administered probiotics can survive and temporarily colonise the gut. They can also stimulate the numbers of other lactic acid bacilli in the gut, and reduce the numbers of pathogens. However consuming live organisms is not the only way to influence gut microbiota. Dietary prebiotics are selectively fermented ingredients that can change the composition and/or activity of the gastrointestinal microbiota in beneficial ways. Dietary components that reach the colon, and are available to influence the microbiota include poorly digestible carbohydrates, such as non-starch polysaccharides, resistant starch, non-digestible oligosaccharides (NDOs) and polyphenols. Mixtures of probiotic and prebiotic ingredients that can selectively stimulate growth or activity of health promoting bacteria have been termed ‘synbiotics’. All of these approaches can influence gut microbial ecology, mainly to increase bifidobacteria and lactobacilli, but metagenomic approaches may reveal wider effects. Characterising how these changes produce physiological benefits may enable broader use of these tactics in health and disease in the future. The current status of probiotic products commercially available worldwide is less than ideal. Prevalent problems include misidentification of ingredient organisms and poor viability of probiotic microorganisms leading to inadequate shelf life. On occasions these problems mean that some commercially available products cannot be considered to meet the definition of a probiotic product. Given the potential benefits of manipulating the human microbiota for beneficial effects, there is a clear need for improved regulation of probiotics. The potential importance of the human microbiota cannot be overstated. ‘We feed our microbes, they talk to us and we benefit. We just have to understand and then exploit this.’ (Willem de Vos).