Proteomic analysis of plasma exosomes from Cystic Echinococcosis patients provides in vivo support for distinct immune response profiles in active vs inactive infection and suggests potential biomarkers.

The reference diagnostic method of human abdominal Cystic Echinococcosis (CE) is imaging, particularly ultrasound, supported by serology when imaging is inconclusive. However, current diagnostic tools are neither optimal nor widely available. The availability of a test detecting circulating biomarkers would considerably improve CE diagnosis and cyst staging (active vs inactive), as well as treatments and follow-up of patients. Exosomes are extracellular vesicles involved in intercellular communication, including immune system responses, and are a recognized source of biomarkers. With the aim of identifying potential biomarkers, plasma pools from patients infected by active or inactive CE, as well as from control subjects, were processed to isolate exosomes for proteomic label-free quantitative analysis. Results were statistically processed and subjected to bioinformatics analysis to define distinct features associated with parasite viability. First, a few parasite proteins were identified that were specifically associated with either active or inactive CE, which represent potential biomarkers to be validated in further studies. Second, numerous identified proteins of human origin were common to active and inactive CE, confirming an overlap of several immune response pathways. However, a subset of human proteins specific to either active or inactive CE, and central in the respective protein-protein interaction networks, were identified. These include the Src family kinases Src and Lyn, and the immune-suppressive cytokine TGF-ß in active CE, and Cdc42 in inactive CE. The Src and Lyn Kinases were confirmed as potential markers of active CE in totally independent plasma pools. In addition, insights were obtained on immune response profiles: largely consistent with previous evidence, our observations hint to a Th1/Th2/regulatory immune environment in patients with active CE and a Th1/inflammatory environment with a component of the wound healing response in the presence of inactive CE. Of note, our results were obtained for the first time from the analysis of samples obtained in vivo from a well-characterized, large cohort of human subjects.

Necrotic cell death in human amniotic cells infected by Listeria monocytogenes.

Listeria monocytogenes can cause a placental-foetal infection that results in spontaneous abortion, premature labour, stillbirth, or neonatal sepsis and meningitis. Bacteria cross the maternofoetal barrier at the villous syncytiotrophoblast level and subsequently spread from the placenta to the fetus. L. monocytogenes is able to induce different kinds of death in a variety of cells. Murine hepatocytes, murine T and human B lymphocytes, and murine dendritic cells die by apoptosis, whereas bacterial infection of murine and human macrophages leads mainly to necrotic cell death. As we previously described the efficient infection and growth of L. monocytogenes in a human amniotic cell line, we investigated the fate of these cells in order to analyse the mode of cell death. Our results provide biochemical and morphological evidence of necrotic death induced by L. monocytogenes infection.

Invasive pathway of Listeria ivanovii in human amnion-derived WISH cells.

Among Listeria genus, only two species, Listeria ivanovii and Listeria monocytogenes, are pathogenic. L. ivanovii is almost only associated with infections in animals, mainly sheep and cattle, and has rarely been associated with human infections, whereas L. monocytogenes causes severe illnesses in both humans and animals. To further investigate the pathogenetic features of L. ivanovii in humans, we undertook a study in which the intracellular behaviour of this pathogen was analysed in WISH cells, a cell line derived from human amniotic tissue, and compared to that of L. monocytogenes. Using microbiological, biochemical, and ultrastructural approaches, we demonstrate that L. ivanovii can adhere to and invade human amniotic cells, lyse the phagosomal membrane, polymerize host cell actin, and spread from cell to cell more efficiently than L. monocytogenes. However, although L. ivanovii is capable of specifically infecting and replicating in human amnion cells, its survival in cytoplasm is limited compared to that of L. monocytogenes.

Clonality among Enterococcus faecium clinical isolates.

In the course of a survey to determine the epidemiology of enterococcal infections in Italy, a sudden increment, in a 1-year time, was noted in the number of glycopeptide resistant Enterococcus faecium isolated from different wards of the University Hospital in Rome, Italy. The isolates were characterized for clonal relatedness by comparing SmaI gel electropherotypes, presence of vancomycin-resistance genes, and expression of virulence factors. PFGE identified in a single pulsed type all the glycopeptide-resistant isolates but one. Resistance to high levels of aminoglycosides was expressed by these same isolates, which also included a majority of non biofilm-forming strains. Two esp gene-carrying strains were also identified in different PFGE types. Data indicates that a specific clone acquired, in the clinical setting, the genetic determinant for glycopeptide resistance, thus improving environmental adaptation and favoring its persistence and spread.

Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci.

Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, source-specific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy.

Effect of iron limitation on slime production by Staphylococcus aureus.

The aim of this study was to examine the effect of growth conditions on slime production by Staphylococcus aureus clinical isolates. The addition of glucose to the medium enhanced slime production in the majority of Staphylococcus aureus isolates cultured from infections associated with orthopaedic prostheses. Iron limitation also stimulated this ability even in the absence of the additional carbohydrate source. Staphylococcus aureus isolates were classified as Group 1 [strains producing slime only in trypticase soy broth supplemented with 1% glucose (TSBG) or in iron-limited trypticase soy broth (TSB/Fe-)]; Group 2 (slime + only in TSB/Fe-); or Group 3 (slime+ only in TSBG). Seven repeatedly slime-negative strains were stimulated to produce slime by subpassaging in iron-limited medium. Low iron levels, usually found in vivo, could stimulate slime production by Staphylococcus aureus and support chronic infections associated with orthopaedic prostheses.

Enterococcus spp. produces slime and survives in rat peritoneal macrophages.

Enterococcal clinical isolates were investigated for the ability to form biofilm on inert surfaces, as a measure of slime production, in an attempt to find new possible virulence factors for these microorganisms. This property was commonly found among Enterococcus faecalis. Also E. faecium isolates were able to form biofilm, although to a lesser extent; for this species, however, biofilm formation seemed more frequently associated with isolates from infection rather than with environmental strains or isolates from healthy individuals. Biofilm formation was strongly affected by the presence of an additional carbohydrate source in the medium, or by iron deprivation, indicating a role of slime for survival in stressful conditions. Slime-producing E. faecalis were able to survive inside peritoneal macrophages for extended periods compared to slime-negative strains or to slime-positive bacteria grown in conditions depressing slime production. In particular, slime-producing and slime-negative cells showed a decrease of 1 and 2 log units, respectively, at 1 h after infection; slime-negative cells were then rapidly killed, with clearance of bacterial cells at 24 h. Slime-producing bacteria persisted up to 48 h, which was the last time point examined, as after that time viability of both infected and non-infected macrophages started to decline. Scanning electron microscopy observations showed the presence of abundant amorphous extracellular material, of possible polysaccharide nature, embedding bacterial cells to form a multilayered biofilm. Even in conditions not supporting biofilm formation, bacterial cells appeared capsulated, suggesting that capsule and slime might represent different structures. Genes belonging to the epa locus or to a putative icaA homolog did not seem to be involved in synthesis and export of slime.