Prevalence and Antimicrobial Resistance of Staphylococcus aureus and Coagulase-Negative Staphylococcus/Mammaliicoccus from Retail Ground Meat: Identification of Broad Genetic Diversity in Fosfomycin Resistance Gene fosB.

Staphylococcus is a major bacterial species that contaminates retail meat products. The objective of this study was to clarify the prevalence, antimicrobial resistance and genetic determinants of Staphylococcus/Mammaliicoccus species in retail ground meat in Japan. From a total of 146 retail ground meat samples (chicken, pork, mixed beef/pork) purchased during a 5-month period, 10 S. aureus and 112 isolates of coagulase-negative staphylococcus (CoNS)/Mammaliicoccus comprising 20 species were recovered. S. aureus isolates were classified into five genetic types, i.e., coa-IIa/ST5, coa-VIc/ST352 (CC97), coa-VIIb/ST398, coa-Xa/ST15, and coa-XIc/ST9, which were all related to those of livestock-associated clones. All the staphylococcal isolates were mecA-negative and mostly susceptible to all the antimicrobials tested, except for ampicillin among S. aureus (resistance proportion; 50%). Among CoNS, the fosfomycin resistance gene fosB was prevalent (30/112; 26.8%), primarily in S. capitis, S. warneri, and S. saprophyticus. Phylogenetic analysis of fosB revealed the presence of seven clusters, showing broad diversity with 65-81% identity among different clusters. In the CoNS isolates from ground meat samples, fosB was assigned into three clusters, and S. saprophyticus harbored the most divergent fosB with three genetic groups. These findings suggested the circulation of multiple fosB-carrying plasmids among some CoNS species.

Label retention identifies a multipotent mesenchymal stem cell-like population in the postnatal thymus.

Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRß,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments.

DKK1 mediated inhibition of Wnt signaling in postnatal mice leads to loss of TEC progenitors and thymic degeneration.

BACKGROUND: Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of DeltaNP63(+) Foxn1(+) and Aire(+) TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments. CONCLUSIONS/SIGNIFICANCE: Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.

The Wnt signaling antagonist Kremen1 is required for development of thymic architecture.

Wnt signaling has been reported to regulate thymocyte proliferation and selection at several stages during T cell ontogeny, as well as the expression of FoxN1 in thymic epithelial cells (TECs). Kremen1 (Krm1) is a negative regulator of the canonical Wnt signaling pathway, and functions together with the secreted Wnt inhibitor Dickkopf (Dkk) by competing for the lipoprotein receptor-related protein (LRP)-6 co-receptor for Wnts. Here krm1 knockout mice were used to examine krm1 expression in the thymus and its function in thymocyte and TEC development. Krm1 expression was detected in both cortical and medullary TEC subsets, as well as in immature thymocyte subsets, beginning at the CD25+CD44+ (DN2) stage and continuing until the CD4+CD8+(DP) stage. Neonatal mice show elevated expression of krm1 in all TEC subsets. krm1(-/-) mice exhibit a severe defect in thymic cortical architecture, including large epithelial free regions. Much of the epithelial component remains at an immature Keratin 5+ (K5) Keratin 8(+)(K8) stage, with a loss of defined cortical and medullary regions. A TOPFlash assay revealed a 2-fold increase in canonical Wnt signaling in TEC lines derived from krm1(-/-) mice, when compared with krm1(+/+) derived TEC lines. Fluorescence activated cell sorting (FACS) analysis of dissociated thymus revealed a reduced frequency of both cortical (BP1(+)EpCAM(+)) and medullary (UEA-1(+) EpCAM(hi)) epithelial subsets, within the krm1(-/-) thymus. Surprisingly, no change in thymus size, total thymocyte number or the frequency of thymocyte subsets was detected in krm1(-/-) mice. However, our data suggest that a loss of Krm1 leads to a severe defect in thymic architecture. Taken together, this study revealed a new role for Krm1 in proper development of thymic epithelium.

Near-infrared Fourier transform Raman spectroscopic analysis of proteins, water and lipids in intact normal stratum corneum and psoriasis scales.

Previous biochemical studies demonstrated differences in the structure of lipids in normal skin and psoriatic lesions. Raman spectroscopy provides a unique possibility of studying the molecular structure of proteins, lipids and water in intact skin. Near-infrared Fourier transform Raman spectroscopy was used to study changes in molecular structure and conformation of proteins and lipids of stratum corneum in healthy persons and patients with psoriasis. In vitro Raman spectra were obtained from intact psoriatic scales in 11 patients and from normal stratum corneum of forearm, elbow and heel in a group of age-matched healthy persons. The spectra of stratum corneum differed between psoriasis and normal skin but not between investigated regions. No major changes of lipid band positions in Raman spectra were found, but the crystalline lipid structure was disrupted in psoriatic scales (assessed as the ratio of the symmetric methylene C-H stretching-mode intensities, S(lat)). Major spectral differences were seen in the molecular structure of the proteins. In the spectra of psoriatic scales, the peak position of the amide I band, in comparison with the normal skin, was shifted to higher wavenumbers, suggesting unfolding of proteins. Moreover, alterations in the disulfide stretch bonds of proteins were found in psoriasis scales, resulting in a less energetically favourable gauche-gauche-trans conformation (band around 520 cm(-1)). Psoriatic scales and normal stratum corneum did not statistically differ in their water content. The findings further define the molecular abnormalities in the stratum corneum in psoriasis.

Increased microorganisms DNA levels in peripheral blood monocytes from psoriatic patients using PCR with universal ribosomal RNA primers.

It has long been suspected that systemic and focal infections cause or exacerbate psoriatic lesions. We previously showed that peripheral blood monocytes in psoriatic patients are activated and overproduce inflammatory cytokines. In addition, it has been reported that macrophages activated by ingesting microorganisms release tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Therefore we hypothesized that the monocytes in psoriatic patients may be activated by ingesting microorganisms and overproduce inflammatory cytokines. We examined the detection of microorganism DNA in monocytes from 15 patients with psoriasis vulgaris and from 12 healthy controls. DNA was extracted from monocytes, and a polymerase chain reaction (PCR) assay was performed for the detection using universal primers from conserved regions of the bacterial 16S ribosomal RNA gene or the fungal 18S rRNA gene. At the same time, we calculated the psoriasis area and severity index (PASI) scores and analyzed their correlations with the microorganisms DNA levels. The results showed that bacterial 16S DNA levels in monocytes were significantly higher in psoriatic patients than in controls. The fungal 18S DNA levels were also higher in psoriatic patients than in controls, but the differences were not significant. Although the microorganisms DNA levels in monocytes of psoriatic patients were high, there was no correlation between the bacterial DNA levels in monocytes of the psoriatics and PASI scores. Our study suggests that monocytes in psoriatic patients engulf more bacteria than there in controls, causing an activation of monocytes and triggering the formation of new lesions in the initial stages of psoriasis.