Source tracking on a dairy farm reveals a high occurrence of subclinical mastitis due to hypervirulent Listeria monocytogenes clonal complexes.

AIMS: An extensive source investigation was conducted on a dairy farm with neurolisteriosis and subclinical mastitis cases to identify infection source and potential transmission routes of Listeria monocytogenes. METHODS AND RESULTS: A total of 36 L. monocytogenes isolates were obtained from animal clinical cases (neurolisteriosis and udder infection) and the farm environment (silage, faeces, water). Isolates were typed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Their virulence potential was assessed using the gentamicin protection assay and WGS-based identification of virulence genes. PFGE and WGS revealed a high genetic diversity of L. monocytogenes. An epidemiological link was confirmed for isolates from (i) several subclinical mastitis cases, (ii) silage and subclinical mastitis cases and (iii) different water sources. The neurolisteriosis isolate belonged to clonal complex (CC) 1, but infection source was not identified. A high occurrence (9/47 cows; 19·1%) of subclinical mastitis was observed with isolates belonging to CC2, CC4 and CC11. CONCLUSIONS: The dairy farm environment was contaminated with diverse L. monocytogenes strains, including genotypes associated with human disease. Several isolates harboured genetic determinants associated with increased infectious potential in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that subclinical listerial mastitis should not be neglected as a potential source of milk contamination. The presence of hypervirulent CCs in subclinical mastitis cases calls for the implementation of improved mastitis detection.

Antimicrobial Susceptibility Patterns of Clostridium difficile Isolates from Family Dairy Farms.

A significant risk factor for developing Clostridium difficile infection (CDI) in humans and animals is associated with the antimicrobial use. It has often been hypothesized that farm animals could be the source for human infection with Clostridium difficile (CD). In the European Union, family-run dairy farms are the predominant farming model, which are more interlinked within the community compared to large-scale intensive dairy or beef farms. Therefore, it is important to investigate antimicrobial susceptibility patterns of CD in such environment. A total of 159 CD isolates from 20 family dairy farms were tested with a customized broth microdilution plate for their antimicrobial resistance. Seventeen antimicrobials were selected (amoxicillin, ceftriaxone, clindamycin, daptomycin, erythromycin, fusidic acid, imipenem, levofloxacin, linezolid, metronidazole, moxifloxacin, oxacillin, rifampicin, tetracycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin), which are commonly used for treatment of CDI in veterinary and human medicine, or were previously applied in CD epidemiological studies. Antimicrobials, which are used for treatment of CDI in humans (metronidazole, vancomycin, fusidic acid, tigecycline, linezolid) inhibited CD growth in vitro. Most CD isolates were resistant to erythromycin (93.1%), daptomycin (69.2%) and clindamycin (46.5%). High multiple-resistance was found in CD ribotype 012 (n = 5, 100%), some CD SLO 060 (n = 4, 25%) and one CD 033 (n = 1, 1.1%). High multiple-resistance in this study was linked with CD ribotypes and not with the origin of CD. The low prevalence of these ribotypes (6.3%; 10/159) indicates that family-run dairy farms are an unlikely source of CD with multiple-resistance to antimicrobials.

Detection of methicillin-resistant Staphylococcus aureus carrying the mecC gene in human samples in Slovenia.

SUMMARY Following the recognition of a mecC MRSA isolate from a patient hospitalized in the northeastern region of Slovenia, a national collection of 395 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates from 2006 to 2013 was screened. An additional six mecC MRSA strains were found and characterized as spa types t843, t9397 and t10009, and multilocus sequence type ST130. The low oxacillin minimum inhibitory concentrations and absence of the mecA gene make recognition of these MRSA strains problematical for diagnostic laboratories. In such strains the presence of mecC should be determined.

Interrelationship between peripheral arterial occlusive disease, carotid atherosclerosis and flow mediated dilation of the brachial artery.

AIM: Atherosclerosis is considered a systemic disease. Therefore, in patients with atherosclerotic disease effects on various sections of the arterial system are expected. The aim of our study was to determine whether patients with evident peripheral arterial occlusive disease (PAOD) of the lower limbs have any subclinical functional or structural arterial wall changes in other sections of the arterial system. METHODS: The study included 54 patients with PAOD, Fontaine stage II and a claudication distance from 50 to 500 m (average 250+/-170 m). Their mean age was 64. None of them had any symptoms or signs of coronary or cerebrovascular atherosclerosis (CVD). The control group consisted of 50 healthy volunteers with a mean age of 64 years without any risk factors of atherosclerosis. In all subjects the carotid intima-media thickness (IMT), was measured, the presence of atherosclerotic plaques in the carotid artery (CA) was registered and the endothelium-dependent dilation capability of the brachial artery (BA) during reactive hyperemia was measured using the B-mode ultrasound technique. RESULTS: The average IMT was significantly greater in PAOD patients than in controls (0.8+/-0.2 mm vs 0.6+/-0.1 mm, p<0.001). In patients atherosclerotic plaques in the CA were also more numerous than in controls (38 vs 4, p<0.001). The IMT of patients was related to body mass index (BMI), ankle-brachial pressure index (ABI), LDL cholesterol and to the number of atherosclerotic plaques. In PAOD patients flow-mediated dilation of the BA was significantly lower than in controls (7.2+/-4.9% vs 12.3+/-2.1%, p<0.001). The dilation capability of the BA was linearly related to the BMI, ABI and IMT. CONCLUSION: The results of our study show that PAOD patients without clinical evidence of CVD have morphological changes of the CA, increased IMT and numerous atherosclerotic plaques. Furthermore, in PAOD patients flow-mediated endothelium-dependent dilation of the peripheral arteries is decreased. These results support the hypothesis that atherosclerosis is a generalized disease, leading to functional and structural changes in several segments of the arterial system.

Isolation of invasion-associated cDNAs in melanoma.

Metastasis and invasion are key steps in the systemic spread of tumor cells. To identify the genes involved in this process we recently selected highly invasive and weakly invasive cell clones from a melanoma cell line. Both cell clones showed a stable phenotype over more than 40 passages and previous analyses revealed a fivefold difference in their invasive potential in vitro and in tumorigenesis in vivo. To compare gene expression of the two cell clones a cDNA array system (Clontech human cancer cDNA array) was used. Exact quantification of differentially expressed genes in each cell clone was performed by real-time RT-PCR. An evaluation of the array data revealed a total of 36 genes that were more than 1.5-fold differentially expressed, and 26 (72%) of these showed a differential expression pattern by quantitative RT-PCR. Previously known differences in expression patterns, including loss of p16 and HLA I, or equal expression of p73, and RAR alpha, beta and gamma were confirmed by the array data. In addition, reduced expression levels of several cytoskeletal proteins, such as vimentin, gamma-actin, desmin and cytokeratins, in the highly invasive cell clone were reproducibly identified. Other genes strongly upregulated in the highly invasive cell clone included jagged 2, STAT1, tPA and c-myc, whereas MDA-6 (p21), caspase 2 and semaphorin were found to be downregulated. In conclusion, comparative hybridization of cDNA arrays identified a series of novel invasion-associated changes in gene expression and confirmed previously known expression patterns.

The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold.

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain-like fold in solution, with two perpendicular, antiparallel, three- and five-stranded beta-sheets. In contrast to known structures with the SH3 domain fold, MIA is a single-domain protein, and contains an additional antiparallel beta-sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain-like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin alpha4beta1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.

Characterization of a transcription factor binding site, specifically activating MIA transcription in melanoma.

We have previously isolated the protein MIA, which is secreted from melanoma cells, and identified highly restricted expression patterns in melanocytic tumors. Preliminary studies of the human MIA gene provided evidence that the promoter is specifically activated in melanoma cells but is silent in nonmelanocytic cells and benign melanocytes. In this study we aimed to identify cis-regulatory promoter elements that mediate promoter activation during malignant transformation of melanocytes. We therefore subcloned 1.4 kb of the murine MIA promoter and a series of 5' deletion constructs into a luciferase reporter plasmid and identified the most active cis-regulatory element between nucleic acids -230 and -130. Cloning oligomeric fragments of this promoter region in front of a minimal TK promoter revealed a 30 bp enhancer element mediating expression of the reporter gene in melanoma cells but not in melanocytes or nonmelanocytic cells. Gel mobility shift assays and southwestern blots led to the identification of specific DNA-protein complexes in melanoma cells. Fine mutational analysis of the cis-regulatory promoter element showed that two critical nucleic acid residues are essential for both transcriptional activity and formation of the band shift complexes. By an initial small-scale affinity purification we were able to isolate a protein approximately 32 kDa in size from melanoma cells, which we refer to as MATF ("melanoma-associated transcription factor"). Our study identified for the first time MATF, a transcription factor that is upregulated or activated during malignant transformation of melanocytes.

[MIA ("melanoma inhibitory activity"). Biological functions and clinical relevance in malignant melanoma]. / MIA ("melanoma inhibitory activity"). Biologische Funktionen und klinische Relevanz beim malignen Melanom.

The protein MIA was identified and isolated from the tissue culture supernatant of melanoma cells in vitro by its ability to inhibit thymidine incorporation by melanoma cell lines. After purification and partial sequencing of the peptide, a fragment of the MIA cDNA was cloned by RT-PCR. This cDNA fragment was used to screen phage libraries and subsequently fully encoding human and murine MIA cDNA and genomic DNA clones were obtained. The MIA gene spans a region of approximately 2 kb and is divided into 4 exons. Mapping the MIA gene revealed that the human gene is located on chromosome 19 and the murine gene on chromosome 7. The MIA open reading frame spans 131 (human) or 130 (murine) amino acids. The first 24 (human) or 23 (murine) amino acids represent a signal sequence directing the secretion of MIA into the extracellular compartment. The mature, secreted MIA consists of 107 amino acids and its MW is approximately 11 kDa. Preliminary structural data suggests that MIA is a small globular protein stabilized by two intramolecular disulfide bonds. Expression studies of protein und mRNA levels indicate that MIA is expressed specifically by malignant melanoma cells and chondrocytes. This points to a highly restricted expression pattern which is controlled by the MIA promoter. In addition, MIA provides a clinically useful parameter in patients with malignant melanoma. Enhanced values were measured in the serum of all patients with metastatic melanoma (stage III and IV). In vitro and in vivo experiments using recombinant MIA protein revealed that MIA specifically inhibits attachment of melanoma cells to fibronectin and laminin. Further analysis indicated a direct binding between MIA and the matrix proteins. This finding provides an explanation for the capability of MIA to inhibit proliferation of melanoma cells in vitro. Our studies suggest a putative function of MIA in regulated detachment of melanoma cells from the extracellular matrix which is an important step in metastasis.