Dogs as carriers of virulent and resistant genotypes of Clostridioides difficile.

While previous research on zoonotic transmission of community-acquired Clostridioides difficile infection (CA-CDI) focused on food-producing animals, the present study aimed to investigate whether dogs are carriers of resistant and/or virulent C. difficile strains. Rectal swabs were collected from 323 dogs and 38 C. difficile isolates (11.8%) were obtained. Isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing (WGS) and a DNA hybridization assay. Multilocus sequence typing (MLST), core genome MLST (cgMLST) and screening for virulence and antimicrobial resistance genes were performed based on WGS. Minimum inhibitory concentrations for erythromycin, clindamycin, tetracycline, vancomycin and metronidazole were determined by E-test. Out of 38 C. difficile isolates, 28 (73.7%) carried genes for toxins. The majority of isolates belonged to MLST sequence types (STs) of clade I and one to clade V. Several isolates belonged to STs previously associated with human CA-CDI. However, cgMLST showed low genetic relatedness between the isolates of this study and C. difficile strains isolated from humans in Austria for which genome sequences were publicly available. Four isolates (10.5%) displayed resistance to three of the tested antimicrobial agents. Isolates exhibited resistance to erythromycin, clindamycin, tetracycline and metronidazole. These phenotypic resistances were supported by the presence of the resistance genes erm(B), cfr(C) and tet(M). All isolates were susceptible to vancomycin. Our results indicate that dogs may carry virulent and antimicrobial-resistant C. difficile strains.

Diversity of methicillin-resistant coagulase-negative Staphylococcus spp. and methicillin-resistant Mammaliicoccus spp. isolated from ruminants and New World camelids.

Information about livestock carrying methicillin-resistant coagulase-negative staphylococci and mammaliicocci (MRCoNS/MRM) is scarce. The study was designed to gain knowledge of the prevalence, the phenotypic and genotypic antimicrobial resistance and the genetic diversity of MRCoNS/MRM originating from ruminants and New World camelids. In addition, a multi-locus sequence typing scheme for the characterization of Mammaliicoccus (formerly Staphylococcus) sciuri was developed. The study was conducted from April 2014 to January 2017 at the University Clinic for Ruminants and the Institute of Microbiology at the University of Veterinary Medicine Vienna. Seven hundred twenty-three nasal swabs originating from ruminants and New World camelids with and without clinical signs were examined. After isolation, MRCoNS/MRM were identified by MALDI-TOF, rpoB sequencing and typed by DNA microarray-based analysis and PCR. Antimicrobial susceptibility testing was conducted by agar disk diffusion. From all 723 nasal swabs, 189 MRCoNS/MRM were obtained. Members of the Mammaliicoccus (M.) sciuri group were predominant (M. sciuri (n = 130), followed by M. lentus (n = 43), M. fleurettii (n = 11)). In total, 158 out of 189 isolates showed phenotypically a multi-resistance profile. A seven-loci multi-locus sequence typing scheme for M. sciuri was developed. The scheme includes the analysis of internal segments of the house-keeping genes ack, aroE, ftsZ, glpK, gmk, pta1 and tpiA. In total, 28 different sequence types (STs) were identified among 92 selected M. sciuri isolates. ST1 was the most prevalent ST (n = 35), followed by ST 2 (n = 15), ST3 and ST5 (each n = 5), ST4 (n = 3), ST6, ST7, ST8, ST9, ST10 and ST11 (each n = 2).

Gut microbiota of the European Brown Hare (Lepus europaeus).

Diseases of the gastrointestinal tract due to changes in the bacterial flora have been described with increasing incidence in the European brown hare. Despite extensive demographic and phylogeographic research, little is known about the composition of its gut microbiota and how it might vary based on potential environmental or host factors. We analysed the intestinal and faecal microbiota of 3 hare populations by Illumina MiSeq 16S rRNA gene amplicon sequencing. The phyla and OTU abundance composition differed significantly between intestinal and faecal samples (PERMANOVA: P = 0.002 and P = 0.031, respectively), but in both sample types Firmicutes and Bacteroidetes dominated the microbial community composition (45.51% and 19.30% relative abundance). Intestinal samples contained an enrichment of Proteobacteria compared with faecal samples (15.71-fold change, P < 0.001). At OTU level, a significant enrichment with best BLAST hits to the Escherichia-Shigella group, Eubacterium limosum, Sphingomonas kyeonggiensis, Flintibacter butyricus and Blautia faecis were detected in intestinal samples (P < 0.05). In our statistical model, geographic location and possibly associated environmental factors had a greater impact on the microbiota composition than host factors. Population had a significant effect on the composition of abundant intestinal and faecal OTUs, and on the abundance of potential pathogenic bacteria of the family Enterobacteriaceae, regularly associated with intestinal dysbiosis in hares, in faecal samples. Our study is the first to describe the microbiota in brown hares and provides a foundation to generate hypothesis aiming to test the role of gut health in population fluctuations of the species.

Diversity of methicillin-resistant Staphylococcus aureus (MRSA) isolated from Austrian ruminants and New World camelids.

The aim of this study was to determine the prevalence, the antimicrobial resistance patterns and the genetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) from Austrian ruminants and New World camelids that were treated at the University of Veterinary Medicine, Vienna. Between April 2014 and January 2017, 723 nasal swabs originating from ruminants and New World camelids were examined. MRSA isolates were characterized by mecA/mecA1/mecC PCRs and by DNA microarray analysis. They were genotyped by spa typing, dru typing, MLST and MLVA. Glycopolymer fingerprinting by FTIR spectroscopy was also performed. Antimicrobial susceptibility testing was conducted by agar disk diffusion. Twelve MRSA isolates were mecA-positive, whereas three were mecC-positive. The MRSA isolates carried five different SCCmec elements, and belonged to three sequence types (ST45, ST130, ST398). The MRSA isolates displayed seven different resistance phenotypes. The present study describes for the first time mecC-carrying MRSA isolates originating from domesticated animals in Austria. More systematic studies are needed to unravel the role of ruminants and New World camelids as reservoirs for MRSA as a potential risk for zooanthropogenic transmission.

Pyogranulomatous panniculitis in ferrets (Mustela putorius furo) with intralesional demonstration of Pseudomonas luteola.

One ferret (Mustela putorius furo) from Finland and two ferrets from Austria, aged 1-4.5 years and of both genders, were presented with pyogranulomatous subcutaneous inflammation affecting the inguinal, preputial and femoral regions, respectively. Histologically, microorganisms were detected within the lesions. The organisms had a capsule that stained positively by the periodic acid-Schiff reaction. Pseudomonas spp. were cultured from the lesions in two cases. In the third case, electron microscopy revealed a prokaryotic organism surrounded by an electron lucent matrix. 16S rRNA gene sequencing showed highest sequence homology to Pseudomonas luteola in all three cases. In combination with recent reports of pleuropneumonia in ferrets due to P. luteola infection, these cases might indicate a predisposition of ferrets for infection by these bacteria.

Camelimonas abortus sp. nov., isolated from placental tissue of cattle.

A gram-negative, rod-shaped, non-spore-forming bacterium, isolated from placental tissue of a cow, was investigated for its taxonomic position. On the basis of 16S rRNA gene sequence similarities, strain UK34/07-5(T) was shown to belong to the class Alphaproteobacteria, closely related to the type strain of Camelimonas lactis (96.0 % sequence similarity). The polyamine pattern showed the major compound spermidine and moderate amounts of putrescine. The major quinone was ubiquinone Q-10. The polar lipid profile was composed of the major compounds phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine and moderate amounts of diphosphatidylglycerol, three unidentified aminolipids and an unidentified phospholipid. The profile of major fatty acids, consisting of C(19 : 0) cyclo ω8c and C(18 : 1)ω7c, with C(18 : 0) 3-OH as the hydroxylated fatty acid, was very similar to that of C. lactis M 2040(T). The results of DNA-DNA hybridization and physiological and biochemical tests allowed both genotypic and phenotypic differentiation of the isolate from C. lactis. The relatively low 16S rRNA gene sequence similarity of 96.0 % to C. lactis M 2040(T) and marked differences in the polar lipid profiles as well as the results of physiological tests and the DNA-DNA hybridization data support the creation of a novel species, for which the name Camelimonas abortus sp. nov. is proposed, with the type strain UK34/07-5(T) ( = CIP 110303(T)  = CCUG 61094(T)  = DSM 24741(T)  = CCM 7941(T)).

Strain-specific detection of two Aureobasidium pullulans strains, fungal biocontrol agents of fire blight by new, developed multiplex-PCR.

AIM: The aim of this study was to develop a specific and sensitive identification method for two Aureobasidium pullulans biocontrol strains, CF10 and CF40, based on a sequence-characterized amplified region (SCAR) derived from RAPD - and multiplex-RAPD PCR analysis. METHODS AND RESULTS: The random amplified polymorphic DNA (RAPD) and multiplex RAPD-PCR techniques were used for a preliminary screening of A. pullulans genetic variability among 200 isolates. This approach allowed the selection of ten fragments present solely in strains CF10 and CF40. The RAPD fragments were cloned, sequenced and used to design two SCAR primers. Two primer pairs obtained from SCH3RAPD fragment of CF 40 and 6RAPD of CF10 were highly specific and sensitive. CONCLUSIONS: In this study, we developed strain-specific multiplex-PCR based on sequence-characterized amplified region (SCAR) markers to simultaneously detect both strains in a single PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex-PCR provides a valuable tool for specific and sensitive identification of CF10 and CF40, and could be used in studies on the efficacy and persistence of introduced strains of A. pullulans for fire blight control.

The carcinostatic and proapoptotic potential of 4-hydroxynonenal in HeLa cells is associated with its conjugation to cellular proteins.

BACKGROUND: Previous studies have shown that the lipid peroxidation product 4-hydroxynonenal (HNE) acts as a cell growth modulator if used at low, physiological concentrations being strongly cytotoxic at higher concentrations for a number of cells. These effects of HNE also appeared to be mutually dependent on the effects of serum growth factors. The aim of this investigation was to study the concentration-dependent response of human cervical carcinoma (HeLa) cells in vitro with respect to the intracellular uptake of exogenous HNE, the cellular energy metabolism, DNA synthesis, overall gene expression and susceptibility to apoptosis. MATERIALS AND METHODS: MTT assay was applied as an index of energy metabolism and the replicative activity was quantitated by the 3H-thymidine incorporation assay. The occurence and intracellular distribution was studied with monoclonal antibodies directed against HNE-protein conjugates. Binding of HNE to serum proteins was determined with the same antibodies by Western blotting. Differential gene expression was studied by differential display RT-PCR while a novel photometric assay, denoted Titer-TACS, was used for in situ detection and quantitation of apoptosis in monolayer cell cultures. RESULTS: A physiological concentration of HNE (1 microM) had hardly any effect on the parameters of the replicative activity and the energy metabolism. No morphological changes were observed and the number of HNE-positive cells was not significantly different when compared to the untreated control cells, while most of the aldehyde appeared to be bound to serum proteins (albumin fraction). A ten-fold higher concentration (10 microM) was found to be cytostatic. Spindle-shaped cells with a picnotic nucleus were observed occasionally, as well as membrane blebs, which were HNE-positive. The number of HNE-positive cells was significantly increased compared both to the control cells and cells treated with 1 microM HNE, but in the presence of serum the effects of 10 microM HNE were negated due to its binding to the serum proteins. Finally, 100 microM HNE was cytotoxic for the HeLa cells. Most of the cells were picnotic, together with a few spindle-shaped or oval cells. The staining for HNE was diffuse and strong (90% of the cells were HNE-positive) while even binding of the aldehyde to serum proteins did not prevent its cytotoxic effects. This concentration of HNE caused acute stress response of the cells resulting in the decreased expression of several as yet unidentified genes. The altered pattern of gene expression was followed by programmed cell death, i.e. an increased number of apoptotic cells after treatment with low (1 and 10 microM) concentrations of HNE. A rebound effect was observed, i.e. a decrease of apoptotic cells after 24 hours followed by an overshooting increase after 48 hours. CONCLUSIONS: For HeLa carcinoma cells there appears to be a concentration range of HNE where it does not cause necrosis but preferentially apoptosis. At this concentration range HNE is cytochemically detectable within the cells as a protein conjugate. It is proposed that a possible differential sensitivity of cancer cells and their normal counterparts to the cytostatic activity of HNE should be explored.

An overview on anticancer activities of the Viscum album extract Isorel.

The activity principle of the mistletoe (Viscum album L.) phytotherapeutics could be considered as combined cytotoxic and "biological response modifying" activities (increasing host defense against cancer) that result from the activities of the plant lectins and the other biologically relevant substances. We found before that the aqueous extract Isorel, produced by Novipharm GmbH (Pörtschach, Austria) from the entire plant (planta tota) of fresh mistletoe under standardized conditions with bioassay validated batch consistency, can be valuable in experimental adjuvant cancer therapy increasing efficiency of cyclophosphamide chemotherapy. In current study we found that Isorel increases the reactivity of the tumor-bearing mice lymphocytes to the mitogens (ConA and LPS) in vitro, thus indicating its immune stimulating effects for the cancer-immunosuppressed lymphocytes. Moreover, Isorel inhibited the incorporation of 3H-labelled amino acids (protein synthesis) in various malignant cell lines. For the growth inhibition mostly higher MW components were responsible, although even less than 500 Da components were also active. We further analyzed the effects of drug application in vicinity of tumor (murine mammary carcinoma) and compared it with systemic effects. The animals carried mammary carcinoma in both hind limbs and were also injected with tumor cells i.v. to develop artificial lung metastases. Isorel was applied only at the right side (in the limb distal from the tumor) and caused persistent and almost complete inhibition of the tumor growth for 2/7 animals. Anticancer effects were less pronounced on the contralateral side tumors, although tumor growth rate was transiently reduced for some mice. Histology revealed that Isorel treatment, both at the side of tumor and systemically, increased the incidence of apoptosis and necrosis in the tumors, while reduction of mitosis was noticed only for the tumors in vicinity of the tumor exposed to Isorel. Finally, animals treated with Isorel had, on the average, three times less lung metastases than the controls. Thus, we conclude that both local and systemic effects of the application of Isorel could be of benefit for the tumor-bearing organism resulting in immunomodulation combined with tumor growth inhibition and reduction of metastases. According to the in vitro results, antitumorous effects could be the result not only of the mistletoe lectins and the other high MW factors, but also of the very low MW (< 500 Da) substances that deserve further analyses.

4-Hydroxynonenal as a second messenger of free radicals and growth modifying factor.

Immunohistochemical analysis of the distribution of the lipid peroxidation product 4-hydroxynonenal (HNE) in the brain of baboons exposed to experimental hemorrhagic traumatic shock or sepsis showed that systemic oxidative stress and the thereby generated HNE affect the blood:brain barrier and the regulation of cerebral blood flow determining secondary brain damage. Similarly, HNE was determined during ischemia in the brain blood vessels of rats exposed to ischemia/reperfusion injury of the brain. After reperfusion, HNE disappeared from the blood vessels but remained in neurones and in glial cells. Since HNE modulates cell proliferation and differentiation (including proto-oncogene expression), it is postulated that HNE might have prominent local and systemic effects that are not only harmful but beneficial, too, determining the outcome of various pathophysiological conditions based on oxidative stress.

Post-traumatic hormonal disturbances: prolactin as a link between head injury and enhanced osteogenesis.

Traumatic brain injury (TBI) combined with fractures of long bones or large joints is often associated with enhanced osteogenesis (early fracture healing accompanied by hypertrophic callus formation and/or heterotopic ossifications). Humoral factors that cause enhanced osteogenesis in patients with TBI are not yet identified. The aim of this study was to reveal if post-traumatic change(s) of hormone levels in patients with TBI and bone fractures could be associated with the phenomenon of enhanced osteogenesis. The blood values of adrenocorticotropic hormone (ACTH), cortisol, growth hormone (GH), parathyroid hormone (PTH) and prolactin (PRL) were studied weekly over a period of three months after injury in patients with bone fractures only, those with TBI only or combined bone fractures and TBI (patients exerting enhanced osteogenesis). Stress-hormones, ACTH and cortisol, or the hormones related to the bone growth (GH and PTH) did not show any particular post-traumatic changes in the blood of patients with combined injury that could be associated with the enhanced osteogenesis. On the other hand, patients with combined bone fractures and TBI accompanied by enhanced osteogenesis had significantly elevated PRL levels in blood during the 5th week of the post-traumatic period. Thus, the maximal PRL values were measured at the time when in this group of patients fractures were in consolidation and hypertrophic callus or heterotopic ossifications were developing (as verified by x-ray imaging). Hence, PRL does not only influence physiology of the bone metabolism but also seems to be one of the humoral factors involved in the phenomenon of enhanced osteogenesis in patients with TBI.

Spleen peptides (Polyerga) inhibit development of artificial lung metastases of murine mammary carcinoma and increase efficiency of chemotherapy in mice.

There are numerous attempts to find novel anticancer drugs or to improve therapeutic protocols based on application of chemotherapeutic agents and immunomodulators (biological response modifiers, cytokines, various plant or bacterial products). Among the preparations that have beneficial effects for the cancer bearing organism are preparations of spleen peptides (Polyerga). Hence, we analyzed if treatment with spleen oligopeptides GP-1 (active substance for the manufacture of Polyerga ampoules' solution injected as 0.5 microgram/kg every second day) if given alone or combined with chemotherapy (Endoxan 50 mg/kg single i.p. dose) of mice bearing artificial lung metastases of mammary carcinoma will have an impact on the metastases count and survival of the animals. The results obtained have shown that chemotherapy reduced metastases count and increased survival of the tumor bearing mice, while the use of GP-1 alone did not affect metastases development. However, combined GP-1 treatment and chemotherapy were more efficient in prevention of the metastases development than chemotherapy alone. Thus, in mice treated with GP-1 and Endoxan, the average metastases count was four times lower than in the mice treated by chemotherapy only, while 2/12 animals were without tumor nodules in the lungs. Finally, all the animals treated by chemotherapy alone died until the 42nd day after tumor transplantation, while at the same time, only 5/10 animals died receiving combined therapy. Thus, these results give an experimental support for the use of the spleen peptides in biotherapy (or combined therapy) of cancer.

Comparison of the effects of Viscum album lectin ML-1 and fresh plant extract (Isorel) on the cell growth in vitro and tumorigenicity of melanoma B16F10.

Numerous findings indicate that specific plant lectins acting against cancer could be major active components of Viscum album extracts, although activity of low molecular weight components (peptides, carbohydrates and alkaloids) might be as essential for the beneficial activity of the plain plant extracts, too. Thus, active principle of Viscum album extracts is still not understood, and is difficult to be analysed because of the complex composition of the extracts and uncertainty of the standardised effectiveness (batch consistency) of the extracts. The aims of this study were to compare the concentration dependent effects of the pure mistletoe lectin (ML-1) with the fresh plant Viscum album extract (Isorel) and its different MW components on the in vitro growth of ConA stimulated lymphocytes, on the growth and tumorigenicity (artificial lung metastases development) of murine melanoma B16F10 cells, and to compare concentration dependent effects of the different types of the Viscum album extracts in vitro (applying novel type of MTT assay). The results obtained indicate that the effects of Isorel used at high dose could be result of toxic activity of the mistletoe lectins ("ML-1 like" activity). Unlike ML-1, if used at low concentrations, Isorel selectively inhibited tumor cells, due the activity of the low MW components. On the other hand, the number of tumor nodules was reduced (in comparison to the control) equally in the lungs of mice injected with B16F10 cells pre-treated in vitro with the plain Viscum album extract or any of its modifications or ML-1. Hence, it is supposed that the beneficial therapeutic effects of Isorel might result from the combined biological activity of the high and the low MW components not lectins only. Similarly, in MTT assay low concentrations of all types of the Viscum album extract showed stronger inhibiting activity for B16F10 and HeLa cells than pure ML-1. According to these results we propose a standardisation of aqueous Viscum album extracts by comparing their and ML-1 concentration dependent activity on the tumor cells in vitro applying MTT bioassay described which should be relevant for further evaluation of their active principle and for improvement of biotherapy of cancer.