Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.

Molecular characteristics of Clostridium difficile isolates from human and animals in the North Eastern region of India.

A total of 1034 samples were collected from different sources and C. difficile was isolated from 18 (9.04%) of 199 human, 9 (4.89%) of 184 cattle, 29 (12.44%) of 233 pig, and from 23 (13.94%) of 165 poultry samples. Variations were observed on the rate of isolation according to age and clinical conditions (diarrhoea). None of the samples from cow, sheep, goat, local chicken, and wild animals yielded any C. difficile. Out of those isolates, 8, 2, 19 and 6 isolates from human, cattle, pig and poultry, respectively were toxigenic. The toxigenic isolates carried both tcdA, and tcdB (A+B+) and most of the human and the pig isolates were also positive for binary toxin genes (cdtA and cdtB). The A+B+ isolates belonged to three different toxinotypes (0, VI and XXXIII). Human and pig A+B+ isolates belonged to three (045, 126 and ACD 019) and four (046, 087, 126 and ACD 011) different ribotypes, respectively and the ribotypes of two cattle isolates were 014 and ACD 010. Six A+B+ avian isolates belonged to six different ribotypes (014, 087, SLO 134, SLO 160, ACD 012, ACD 014). The non-toxigenic isolates from human, cattle, pig and poultry were grouped into 7, 4, 4 and 7 different ribotypes, respectively. PFGE analysis could not differentiate similar ribotypes/toxinotypes of toxigenic isolates. All the toxigenic isolates showed cytopathic effect on Vero and Hela cell monolayers at 1:100 dilutions of cell-free culture supernatants within 18-20 h of inoculation.

Isolation and characterization of Clostridium difficile from pet dogs in Assam, India.

One hundred and seventeen faecal samples from pet dogs (pup = 21 and adult = 96) brought for treatment to a veterinary clinic were examined for Clostridium difficile. A total of 16 (13.67%) samples were positive. Nine (56.25%) isolates were obtained from 17 adult dogs undergoing antibiotic treatment and this was significantly higher (p < 0.01) as compared to isolates from dogs without antibiotic treatment. Ten isolates (62.5%) were toxigenic (all toxinotype 0) and six were non-toxigenic. None of the isolates were positive for binary toxin genes. PCR ribotyping revealed three different ribotypes (012, 014 and 046) among A(+)B(+) isolates and five different ribotypes (010, SLO 131, and ACD 001 to ACD 003) among A(-)B(-) isolates. The PFGE analysis of toxigenic isolates revealed three different pulsotypes corresponding to the PCR ribotypes.

Survey of community-associated-methicillin-Resistant Staphylococcus aureus in Slovenia: identification of community-associated and livestock-associated clones.

The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Slovenia is poorly documented. The aim of this study was to investigate susceptibility patterns, virulence gene profile and clonality among MRSA isolates with positive screened resistance phenotype for CA-MRSA collected from patients in Slovenia, from January 2010 to December 2010. We included only MRSA isolates that were resistant to cefoxitin and oxacillin, and susceptible to at least two of the following four antibiotics: ciprofloxacin, erythromycin, clindamycin or gentamicin (presumptive CA-MRSA). Altogether 151 isolates fulfilled our screening phenotypic definition, 126 MRSA isolates were classified as CA-MRSA and 25 as HA-MRSA. Thirty-six per cent of them were resistant to ciprofloxacin, 24% to clindamycin, 33% to erythromycin and 13% to gentamicin. The mecA gene was detected in 150 isolates, while the mecC gene only in 1 isolate. The MRSA isolates were classified to 19 different clones. The most prevalent sequence types were ST5 (26.4%), ST45 (25.2%), ST22 (10.6%), ST398 (9.9%), ST8 (5.9%), ST7 (4.6%), ST1 (3.9%), ST152/377 (3.3%), ST228 (2.6%) and ST2883 (1.3%). The ST6, ST9, ST30, ST72, ST88, ST111, ST130, ST225 and ST772 were identified sporadically. The Panton-Valentine leukocidin (PVL) gene was detected in 13 (8.6%) isolates that belonged to ST5, ST7, ST8, ST22, ST72, ST88, ST 152/377 and ST772. Our results show high variability of CA-MRSA circulating in Slovenia and also the presence of LA-MRSA clones.

Exploring ComQXPA quorum-sensing diversity and biocontrol potential of Bacillus spp. isolates from tomato rhizoplane.

Bacillus subtilis is a widespread and diverse bacterium t exhibits a remarkable intraspecific diversity of the ComQXPA quorum-sensing (QS) system. This manifests in the existence of distinct communication groups (pherotypes) that can efficiently communicate within a group, but not between groups. Similar QS diversity was also found in other bacterial species, and its ecological and evolutionary meaning is still being explored. Here we further address the ComQXPA QS diversity among isolates from the tomato rhizoplane, a natural habitat of B. subtilis, where these bacteria likely exist in their vegetative form. Because this QS system regulates production of anti-pathogenic and biofilm-inducing substances such as surfactins, knowledge on cell-cell communication of this bacterium within rhizoplane is also important from the biocontrol perspective. We confirm the presence of pherotype diversity within B. subtilis strains isolated from a rhizoplane of a single plant. We also show that B. subtilis rhizoplane isolates show a remarkable diversity of surfactin production and potential plant growth promoting traits. Finally, we discover that effects of surfactin deletion on biofilm formation can be strain specific and unexpected in the light of current knowledge on its role it this process.

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.

Occurrence of toxigenic Clostridium difficile in edible bivalve molluscs.

Clostridium difficile is an anaerobic bacterium commonly considered to be responsible for antibiotic-associated gastrointestinal diseases, ranging from diarrhea of varying severity to pseudomembranous colitis. The aim of this study was to assess the occurrence of C. difficile in marine edible bivalve molluscs, which, as filter feeding organisms, are able to accumulate particles suspended in water, including microorganisms. Samples of Mytilus galloprovincialis, Tapes philippinarum, and Venus verrucosa were collected from mussel farms and fishmongers in the province of Naples (Southern Italy). C. difficile was found in 49% of the 53 samples investigated. Sixteen isolates were grouped in 12 known different PCR ribotypes (001, 002, 003, 010, 012, 014/020, 018, 045, 070, 078, 106, and 126), whereas 10 additional isolates were grouped in 8 new PCR riboprofiles. Two toxinotypes (0 and V) were found. Fifty eight percent of the isolates were toxigenic. These findings indicate that toxigenic C. difficile strains can be isolated in bivalve molluscs. Marine filter feeding organisms, therefore, may be considered as reservoir of toxigenic strains of C. difficile. The ingestion of raw or poorly cooked contaminated seafood and the high temperature resistance of the spore-forming C. difficile could represent an important source of exposure and pose human health concern.

Calcium dependencies of regulated exocytosis in different endocrine cells.

Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca(2+) concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca(2+) sensitivity in endocrine cells, accurate and fast determination of Ca(2+) dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that leads to a robust exocytotic activity. Slow increases in the [Ca(2+)](i) revealed exocytotic phases with different Ca(2+) sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca(2+) sensitivity 'classes' of secretory vesicles have been observed: a highly Ca(2+)-sensitive, a medium Ca(2+)-sensitive and a low Ca(2+)-sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca(2+) sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca(2+) sensor protein.

Use of different molecular typing methods for the study of heterogeneity within Clostridium difficile toxinotypes V and III.

Clostridium difficile strains of toxinotypes III (n = 13) and V (n = 45) were typed by agarose gel-based PCR ribotyping, capillary gel electrophoresis-based PCR ribotyping and PFGE using two different restriction enzymes, SmaI and SacII. With conventional agarose gel-based PCR ribotyping, toxinotype III strains were distributed among six different PCR ribotypes and toxinotype V strains into three different PCR ribotypes. Capillary gel electrophoresis-based ribotyping was more discriminatory for toxinotype V strains, with six different ribotypes found. With PFGE using SmaI, all toxinotype III strains grouped together into a single pulsotype. Using SacII, ribotype 027 strains grouped together with >90 % similarity and were <83 % similar to other ribotypes of toxinotype III strains. Within ribotype 078, seven (SmaI) and eight (SacII) different pulsotypes were found, whilst ribotype 126 strains belonged to one (SmaI) and two (SacII) pulsotypes. Within ribotype 066, it was possible to distinguish between pig and human isolates. Using SacII, a further distinction could also be made between pig isolates from two different farms. PFGE (SmaI and SacII) clustered strains according to their toxinotype; however, correlation of PFGE and ribotyping was better with SacII. These data suggest that toxinotype III strains are a more heterogeneous group than toxinotype V strains and that SacII is more discriminatory than SmaI. Alternatively, the use of both enzymes simultaneously could improve PFGE typing of C. difficile.

Correlation of multidrug resistance, toxinotypes and PCR ribotypes in Clostridium difficile isolates from Kuwait.

Clostridium difficile is a common cause of nosocomial diarrhea. its role in community-acquired diarrhea is also becoming an important public health concern. Hardly any studies have correlated strain ribotypes, toxinotypes and multidrug resistant (MDR) profiles. To investigate these characteristics, 65 C. difficile isolates obtained from stool samples of patients whose cultures were negative on admission but became positive after 48 h of admission to the ICUs of our hospitals were studied to determine the prevalent ribotypes, toxinotypes and their relationship with the MDR profiles using ELISA/cytotoxicity assays, PCR and Etest methods. The toxin-producing strains were toxinotyped by the PCR-RFLP technique. Of the 65 isolates, 42 (64.6%) were toxigenic (T). The isolates were of diverse ribotypes but types 097, 078, 056 and 039 (NT) were predominant. thirty (71.4%) of 42 T and 13 (56.5%) of 23 NT strains were multiresistant to 3 or more antibiotics. Only 3 toxinotypes (0, "V-like" and XII) were encountered. Of the 42 t strains, 30 (71.4%) were of toxinotype 0, and 12 belonged to variant toxinotypes: 4 (9.4%) to toxinotype XII and 8 (19%) to "V-like" toxinotype in which amplified B1 PCR fragments was amplified as expected for toxinotype V but the A3 PCR fragment could not be amplified. The 43 mDR strains were assigned to 3 arbitrary resistance groups; groups 1, 11 and III. the most prevalent isolates (37; 86.1%) were in group II. Of the predominant T ribotypes (097, 078 and 056), c. 62% clustered in group II. Although the number of strains toxinotyped was small, ribotyping and toxinotyping correlated well with the published literature, except for 078 with a novel "V-like" toxinotype. Antibiogram was not as clear-cut.

The physiology of rodent beta-cells in pancreas slices.

Beta-cells in pancreatic islets form complex syncytia. Sufficient cell-to-cell electrical coupling seems to ensure coordinated depolarization pattern and insulin release that can be further modulated by rich innervation. The complex structure and coordinated action develop after birth during fast proliferation of the endocrine tissue. These emergent properties can be lost due to various reasons later in life and can lead to glucose intolerance and diabetes mellitus. Pancreas slice is a novel method of choice to study the physiology of beta-cells still embedded in their normal cellulo-social context. I present major advantages, list drawbacks and provide an overview on recent advances in our understanding of the physiology of beta-cells using the pancreas slice approach.

Long-term Hg pollution induced Hg tolerance in the terrestrial isopod Porcellio scaber (Isopoda, Crustacea).

The aim of our work was to assess the pollution-induced community tolerance (PICT) of isopod gut microbiota and pollution-induced isopod population tolerance (PIPT). Animals collected from a chronically Hg polluted and an unpolluted location were exposed for 14 days to 10microg Hg/g dry food under laboratory conditions. The lysosomal membrane stability, hepatopancreas epithelium thickness, feeding activity and animal bacterial gut microbiota composition were determined. The results confirm the hypothesis that the response to short-term Hg exposure differs for animals from the Hg polluted and the unpolluted field locations. The animals and their gut microbiota from the Hg polluted location were less affected by Hg in a short-term feeding experiment than those from the unpolluted environment. We discuss the pollution-induced population tolerance of isopods and their gut microbiota as a measure of effects of long-term environmental pollution. The ecological consequences of such phenomena are also discussed.

Is Clostridium difficile-associated infection a potentially zoonotic and foodborne disease?

Clostridium difficile has received much attention in recent years because of the increased incidence and severity of nosocomial disease caused by this organism, but C. difficile-associated disease has also been reported in the community, and C. difficile is an emerging pathogen in animals. Early typing comparisons did not identify animals as an important source for human infection, but recent reports have shown a marked overlap between isolates from calves and humans, including two of the predominant outbreak types, 027 and 017. C. difficile has also been found in retail meat samples, suggesting that food could be involved in the transmission of C. difficile from animals to humans.

Distinct role of Rab3A and Rab3B in secretory activity of rat melanotrophs.

Members of the Rab3 (A-D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share approximately 80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant.

KATP-channels in beta-cells in tissue slices are directly modulated by millimolar ATP.

In pancreatic beta-cells, inhibition of K(ATP)-channels plays a pivotal role in signal transduction of glucose-induced insulin release. However, the extreme sensitivity of K(ATP)-channels to its ligand ATP as found in inside-out patches is not directly compatible with modulation of these channels at physiological [ATP](i). We studied K(ATP)-channel sensitivity to ATP in beta-cells in dispersed culture and in fresh pancreatic tissue slices. Physiological [ATP](i) blocks more than 99% of K(ATP)-channels in cultured beta-cells, while only 90% in beta-cells in slices, indicating reduced sensitivity to ATP in the fresh slices. Applying cytosolic factors like ADP, phosphatidylinositol-4,5-bisphosphate (PIP(2)) or oleoyl-CoA did not restore the K(ATP)-channel sensitivity in cultured beta-cells. Our data suggest that interaction between SUR1 and Kir6.2 subunit of the K(ATP)-channel could be a factor in sensitivity modulation. Tissue slices are the first beta-cell preparation to study direct K(ATP)-channel modulation by physiological [ATP](i).

Anaerobic bacteria in the gut of terrestrial isopod Crustacean Porcellio scaber.

Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and Enterococcus were retrieved, as well as sequences from 'A1 subcluster' of the wall-less mollicutes. Bacteria from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable, i.e. predominantly oxic, conditions.

A European survey of diagnostic methods and testing protocols for Clostridium difficile.

OBJECTIVE: To conduct a survey of the methods used in clinical microbiology laboratories in Europe to diagnose infection with Clostridium difficile. METHODS: A questionnaire was devised and sent to a co-ordinating member of the Study Group in each of eight European countries. This co-ordinator was in charge of forwarding the questionnaire to hospital laboratories arbitrarily selected. The number of laboratories in each country was determined on the basis of one laboratory for 10,000 beds of hospitalization. This questionnaire covered different aspects pertaining to Clostridium difficile associated to diarrhea (CDAD) diagnosis such as circumstances of request, criteria used for undertaking C. difficile investigations, methods used for the diagnosis, etc. RESULTS: A total of 212 questionnaires were completed and submitted for analysis: 87.7% of laboratories reported routinely performing C. difficile diagnostic tests. Methods used included toxin detection (93%), culture (55%), and glutamate dehydrogenase (GDH) detection (5.9%). Among the laboratories detecting toxins, different enzyme immunoassays (EIA) and cytotoxicity assays were used in 79% and 17.3% of cases, respectively. Among the different strategies reported, 4.8% were considered suboptimal for the diagnosis of C. difficile infections, but marked discrepancies could be observed between countries. The overall incidence (median) of CDAD was estimated at 1.1 for 1,000 patient admissions. CONCLUSION: The results of this study suggest marked discrepancies between laboratories and also between countries regarding the criteria by which C. difficile is investigated for, and the methods and the strategies that are used for the diagnosis of C. difficile. These discrepancies could be explained by the lack of clear guidelines for C. difficile diagnosis in each country, and by the importance that physicians attach to C. difficile. Precise guidelines for C. difficile diagnosis would be the first step to make possible accurate comparison of the incidence and the epidemiology of CDAD from one hospital to another or from one country to another.

Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells.

Synaptotagmin I (Syt I), a low-affinity Ca(2+)-binding protein, is thought to serve as the Ca(2+) sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca(2+)], suggesting that Syt I may play a more complex role in determining the high-affinity Ca(2+) dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca(2+)-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca(2+)-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca(2+)-dependent exocytosis by cytosol dialysis with solutions containing 1 microM [Ca(2+)] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca(2+)-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca(2+)].

Binary toxin producing Clostridium difficile strains.

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.

Cardiovascular physiology: simulation of steady state and transient phenomena by using the equivalent electronic circuit.

By using commercially available software it is readily possible to design electronic circuits and to analyze them. By introducing the concept of equivalent quantities a simulation of various physiological phenomena is possible. This includes the steady state as well as various complex transient phenomena. This paper describes the use of an equivalent electronic circuit in simulating the cardiovascular system. It allows a stepwise upgrading. The first step is a one-ventricle circuit similar to the Starling heart-lung preparation. The final step is an equivalent circuit allowing simulation of various normal as well as pathological states (e.g. effects of heart rate, negative intrathoracic pressure, exercise, hemorrhage, heart failure, and hypertension). The degree of disturbance can be set by adjusting the value of single components. Following this, the optimal type of compensation (e.g. the increase in blood volume in failure of the right ventricle; systemic venoconstriction in failure of the left ventricle) of the basic disturbance can be searched for, activated and the consequences studied. The described approach has been found a useful tool in teaching physiology and pathophysiology for postgraduate medical students.