Virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method

We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars

Porcine E. coli: Virulence-Associated Genes, Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

We established an automated screening method to characterize adhesion of Escherichia colito intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coliisolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity thanE. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars

Comparative molecular, epidemiologic and phylogenetic analyses concerning the adaptation of Escherichia coli in wild boars and domestic pigs

Ziel der vorliegenden Arbeit war es, erstmalig Daten über die coliforme Mikroflora der Wildschweine sowie grundlegende Erkenntnisse über die Diversität von E. coli-Populationen und über das natürliche Vorkommen von Virulenz- und Kolonisationsgenen in nicht vom Menschen beeinflussten Schweinepopulationen zu erhalten. Daher wurden kommensale E. coli aus dem Darm von gesunden Wildschweinen geno- und phänotypisch untersucht. Die Ergebnisse wurden mit bereits publizierten Daten kommensaler E. coli von Hausschweinen verglichen. Im ersten Teil der Arbeit konnte gezeigt werden, dass die Diversität der E. coli Stämme für jedes Wildschwein individuell und hoch variabel, jedoch geringer als die Diversität von Hausschwein E. coli war. Über die Hälfte der untersuchten Wildschwein E. coli wiesen zudem mindestens eines der für schweinepathogene E. coli typischen Virulenzgene auf. Das Toxingen astA war in fast der Hälfte aller Stämme nachweisbar. Alle vier ECoR Gruppen waren in jedem der untersuchten Darmabschnitte vertreten. Die geno- und phänotypische Charakterisierung der Wildschwein E. coli im zweiten Teil der Arbeit belegte, dass für schweinepathogene E. coli typische Virulenzgene bei Wildschwein E. coli nur sporadisch nachgewiesen werden konnten, wohingegen einige für extraintestinal-pathogene E. coli (ExPEC) typische, virulenzassoziierte Gene (mat, fyuA, sitD chr., astA, kpsMTII, ibeA, malX) häufiger als bei kommensalen Hausschwein E. coli auftraten. Der Großteil der untersuchten Isolate gehörte den ECoR Gruppen A und B1 an, wobei die ECoR Gruppe B2, deren Vertreter deutlich mehr virulenzassoziierte Gene aufwiesen, häufiger als bei kommensalen E. coli von Hausschweinen vorkam. Außerdem wiesen E. coli von Wildschweinen gegenüber denen von Hausschweinen durchschnittlich höhere Adhäsionsraten an IPEC J2 Zellen und eine effizientere Verwertung der 49 getesteten Kohlenhydrate auf. Diese Ergebnisse legen nahe, dass sich E. coli von Wildschweinen in einigen Eigenschaften von Hausschwein E. coli unterscheiden. Das bessere Adhäsionsvermögen, die effizientere Kohlenhydratverwertung, die geringere Diversität sowie das häufigere Vorkommen von Kolonisationsgenen und der ECoR Gruppe B2 könnten eine Folge des durch den Menschen eingeleiteten Domestizierungsprozesses und der heutigen konventionellen Schweinehaltung sein.The main objectives of the present study were to obtain preliminary information about the E. coli microflora from wild boars, as well as to get basic knowledge about the diversity and natural occurrence of selected virulence and antimicrobial resistance genes of E. coli isolated from pig populations, which are unaffected by humans as far as possible. For this purpose, commensal intestinal E. coli from healthy wild boars were analyzed geno- and phenotypically to compare the results with already published data from commensal E. coli from domestic pigs. The first part of this study showed that the diversity of the E. coli microflora from wild boars was unique and highly variable but lower than the diversity of E. coli from domestic pigs. In addition, more than half of the isolated E. coli strains exhibited at least one of the virulence genes which are characteristic for porcine pathogenic E. coli. Almost half of the isolated E. coli strains possessed the gene astA coding for a heat stable cytotoxin. Members of each of the four ECoR groups were found in all selected sections of the intestine. The geno- and phenotypical characterization of E. coli from wild boars in the second part of this study could prove that for porcine pathogenic intestinal E. coli typical virulence genes are only sporadically distributed among E. coli from wild boars, whereas some of the virulence associated genes, which are typical for extraintestinal pathogenic E. coli (ExPEC) (mat, fyuA, sitD chr., astA, kpsMTII, ibeA, malX) occurred more frequently in E. coli from wild boars than in those from domestic pigs. The majority of the isolated E. coli were members of the ECoR-groups A and B1, whereas the ECoR group B2, whose members clearly exhibited more virulence associated genes, was represented more frequently than in commensal E. coli from domestic pigs. Furthermore, E. coli from wild boars showed higher adhesion rates to IPEC-J2-cells on average and a more efficient utilization of 49 tested carbohydrates than those from domestic pigs. These results suggest that there exist differences between the E. coli microflora from wild boars and those from domestic pigs. The enhanced adhesion capacity, more efficient utilization of carbohydrates, lower diversity and more frequent occurrence of colonization genes and of E. coli representing the ECoR group B2 could be a consequence of the domestication process initiated by humans and conventional pig husbandry

MSM13/3 raw data of EM120 multibeam echosounder (bathymetry, beam time series & water column)

Between 25.10. and 18.11.2009, Multibeam data based on the KONGSBERG Simrad EM120 were acquired during RV Maria S. Merian cruise MSM13/3.The investigation of diversity and community distribution of chemosynthetic ecosystems related to fluid escape structures at the eastern and central provinces of the Nile fan (Eastern Mediterranean Sea) was the main research target. Additionally element transport rates and fluid export related to these fluid seeps was further explored. This cruise was in corporation with the DIWOOD program. Therefore wood, which was deployed in 2006/2007, was recovered to investigate the influence of wood degradation on ecosystems. To collect gas/fluid and sediment samples, the ROV QUEST4000 (MARUM) was utilized. During the cruise hydroacoustic systems (Multibeam and Parasound) were recorded to generate high-resolution maps of the research areas as well as to identify and to quantify seepage structures. CI Citation: Paul Wintersteller ([email protected]) as responsible party for bathymetry raw data ingest and approval. Description of the data source: During the MSM13-3 cruise, the hull-mounted KONGSBERG EM120 multibeam echosounder (MBES) was utilized for bathymetric mapping in water depth beyond 1000 m as it allows accurate bathymetric mapping up to full ocean depth. Two linear transducer arrays in Mills Cross configuration transmit a nominal sonar frequency of 12 kHz with an maximum beam width of 130° across track and 1° along track. 191 individual beams were created per ping. The actual footprint of a beam has a dimension of 1° by 2°. On flat bottom, the achievable swath width can reach up to six times the water depth. The angular coverage sector and beam pointing angles were set to vary automatically with depth according to achievable coverage. For further information on the system, consult https://www.km.kongsberg.com/. Responsible person during this cruise / PI: Paul Wintersteller ([email protected]), Heiko Sahling and Miriam Römer ([email protected]). Chief Scientist: Antje Boetius CR: https://www.tib.eu/de/suchen/download/?tx_tibsearch_search%5Bdocid%5D=awi%3Adoi~10.2312%252Fcr_msm13_3&cHash=91ccabc6630ae9bbd4b3fc995f742237#download-mark CSR: https://www2.bsh.de/aktdat/dod/fahrtergebnis/2009/20100012.ht

Effects of intramuscularly administered enrofloxacin on the susceptibility of commensal intestinal Escherichia coli in pigs (sus scrofa domestica)

Abstract Background In the European Union, various fluoroquinolones are authorised for the treatment of food producing animals. Each administration poses an increased risk of development and spread of antimicrobial resistance. The aim of this study was to investigate the impact of parenteral administration of enrofloxacin on the prevalence of enrofloxacin and ciprofloxacin susceptibilities in the commensal intestinal E. coli population. Methods E. coli isolates from faeces of twelve healthy pigs were included. Six pigs were administered enrofloxacin on day 1 to 3 and after two weeks for further three days. The other pigs formed the control group. MIC values were determined. Virulence and resistance genes were detected by PCR. Phylogenetic grouping was performed by PCR. Enrofloxacin and ciprofloxacin were analysed in sedimentation samples by HPLC. Results Susceptibility shifts in commensal E. coli isolates were determined in both groups. Non-wildtype E. coli could be cultivated from two animals of the experimental group for the first time one week after the first administration and from one animal of the control group on day 28. The environmental load with enrofloxacin in sedimentation samples showed the highest amount between days one and five. The repeated parenteral administration of enrofloxacin to pigs resulted in rapidly increased MIC values (day 28: MIC up to 4 mg/L, day 35: MIC ≥ 32mg/L). E. coli populations of the control group in the same stable without direct contact to the experimental group were affected. Conclusion The parenteral administration of enrofloxacin to piglets considerably reduced the number of the susceptible intestinal E. coli population which was replaced by E. coli strains with increased MIC values against enrofloxacin. Subsequently also pigs of the control were affected suggesting a transferability of strains from the experimental group through the environment to the control group especially as we could isolate the same PFGE strains from both pig groups and the environment

Methane fluxes and carbonate deposits at eastern Mediterranean cold seeps

High acoustic seafloor-backscatter signals characterize hundreds of patches of methane-derived authigenic carbonates and chemosynthetic communities associated with hydrocarbon seepage on the Nile Deep Sea Fan (NDSF) in the Eastern Mediterranean Sea. During a high-resolution ship-based multibeam survey covering a ~ 225 km**2 large seafloor area in the Central Province of the NDSF we identified 163 high-backscatter patches at water depths between 1500 and 1800 m, and investigated the source, composition, turnover, flux and fate of emitted hydrocarbons. Systematic Parasound single beam echosounder surveys of the water column showed hydroacoustic anomalies (flares), indicative of gas bubble streams, above 8% of the high-backscatter patches. In echosounder records flares disappeared in the water column close to the upper limit of the gas hydrate stability zone located at about 1350 m water depth due to decomposition of gas hydrate skins and subsequent gas dissolution. Visual inspection of three high-backscatter patches demonstrated that sediment cementation has led to the formation of continuous flat pavements of authigenic carbonates typically 100 to 300 m in diameter. Volume estimates, considering results from high-resolution autonomous underwater vehicle (AUV)-based multibeam mapping, were used to calculate the amount of carbonate-bound carbon stored in these slabs. Additionally, the flux of methane bubbles emitted at one high-backscatter patch was estimated (0.23 to 2.3 × 10**6 mol a**-1) by combined AUV flare mapping with visual observations by remotely operated vehicle (ROV). Another high-backscatter patch characterized by single carbonate pieces, which were widely distributed and interspaced with sediments inhabited by thiotrophic, chemosynthetic organisms, was investigated using in situ measurements with a benthic chamber and ex situ sediment core incubation and allowed for estimates of the methane consumption (0.1 to 1 × 10**6 mol a**-1) and dissolved methane flux (2 to 48 × 10**6 mol a**-1). Our comparison of dissolved and gaseous methane fluxes as well as methane-derived carbonate reservoirs demonstrates the need for quantitative assessment of these different methane escape routes and their interaction with the geo-, bio-, and hydrosphere at cold seeps

(Appendix) Visually determined major and minor diameters of gas bubbles emanating from five sites at the high-backscatter patch C-1

High acoustic seafloor-backscatter signals characterize hundreds of patches of methane-derived authigenic carbonates and chemosynthetic communities associated with hydrocarbon seepage on the Nile Deep Sea Fan (NDSF) in the Eastern Mediterranean Sea. During a high-resolution ship-based multibeam survey covering a ~ 225 km2 large seafloor area in the Central Province of the NDSF we identified 163 high-backscatter patches at water depths between 1500 and 1800 m, and investigated the source, composition, turnover, flux and fate of emitted hydrocarbons. Systematic Parasound single beam echosounder surveys of the water column showed hydroacoustic anomalies (flares), indicative of gas bubble streams, above 8% of the high-backscatter patches. In echosounder records flares disappeared in the water column close to the upper limit of the gas hydrate stability zone located at about 1350 m water depth due to decomposition of gas hydrate skins and subsequent gas dissolution. Visual inspection of three high-backscatter patches demonstrated that sediment cementation has led to the formation of continuous flat pavements of authigenic carbonates typically 100 to 300 m in diameter. Volume estimates, considering results from high-resolution autonomous underwater vehicle (AUV)-based multibeam mapping, were used to calculate the amount of carbonate-bound carbon stored in these slabs. Additionally, the flux of methane bubbles emitted at one high-backscatter patch was estimated (0.23 to 2.3 × 106 mol a− 1) by combined AUV flare mapping with visual observations by remotely operated vehicle (ROV). Another high-backscatter patch characterized by single carbonate pieces, which were widely distributed and interspaced with sediments inhabited by thiotrophic, chemosynthetic organisms, was investigated using in situ measurements with a benthic chamber and ex situ sediment core incubation and allowed for estimates of the methane consumption (0.1 to 1 × 106 mol a− 1) and dissolved methane flux (2 to 48 × 106 mol a− 1). Our comparison of dissolved and gaseous methane fluxes as well as methane-derived carbonate reservoirs demonstrates the need for quantitative assessment of these different methane escape routes and their interaction with the geo-, bio-, and hydrosphere at cold seeps

Porcine E. coli: Virulence-Associated Genes, Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

We established an automated screening method to characterize adhesion of Escherichia colito intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coliisolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity thanE. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.This article is from PLoS ONE 8, no. 4 (2013): e59242m, doi:10.1371/journal.pone.0059242.</p