Isolation, characterization and strain-specific detection of canine-derived lactobacillus acidophilus

Lactobacilli are commensal gastrointestinal microbes commonly utilized in probiotic products, as they are believed to bestow multiple beneficial health effects to the host. Most well-studied lactobacilli have been isolated from feces. However, fecal isolates do not reflect the microbiota present in the upper gut, since different niches provide different microbial habitats. The fistulated dog model facilitates investigation of the microbiota in fresh intestinal samples without disturbing the physiology of the canine gut. In this study, jejunal lactobacilli from five permanently fistulated beagles were studied. We found that facultative Lactobacillus strains were abundant in the jejunal microbiota, and L. acidophilus was the dominant species. Repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprint profiles of L. acidophilus isolates revealed one predominant strain, named LAB20. Adhesion is an important factor in bacterial colonization of the host gut. In order to adhere, compete, and dominate within the host, numerous bacterial cell-surface factors are required to interact with the host mucosa. In this study, the protein profile of LAB20 was studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and cell structure was observed via transmission electron microscope (TEM). A surface (S) layer protein was revealed from LAB20. S-layer proteins form crystalline arrays of proteinaceous subunits in the outer layer of the cell wall and are involved in mediating bacterial adhesion to host surfaces. Inverse PCR revealed the DNA sequence of the LAB20 S-layer protein, alignment with other lactobacilli S-layer protein genes showed it was a novel one. The discovery of this novel S-layer protein in LAB20 enabled us to develop a strain-specific detection method. Real-time PCR primers targeting the variable region of the S-layer protein gene were used to detect and quantify LAB20 in dog intervention studies. We found that LAB20 persisted in one dog for over 6 weeks after the feeding period (6 × 108 CFU daily for 5 days), whereas the five dogs in the other study maintained high LAB20 numbers only during the feeding period (108 CFU daily for 3 days). Cultivation of fecal samples demonstrated that LAB20 transits through the dog gut and can be identified based on colony morphotype. TEM revealed a putative extracellular polysaccharide (EPS) layer that comprised LAB20’s outermost structure. Using antisense RNA strategy, EPS production was manipulated to investigate its potential impact on the ability of LAB20 to adhere to mucus and epithelial cells. LAB20 displayed significantly higher adhesion in canine cecal mucus relative to the EPS mutant SAA658 and could adhere to Caco-2 and HT-29 epithelial cells. This suggests that wild-type EPS plays an integral role in the adhesion of LAB20 in the host gut. Moreover, LAB20 attenuated lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in HT-29 cells, which indicates that LAB20 could be a probiotic candidate with anti-inflammatory properties. In conclusion, this study investigated the surface structure, persistence, adhesion ability, and probiotic potential of LAB20, the dominant L. acidophilus strain in the canine small intestine. Our results suggest that LAB20 has potential as a canine probiotic.Laktobasillit ovat bakteereita, joita löytyy nisäkkäiden suolistosta ja monista ympäristön ekologisista lokeroista. Joitain Lactobacillus-kantoja käytetään yleisesti probioottisissa tuotteissa tuottaakseen hyödyllisiä vaikutuksia kuluttajalle. Useimmat hyödylliset laktobasillit on eristetty ihmisen ulosteesta. Toisaalta isolaatit ulosteesta eivät heijasta suoliston ylempää mikrobiotaa ja yhden nisäkkään bakteerit eivät vältämättä osaa kolonisoida toisen nisäkkään suolistoa. Tämän takia hyödynnettiin tässä tutkimuksessa fistuloituja koiria, jotta löytääksemme potentiaalisia probiootteja koirille. Ohutsuolen sisällön näytteistä laktobasillit osoittautuivat vallitseviksi kaikissa viidessä tutkitussa koirassa. Lisäksi näiden laktobasillien joukossa yksi L. acidophilus kanta oli vallitseva ja nimettiin LAB20:ksi jatkotutkimuksiin. Tarttuminen suoliston pintaan on tärkeä ominaisuus bakteerien kolonisoidessa isännän ruuansulatuskanavaa. Siksi LAB20 proteomia tutkittiin, yhdessä elektronimikroskoppisen tarkastelun kanssa, ja pintaproteiinikerros (S-kerros) löytyi LAB20 soluseinästä. S-kerrosproteiinin N-terminuksen sekvensointi osoitti sen olevan uniikki S-kerrosproteiini. Ainutlaatuisuuden osoitus S-kerrosproteiinin osalta ja sen geenin sekvenssi LAB20-kannasta mahdollisti kantakohtaisen osoitusmenetelmän kehittämisen. Kvantitatiivisen LAB20-spesifisen PCR:n avulla oli mahdollista osoittaa ja kvantitoida LAB20 koirien syöttökokeissa. Osoittautui, että LAB20 säilyi suolistossa kuusi viikkoa LAB20 syöttämisen jälkeen yhdessä koirassa, ja oli korkealukuisena viidessä muussa koirassa syöttämisen aikana. Mielenkiintoisesti LAB20 ei ainoastaan säilynyt hengissä läpi ruuansulatuksen, mutta se voitiin myös identifioida pesäkemorfotyypin perusteella viljellyistä ulostenäytteistä. Tutkittiin LAB20:n adheesiokyky mukukseen ja suoliston epiteelisoluihin. Osoittautui, että LAB20 tarttui tilastollisesti enemmän koiran mukukseen kuin kontrolli ja kykeni tarttumaan Caco-2 ja HT-29 epiteelisoluihin. Lisäksi LAB20 attenuoi LPS-indusoitua IL-8 tuottoa HT-29 soluissa indikoiden että LAB20 olisi probioottikandidaatti tulehdusreaktioita vastaan. Tässä tutkimuksessa selvitettiin koiran ohutsuolessa vallitsevaa LAB20:n pintarakenteita, säilymistä suolistossa, tarttumiskykyä ja probioottista potentiaalia. Tulokset viittaavat LAB20-kannalla olevan probioottista potentiaalia koirille

The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro

Peer reviewe

Electromagnetic Energy Redistribution in Coupled Chiral Particle Chain-Film System

Abstract Metal nanoparticle-film system has been proved that it has the ability of focusing light in the gap between particle and film, which is useful for surface-enhanced Raman scattering and plasmon catalysis. The rapid developed plasmonic chirality can also be realized in such system. Here, we investigated an electromagnetic energy focusing effect and chiral near-field enhancement in a coupled chiral particle chain on gold film. It shows large electric field enhancement in the gap between particle and film, as well as chiral near field. The enhancement properties at resonant peaks for the system excited by left circularly polarized light and right circularly polarized light are obviously different. This difference resulted from the interaction of circularly polarized light and the chiral particle-film system is analyzed with plasmon hybridization. The enhanced optical activity can provide promising applications for the enhancement of chiral molecule sensor for this chiral particle chain-film system

Accelerated Urban Expansion in Lhasa City and the Implications for Sustainable Development in a Plateau City

Urbanization challenges regional sustainable development, but a slight expansion mechanism was revealed in a plateau city. We have integrated the urban expansion process and analyzed its determinants in Lhasa (Tibet), and we provide insightful suggestions for urban management and planning for Lhasa. The full continuum of the urban expansion process has been captured using time-series of high-resolution remote sensing data (1990–2015). Four categories of potential determinants involved in economic, demographic, social, and government policy factors were selected, and redundancy analysis was employed to define the contribution rates of these determinants. The results illustrate that considerable urban expansion occurred from 1990 to 2015 in Lhasa, with the area of construction land and transportation land increasing at rates of 117.2% and 564.7%, respectively. The urban expansion in the center of Lhasa can be characterized as temperate sprawl from 1990 through 2008, primarily explained by governmental policies and investment, economic development, tourist growth, and increased governmental investment resulting in faster urban expansion from 2008 to 2015, mainly occurring in the east, south, and west of Lhasa. In contrast with other cities of China, central government investment and “pairing-up support” projects have played an important role in infrastructure construction in Lhasa. The miraculous development of the tourism industry had prominent effects on this economic development and urbanization after 2006, due to the running of the Tibetan Railway. An integrative and proactive policy framework, the “Lhasa development model”, having important theoretical, methodological, and management implications for urban planning and development, has been proposed

Additional file 1: of Electromagnetic Energy Redistribution in Coupled Chiral Particle Chain-Film System

Figure S1. The optical properties of the studied system with different gaps between nanoparticles; Figure S2. Chiral near-field enhancement distributions for different resonant peaks; Figure S3. The optical properties of chiral particle-film system with different R; Figure S4. The volume-averaged chiral enhancement spectra between NPs-film in chiral particle-film system with different R; Figure S5. The volume-averaged chiral enhancement spectra between particles in chiral particle-film systems with different R; Figure S6. Optical properties of chiral particle chain-film systems with different particle numbers. (DOCX 1098 kb