28 research outputs found
SPECIFIC DETECTION OF Salmonella spp. WITH MOLECULAR BIOLOGICAL TECHNIQUES
No full textA method for specific detection of Salmonella spp. was developed based on 16S rRNA targeted PCR (polymerase chain reaction). A method based on recognition of specific region of 16S rRNA gene was published previously by Lin and Tsen in 1996. The sequence analysis of recently determined small subunit ribosomal genes from Salmonella spp. showed however, that the 16SF1 and 16SIII primers are not suitable for specific amplification of all Salmonella strains. 16SF1 was modified and a new forward PCR primer was designed. The specificity of the method was tested using 87 Salmonella strains and 30 non-Salmonella strains of the family Enterobacteriaceae and compared with two other previously published methods and found to be 100% specific. The reverse primer was also labelled with a tetramethylrhodamine isothyiocianate fluorescent dye and successfully used as a probe in in situ experiments
Priprava kvantitativne molekularno biološke metode za ugotavljanje števila celic vrste Salmonella enterica
No full textPripravili smo molekularno biološko metodo za hitro štetje bakterijskih celic iz vrste Salmonella enterica, ki temelji na kompetitivni različici verižne reakcije s polimerazo (c-PCR). Uporabili smo začetna oligonukleotida MINF in MINR-Hex, ki sta zagotavljala specifičnost metode. Notranji standard DNK smo pripravili z encimskim razrezom in lepljenjem stranskih fragmentov pomnožka gena za 16S rRNK seva S. Agona (U92197). Notranji standard DNK je imel isti prepoznavni mesti za začetna oligonukleotida kot tarčna DNK, bil pa je za 27 bp krajši. Pomnožke c-PCR smo ločevali s kapilarno elektroforezo in rezultate obdelali z regresijsko analizo. Z opisano metodo in 5 × 10–5 redčenim notranjim standardom smo lahko učinkovito ugotavljali število celic vrste S. enterica v bujonski kulturi v območju med 1,2 × 104 in 6 × 105 celic na reakcijsko mešanico. Z metodo smo v preliminarnem poskusu ugotavljali število celic seva S. Livingstone v bujonski kulturi in rezultate primerjali z rezultati indirektne števne metode. Rezultati opisane metode c-PCR so bili 2,9 krat višji od rezultatov indirektnega štetja kolonij, vendar je glede na številne prednosti opisana metoda kljub temu uporabna za hitro, specifično in dovolj natančno ugotavljanje števila celic vrste S.enterica v naravnih mikrobioloških vzorcih
Vpliv različnih hitrih metod osamitve genomske dnk salmonel na občutljivost PCR
No full textThe purpose of our research was to determine the effect of rapid methods for isolation of Salmonella genomic DNA on PCR sensitivity. Salmonella specific primers ST11 and ST15 were used for DNA amplification. Among tested procedures the best PCR sensitivity was achieved after treating of sample with NaOH and SDS solutions and heating of sample in water bath at 95o C for 15 min. PCR detection limit for Salmonella in pure and mixed cultures was in a range from 104 to 105 cfu ml-1. Described procedure for DNA isolation is rapid and not work-consuming.Namen raziskave je bil proučiti hitre metode osamitve genomske DNK salmonel in določiti njihov vpliv na občutljivost PCR. Za pomnoževanje DNK sta bila uporabljena za rod Salmonella specifična začetna oligonukleotida ST11 in ST15. Izmed preskušenih postopkov je bila najboljša občutljivost PCR v primeru, ko smo spranim celicam dodali mešanico raztopin NaOH in SDS ter vzorec 15 minut segrevali v vodni kopeli pri 95o C. Prag občutljivosti PCR za določanje salmonel tako v čisti kot v mešani kulturi je bil v območju 104-105 cfu ml-1. Opisan postopek osamitve DNK je hiter in izvedbeno ni zahteven
Osamitev in opis vampnih spirohet
No full textIz vzorca vampne vsebine smo osamili tri seve vampnih spirohet. Morfologijo osamljenih sevov smo opazovali s fazno kontrastno in epifluorescentno mikroskopijo ter jih opisali s hitrimi biokemijskimi preskusi (API 50CH) in molekulsko biološkimi preiskavami. Rezultati preiskav so pokazali, da sta si seva SM1 in SM3 zelo podobna in značilno različna od seva SM2. Vsi osamljeni sevi so bili Gram negativni. Celice sevov SM1 in SM3 so bile manjše od celic seva SM2, pri sevih SM1 in SM3 je bila krajša tudi amplituda in valovna dolžina posameznega zavoja. Od 49 ogljikovih hidratov so vsi osamljeni sevi razkrajali sedem ogljikovih hidratov, sev SM2 pa je razkrajal še dodatnih 14. Primerjava literaturnih podatkov razkroja ogljikovih hidratov tipskih sevov T. bryantii in T. saccharophilum je pokazala, da se seva spirohet SM1 in SM3 razlikujeta od opisanih vrst, sev SM2 pa je podoben tipskemu sevu T. saccharophilum. Veliko raznolikost med preiskovanimi sevi pa smo ugotovili s teoretično in eksperimentalno preiskavo RFLP, pri čemer smo gen za 16S rRNK sevov T. bryantii, T. saccharophilum, SM1, SM3 in SM2 razrezali z različnimi restrikcijskimi encimi
Closing gaps for performing a risk assessment on Listeria monocytogenes in ready-to-eat (RTE) foods : activity 3, the comparison of isolates from different compartments along the food chain, and from humans using whole genome sequencing (WGS) analysis
No full textWe would like to thank all the persons and institutes that have provided the project with isolates and accompanying information. Without them, this project would not have been possible. Lin Cathrine T. Brandal, Norwegian Institute of Public Health, Norway Julio Vázquez Moreno and Raquel Abad Torreblanca, Instituto de Salud Carlos III, Spain Marc Lecuit, Institut Pasteur, France Alexandre Leclercq, Institut Pasteur, France Iva Hristova, National Center of Infectious and Parasitic Diseases, Bulgaria Marija Trkov, National Laboratory of Health, Environment and Food, Slovenia Cecilia Jernberg, Public Health Agency of Sweden, Sweden Ariane Pietzka, Austrian Agency for Health and Food Safety, Austria Eelco Franz and Ingrid Friesema, RIVM, The Netherlands Carlo Spanu, University of Sassari Sardinia Ifip, French Institute for Pig and Pork Industry, Maisons-Alfort, France All the NRLs for providing the isolates from the EU baseline study Special thanks to Sylvain Brisse and Alexandra Moura, Institut Pasteur, France, for providing cgMLST data. The authors would also like to thank the EFSA staff members: Maria Teresa da Silva Felicio, Beatriz Guerra, Ernesto Lìebana and Valentina Rizzi as well as the members of the Working Group on Listeria monocytogenes contamination of ready-to-eat foods: Kostas Koutsoumanis, Roland Lindqvist, Moez Sanaa, Panagiotis Skandamis, Niko Speybroek, Johanna Takkinen and Martin Wagner for the support, revisions and suggestions during the development of the present procurement activity and report
Molekularne značajke sojeva Escherichia coli izoliranih iz različitih uzoraka hrane
No full textSince food represents a possible source of pathogenic and antibiotic-resistant Escherichia coli strains, we analyzed 84 isolates from food samples identified in 2007 and 2008 at the National Institute of Public Health in Slovenia. Using polymerase chain reaction (PCR), the isolates were classified into phylogenetic groups and subgroups following the Clermont method. Forty-two (50 %) and thirty (35.7 %) isolates were classified into commensal gut phylogenetic groups A and B1, respectively. Only ten (11.9 %) and two (2.4 %) isolates were assigned to the phylogenetic groups D and B2, which include mainly extraintestinal pathogenic E. coli strains. The strains were further analyzed for the presence of various virulence genes and plasmid-mediated quinolone resistance qnr genes. Virulence genes stx1, stx2, both stx1 and stx2, ehxA and eae associated with Shiga-toxin producing E. coli were detected in one (1.2 %), five (6 %), five (6 %), eight (9.5 %) and three (3.7 %) isolates, respectively. Seventy-four (88.1 %) isolates carried the gene fimH, whereas virulence genes characteristic of extraintestinal pathogenic E. coli, hra, ompTAPEC and iha, were detected in nine (11 %), eight (9.5 %) and six (7 %) isolates, respectively. Genes kpsMTII, sfa, usp and vat were discovered in single isolates, whereas hlyA, bmaE, cnf, hbp and sat, as well as plasmid- mediated quinolone resistance genes qnr, were not detected in the analyzed strains. Our results show that various food items are indeed a source of intestinal and, albeit to a lesser extent, of extraintestinal pathogenic E. coli strains.Hrana predstavlja mogući izvor patogenih i na antibiotik otpornih sojeva bakterije Escherichia coli, pa je u radu analizirano 84 izolata iz uzoraka hrane identificiranih 2007. i 2008. godine na Nacionalnom institutu za javno zdravstvo u Sloveniji. Pomoću metode PCR (lančana reakcija polimeraze) prema Clermontu izolati su razvrstani u filogenetske skupine i podskupine. Četrdeset i dva (50 %) su izolata svrstana u filogenetsku skupinu A, a njih trideset (35,7 %) u skupinu B1. Te dvije skupine uglavnom obuhvaćaju komenzale crijevne mikroflore. Deset je izolata (11,9 %) svrstano u skupinu D, a samo njih dva (2,4 %) u skupinu B2. U tim se skupinama uglavnom nalaze ekstraintestinalni patogeni sojevi E. coli. Sojevi su zatim analizirani ne bi li se utvrdila prisutnost različitih virulentnih gena i plazmidnih gena otpornosti na kinolone (qnr). U jednom je izolatu (1,2 %) otkriven virulentni gen stx1, kod njih pet (6 %) gen stx2, a u sljedećih pet izolata (6 %) oba gena, stx1 i stx2. U osam je izolata (9,5 %) pronađen gen ehxA, a u njih tri (3,7 %) gen eae. Svi su ti geni karakteristični za patotip STEC, koji proizvodi Shiga toksin. Gen fimH pronađen je u sedamdeset i četiri (88,1 %) izolata. Od virulentnih gena tipičnih za ekstraintestinalne patogene sojeve E. coli, gen hra je pronađen u devet (11 %), gen ompTAPEC u osam (9,5 %), a gen iha u šest (7 %) izolata. U jednom su izolatu otkriveni geni kpsMTII, sfa, usp i vat, dok geni hlyA, bmaE, cnf, hpb, sat i plazmidni geni otpornosti na kinolone qnr nisu pronađeni u ispitanim izolatima. Rezultati pokazuju da razni prehrambeni proizvodi zaista predstavljaju izvor intestinalnih, te u manjoj mjeri ekstraintestinalnih patogenih sojeva E. coli
Verotoxigenic Escherichia coli isolated from human samples in Slovenia
No full textBACKGROUND
Shiga toxin-producing E. coli or
Vero cytotoxin-producing E. coli (VTEC) are
characterised by the ability to produce either one
or both cytotoxins referred to as Shiga toxin 1
(Stx1) and Shiga toxin 2 (Stx2). VTEC infection
may result in life-threatening conditions such as
haemolytic uremic syndrome (HUS) and thrombotic
thrombocytopenic purpura (TTP). Due to
different methods of monitoring and identification
of these bacteria in recent years, the existing
data on reported cases of VTEC infections probably
do not reflect reality. Our study of VTEC
strains isolated in different regions of Slovenia,
showed serogroups, major virulence factors and
specific epidemiological data that can serve as a
basis for further laboratory and epidemiological
surveillance of VTEC infections.
METHODS
A total of 66 VTEC strains, isolated
from stool samples of patients with diarrhoea
from the year 1993 to 2009, were collected at
NIPH (National Institute of Public Health). The
data of patient’s age and gender, onset of illness
and clinical manifestation of disease were gathered.
The serogroups of isolated strains were
determined with antisera following manufacturer’s
instructions. The ability to produce verocytotoxins
was tested using the reversed passive
latex agglutination method. The presence
of genes for intimin (eae), enterohaemolysin
(ehxA) and verotoxins (vtx1 and vtx2) were determined
by polymerase chain reaction (PCR).
RESULTS
Infection with VTEC was encountered
throughout the year, but most people were ill
in the summer and autumn months. More than
half of patients (57.6 %) were younger than five
years. Collected VTEC strains belonged to serogroups
O17, O26, O91, O103, O111, O113, O126,
O128, O145, O148 and O157 (the most frequent
were O157 and O26). A high percentage of VTEC strains showed the presence of intimin (86.4 %)
and enterohaemolysin (86.4 %) genes. The gene
for vtx1 was found in 22.7 % of strains, the vtx2 in
57.6 % of strains, while the presence of both genes
was determined in 19.7 % of strains. The presence
of the vtx2 gene was determined in all strains associated
with HUS and TTP, most of them possessed
the eae and ehxA genes too. These patients
were mostly older people and young children.
CONCLUSIONS
Most infections with VTEC occurred
in the warmer months of the year, most
patient were small children. The severity of
VTEC infection is determined by several factors
such as the E. coli serogroup, the type of Shiga
toxin produced and presence of other virulence
genes. The most common serogroups among the
study strains were O157 and O26. VTEC O26 has
been the most commonly isolated serogroup in
recent years, nevertheless more and more different
serological groups began to emerge. In all
strains associated with HUS and TTP, the vtx2
gene was determined. Further typing of verocytotoxin
encoding genes will contribute to assess
the risk for complications with VTEC infection.
The study provides insights about the age of the
patients, seasonal distribution of disease, serogroups
and genotypes of the agent
New Mediterranean biodiversity records (July 2018)
No full textIn the present article, new records are given for 15 species (4 native and 9 alien and 2 cryptogenic), belonging to 6 Phyla (i.e. Chlorophyta, Ctenophora, Cnidaria, Mollusca, Arthropoda, and Chordata), from 10 Mediterranean countries: Morocco: the finding of the crab Callinectes sapidus represents the westernmost one of the species in the Mediterranean; Italy: first records of the nudibranch Polycera hedgpethi from the harbour of La Spezia, and first finding of the invasive ctenophore Mnemiopsis leidyi in the Fiora River; Tunisia: Caulerpa taxifolia var. distichophylla is recorded for the first time, showing an even wider distribution in the Mediterranean; Greece: the finding of the jellyfish Pelagia benovici represents the first record of the species in the Ionian Sea, while the finding of the smallscale codlet Bregmaceros nectabanus in the Ionian Sea is another interesting first report for the area; Malta: the cryptogenic scleractinian coral Oculina patagonica was recorded; Slovenia: the parasitic copepod Demoleus heptapus was recorded from a sixgill bluntnose shark, Hexanchus griseus; Croatia: the Lessepsian cephalaspidean mollusc Haminoea cyanomarginata is recorded for the first time from the area; Bulgaria: the Asian date mussel Arcuatula senhousia was recorded from the Black Sea; Cyprus: the Lessepsian gastropod Viriola sp. [cf. corrugata) was recorded for the first time from the area, while two decapod species were recorded also for the first time from Cyprus, i.e. the caridean shrimp Pasiphaea sivado and the anomuran Munida curvimana; Turkey: the acari Lohmannella falcata is recorded for the first time from Antalya and the Lessepsian fish Priacanthus sagittarius in the Levantine coasts of Turkey (off Hatay/Arsuz) showing that this species has extended its range in a very short time.peer-reviewe
Balance recoverability and control of bipedal robotic walkers with foot slip
No full textLow-friction surfaces present a particular challenge in the field of bipedal walking as they can lead to foot slippage. Human foot slip often leads to falls, which is a major cause of injuries and results in a high societal and economic burden, especially for the elderly population. Since slip cannot always be avoided, it is valuable to understand the dynamics of walking under slip conditions, the likelihood of falling, and the effects of foot-slip on biped walking stability. Furthermore, recovery control strategies can potentially stabilize slipping gait and prevent fall. Such developments would therefore benefit both the robotic and clinical biomechanics walking communities. This dissertation addresses the questions of whether a walker experiencing a foot-slip is in danger of falling or whether it can successfully recover balance and prevent a fall. We present dynamics models which describe the motion of slipping gait and the frameworks to evaluate the stability of gaits with foot-slip. We further present the algorithms which can decide on the appropriate control input to increase stability on multiple levels of control design. The feasibility of the theoretical results is demonstrated both in simulation and by deploying an experimental robotic walker, as well as by comparison with human subject trials.
The first part of this dissertation focuses on modeling of walking under foot-slip conditions. Reduced-order models, such as linear inverted pendulum models, are used to represent the key dynamic behavior of a walker without constraining a particular structure. While models exist for normal walking, they need to be adapted to relax the ubiquitous assumption of the stationary, non-slip contact point between the walker's foot and the ground. We introduce two novel models which are based on inverted pendulum but incorporate an angular moment of inertia as well as allow movement in the vertical direction. With those modifications, the new models have the ability to accurately capture slipping dynamics. Building upon those models, the stability of slipping gaits is assessed using the concept of capturability. We demonstrate how varying the variables such as step duration and ground friction changes the capture regions and influences reliable foot positioning.
Next, a variation of a linear inverted pendulum model is presented which allows for explicit modeling of foot-slip conditions while also retaining the linearity and dimensionality of its dynamics. We analytically solve the dynamics motion equations and present solutions in the form of time-invariant manifolds for both normal walking and foot-slip conditions. By utilizing the solution manifolds, predictions of walker's motion no longer rely on solving differential equations but rather on simplified algebraic equations which define the geometry of hyperbolic solution manifolds. Based on the geometry of solution manifolds, we introduce the concept of slip recoverability. Recoverability partitions the phase portrait space into three distinct regions such that every state can be classified as safe, recoverable with adequate input, and non-recoverable. Based on the results of the recoverability analysis, we introduce a novel optimal control framework that ensures robustness when slip is imminent, and reduces the within-step control effort when fall is not likely. In addition to the within-step control, the proposed controller also prescribes step location for single- or multi-step recovery. The performance of the controller is verified via extensive simulation. To further verify the proposed abstract model, we analyze human walking data including subjects' responses to unexpected slips. An example of successful and unsuccessful slip recovery is used to compare the pose evolution to the prediction of the proposed model.
In order to realize the high-level recovery strategy, a whole-body controller is needed to calculate joint torques. To that extent, the second half of this dissertation deals with whole-body operational space (WBOS) based torque controller. We extend the WBOS-based controller to the case of foot slip. The new frictional WBOS (FWBOS) is formulated such that the dynamics remains consistent with both forward and backward slip of the standing foot. We formulate a task to maintain balance and demonstrate the performance of the FWBOS algorithm through extensive simulation. The whole-body controller is then integrated with the reduced-order model proposed in the previous chapters. The current state of the reduced model is calculated from the full model and yields the solution manifolds. Based on the solution manifolds, the controller predicts the appropriate stepping location to maintain the walker's forward progression. The FWBOS algorithm is then used to plan and design the foot placement. In this way, both step planning and joint torque calculation explicitly consider foot slip. Simulation is carried out to demonstrate the improvement of considering slip at both levels of the integrated controller. Finally, the performance of the proposed control algorithms is demonstrated experimentally using a planar 5-link robot with pointy feet. We present the setup, modeling, system identification, friction compensation, and other aspects of a mechanical system. Walking gaits are demonstrated both for normal walking, as well as on slippery surfaces where foot-slip is present. We implement the FWBOS-based controller and experimentally demonstrate the successful robot walking on a slippery surface.Ph.D.Includes bibliographical reference
Modeling, sensing, and control of human bipedal walking with foot slip
No full textHuman walking is a fundamental motor skill that is developed at an early stage in our lives. Maintaining stable walking capability demands a substantial effort and requires synchronization and coordination of many neurological, sensorimotor and musculoskeletal systems. Moreover, disturbances such as foot slip require even more demanding walking control strategies for successful balance recovery and fall prevention. However, it is challenging to capture and model human motion and reaction to foot slip. Most of the existing slip-and-fall studies focus on clinical human experiments and few use control systems approaches to analyze the slip dynamics and human recovery mechanisms. Further challenges arise as few real-time sensing and robotic assistive technologies are currently available for reliably detecting foot slips and assisting human balance for slip-induced fall prevention. The goal of this dissertation is to advance the understanding and knowledge of slip dynamics with emphasis on four interlaced topics: (i) analyzing and modeling the shoe-floor interaction during foot slip, (ii) developing a novel bipedal modeling framework to capture human walking locomotion with foot slip, (iii) developing a novel linear inverted pendulum (LIP) modeling framework for balance recovery control, and (iv) developing new wearable sensing and robotic assistive devices for real-time detection of foot slip and effective prevention of slip-induced falls. In the first part, we present modeling of foot slip evolution and development based on a quasistatic friction force model. We present a model to obtain the normal force distribution on the shoe-floor contact patch. In addition, we extend the previously developed beam-spring network model and integrate it with the LuGre dynamic friction model. In the second part of the dissertation, we present a new bipedal modeling approach, where we relax the non-slip assumption used in the existing literature. We develop a hybrid bipedal model and the gait controllers to capture and predict human walking with foot slip. In the third part, we present a new two-mass LIP model for human balance control during walking and walking with foot slip. We extend the capture point based control approach and incorporate time-varying locations of the zero moment point and the LIP pivoting location. In the fourth part, we propose a novel real-time foot slip detection method using only wearable inertial measurement units. The developed slip-prediction algorithm is built on a dynamic model for bipedal walking and is also integrated with the human locomotion constraints. A slip indicator is introduced into the algorithm to detect the foot slip shortly after the heel-strike event. All of the above mentioned models, control strategies and devices are validated through the extensive experiments and simulations. In addition, we further design and fabricate a wearable robotic knee assistive device for slip balance recovery and slip-induced fall prevention. This device prototype serves as an enabling tool for future testing of possible robotic assistive fall prevention strategies.Ph.D.Includes bibliographical referencesby Mitja Trko
