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Using the PCR technique, the presence of the seven genotypie virulence markers of Campylobacter has been tested. It was found that all C. coli (92 strains) and C. jejuni (105 strains) possessed the flaA and cadF markers. Several strains were positive for the CDT toxin genes (76.6%, 85.3%, and 83.2% strains with the A, B, and C cdt gene variants, respectively). On the other hand, only 66.0% and 20.8% isolates had the iam and virB genes.
Campylobacter spp. należy do najczęstszych bakteryjnych czynników etiologicznych infekcji pokarmowych u ludzi. Główną przyczyną zakażeń człowieka tymi drobnoustrojami jest spożywanie niedogotowanego mięsa drobiowego, skażonej wody oraz niepasteryzowanych produktów mlecznych. W badaniach epidemiologicznych wykorzystywane są molekularne metody różnicujące, które umożliwiają analizę pokrewieństwa genotypowego między izolatami należącymi do rodzaju Campylobacter, wyosobnionymi z tego samego lub z różnych źródeł. Jedna z nich - ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus - Polymerase Chain Reaction) polega na zastosowaniu w reakcji amplifikacji starterów o długich sekwencjach nukleotydowych, komplementarnych do konserwatywnych fragmentów obecnych w genomie drobnoustrojów rodziny Enterobacteriaceae, jednak różnie w nim rozmieszczonych w zależności do gatunku lub szczepu bakteryjnego. ERIC-PCR umożliwia szybkie różnicowanie izolatów i stanowi przydatne narzędzie służące do analizy genomu prokariotycznego. W ostatnich latach do molekularnego typowania Campylobacter, zwłaszcza C. jejuni i C. coli, stosowana jest analiza długości fragmentów restrykcyjnych (RFLP) amplifikowanego techniką PCR genu flaA. Metoda ta często jest również wykorzystywana w badaniach epidemiologicznych. Celem badań było określenie przydatności technik PCR-RFLP i ERIC-PCR do molekularnego różnicowania szczepów Campylobacter izolowanych z tuszek drobiowych. W wyniku analizy PCR-RFLP otrzymano 5 profili restrykcyjnych, składających się z szeregu fragmentów DNA o różnych masach molekularnych. Natomiast test ERIC-PCR pozwolił na otrzymanie 6 profili molekularnych. Uzyskane rezultaty wskazują na stosukowo dużą zdolność różnicowania obu zastosowanych w pracy metod oraz ich przydatność do genotypowego zróżnicowania szczepów C. jejuni i C. coli.
The aim of this study was to evaluate and compare two genotyping methods for the examination of genetic diversity in C. jejuni and C. coli strains isolated from poultry. Moreover, the molecular identification of putative virulence factors was additionally included to establish a correlation between the isolates. Both typing methods, RAPD-PCR and ERIC-PCR, were found to have a high discriminatory power, D=0.95 and 0.94, respectively. The isolates were clustered to 28 groups by RAPD-PCR and to 29 by ERIC- PCR. Sometimes the ERIC or RAPD assays were able to further discriminate the isolates, which had the same virulence profile generated by the second technique. These PCR-based differentiation methods demonstrated the potential for initial rapid epidemiology studies, and when used in conjunction with distribution of virulence markers provide a high resolution strategy for accurately defining subtypes of Campylobacter.
The occurrence of Campylobacter in poultry meat and subsequent antibiotic resistance profiles of the isolates were investigated. The prevalence of Campylobacter in 101 samples was 87.1%. Of these samples, 54.5% contained Campylobacter coli and 45.5% were contaminated with Campylobacter jejuni. Among the strains tested, resistance to ciprofloxacin and nalidixid acid was the most common, followed by tetracycline and streptomycin. On the other hand, all analysed isolates were susceptible to erythromycin and gentamycin. Moreover, the prevalence of several virulence marker genes among Campylobacter isolates was estimated. All strains showed the presence of the flaA and cadF factors, whereas the iam was identified only in C. jejuni, while the cdtA, cdtB, and cdtC genes were amplified almost in all C. coli isolates.
The presence of the flaA, cadF, cdtB and iam genes of Campylobacter spp. was determined with the PCR method. The materials to investigate were 56 C. jejuni and 23 C. coli strains isolated from clinical samples (children and domestic animals). It was found that all of the Campylobacter spp. isolates from children with diarrhoea and domestic animals had cadF gene, responsible for adherence. The flaA gene was present in all Campylobacter spp. isolates derived from children and cats. Occurrence of flaA gene was confirmed in 100% of C. jejuni strains obtained from dogs. The high prevalence of the cdtB gene associated with toxin production was observed in this study (100%-Campylobacter spp. isolates obtained from dogs and cats, 97.9%-Campylobacter spp. isolates from children). The isolates showed a wide variation for the presence of iam gene. The lowest prevalence (23.5%) was detected in Campylobacter spp. obtained from dogs. The highest rates of iam detection (91.6%) were revealed in C. coli isolates from children.
Określono antagonistyczne właściwości bakterii z rodzaju Lactobacillus, wyizolowanych z kału zdrowych noworodków karmionych mlekiem matki, wobec najczęstszych beztlenowych i mikroaerofilnych etiologicznych czynników zakażeń przewodu pokarmowego. Stwierdzono, że większość z przebadanych szczepów Lactobacillus w warunkach in vitro wykazuje wyraźne antagonistyczne działanie wobec badanych szczepów wskaźnikowych tj. Helicobacter pylori, Campylobacter coli, Campylobacter jejuni i Clostridium difficile.
Survival rates of Campylobacter jejuni ATCC 33 291, Campylobacter jejuni PZH 38 and Campylobacter coli ATCC 43 478 in leg quarters (ca 330g) of chicken broilers after microwave heating (480 and 760 Watt) for 2, 4, 6, 8 and 10 minutes were determined. Heating the meat in a microwave oven (Moulinex, type Dialog cook) at 480 Watts caused a gradual decrease in the number of cells of the studied bacterial strains to total inactivation after 8 – 10 minutes of heating. Increasing the heating power to 760 Watts led to a decreased microwave heating time of 6 – 8 minutes for inactivation of all the cells of the studied C. jejuni/coli strains. These findings clearly indicate the dependence of effectiveness of inactivation of the bacteria studied on microwave heating power, heating duration and bacterial strain.
Campylobacte is an asaccharolytic microorganism which uses amino acids as a source of carbon and energy. CjaC/HisJ is a ligand-binding protein, a component of the ABC transport system. Campylobacter CjaC/HisJ is post-translationally modified by glycosylation. The number of glycosylation motifs present in the CjaC protein is species-specific. C. coli CjaC has two and C. jejuni one motif (E/DXNYS/T) which serves as a glycan acceptor. Although the two C. coli CjaC motifs have identical amino-acid sequences they are not glycosylated with the same efficiency. The efficacy of CjaC glycosylation in Escherichia coli containing the Campylobacter pgl locus is also rather low compared to that observed in the native host. The CjaC localization is host-dependent. Despite being a lipoprotein, CjaC is recovered in E. coli from the periplasmic space whereas in Campylobacter it is anchored to the inner membrane.
We investigated the regulation of the cjaA and cjaB genes of Campylobacter coll. These genes are seemingly arranged into one operon but appear to encode functionally different proteins i.e. an extracytoplasmic solute receptor and a MHS - metabolite: H⁺ symporter transport protein. Analysis of various transcriptional cjaA and/or cjaB lacZ fusion constructs revealed that both genes are arranged in an operon. RACE analysis located the transcription start site of the cjaAB operon 46 bp upstream of the translation start point. β-galactosidase reporter assays yielded much higher activity for the cjaA than the cjaB gene fusion products. RT-PCR showed unequal amounts of mRNA, indicating differential post-transcriptional processing of cjaA and cjaB mRNA possibly related to the presence of inverted repeats in the intergenic region. Phylogenetic analysis grouped CjaB into a new MHS sub-family together with potential transporters with uncharacterised functions of Campylobacter and Helicobacter. Notably, no CjaB family members were identified in ε-Proteobacteria from different ecological niches, such as H. hepaticas and Wolinella succinogenes.
Immunogenic C. coli CjaA protein is a candidate for a chicken anti-Campylobacter subunit vaccine. In order to enhance its immunogenicity an antigenic determinant of the CjaA protein was identified. Thereafter, two copies of the antigenic epitope were cloned in tandem directly or with a flexible hinge between them. All experiments documented that the CjaA protein contained not only linear antigenic epitope/s but also conformational ones.
Z tusz kurcząt izolowano termotolerancyjne szczepy Campylobacter, określano ich przynależność gatunkową, a następnie badano w nich obecność sekwencji DNA kojarzonej z inwazyjnymi właściwościami tych szczepów.
This study analysed the pattern of antibiotic resistance in 251 Campylobacter strains isolated from symptomatic children hospitalized in 4 large paediatric hospitals in Poland from 2000 through 2007. The highest resistance was found for ciprofloxacin (49.5% for C. jejuni and 51.3% for C. coli), followed by tetracycline (17.5% and 18.0%, respectively), and ampicillin (13.2% and 10.2%, respectively). Almost all isolates were susceptible to macrolides. As much as 22.6% of C. jejuni and 25.6% of C. coli were resistant to more than one class of antimicrobial agents. Multidrug resistance (defined as resistance to at least two classes of antimicrobials) rose significantly from 5.1% in 2000-2003 to 34.6% in 2004-2007.
Fifty five thermophilic Campylobacter spp. strains were isolated from the caecum of broilers, cecum and colon of pigs and from human faeces. The strains were identified as Campylobacter jejuni and Campylobacter coli. The more prevalent species in humans and broilers was Campylobacter jejuni, and in pigs Campylobacter coli. In the framework of this study, sensitivity to ciprofloxacin in isolated strains of Campylobacter jejuni and Campylobacter coli was tested by E-test. Resistant to ciprofloxacin were 50.0% of 24 thermophilic Campylobacter strains isolated from humans. In 16 tested strains isolated from broilers, 56.2% were resistant to ciprofloxacin. More resistant species was Campylobacter coli (83.3%). In 15 strains of thermophilic Campylobacter spp. isolated from pigs, the percentage of resistant strains was 26.7%, a relatively high percentage considering the quinolones have not been extensively used in swine farming compared to poultry farming.
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