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At least 1,261 records · Page 70Linked to original sources
PubMed1994

Cultivation and phylogenetic characterization of a newly recognized human pathogenic protozoan.

An intraerythrocytic protozoan (WA1) recently isolated from a patient in Washington State was shown to be morphologically identical to Babesia microti but biologically and genetically distinct. Continuous growth of WA1 was established in stationary erythrocyte cultures. Hybridization of a chemiluminescent Babesia-specific DNA probe to Southern blots of restriction enzyme-digested genomic DNA showed that WA1 could be distinguished from other Babesia species that were antigenically cross-reactive (Babesia gibsoni and babesial parasites from desert bighorn sheep, Ovis canadensis nelsoni) or known to infect humans (B. microti, Babesia divergens, and Babesia equi), or both. A 1436-bp portion of the nuclear small subunit rRNA gene of WA1 was sequenced and analyzed. Genetic distance analysis showed that WA1 is most closely related to the canine pathogen B. gibsoni and lies within a phylogenetic cluster with Theileria species and B. equi. The methodology described will be useful for improved diagnosis and identification of human protozoal pathogens.

Animals↗
PubMed2005

Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay.

BACKGROUND: In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay. METHODS: A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997-2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth. RESULTS: The ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation. CONCLUSIONS: The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen.

Antigens, Bacterial↗
PubMed2006

Detection of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in kimchi by multiplex polymerase chain reaction (mPCR).

We developed an mPCR assay for the simultaneous detection, in one tube, of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes using species-specific primers. The mPCR employed the E. coli O157:H7 specific primer Stx2A, Salmonella spp. specific primer Its, S. aureus specific primer Cap8A-B and L. monocytogenes specific primer Hly. Amplification with these primers produced products of 553, 312, 405 and 210 bp, respectively. All PCR products were easily detected by agarose gel electrophoresis, and the sequences of the specific amplicons assessed. Potential pathogenic bacteria, in laboratory-prepared and four commercially available kimchi products, were using this mPCR assay, and the amplicons cloned and sequenced. The results correlated exactly with sequences derived for amplicons obtained during preliminry tests with known organisms. The sensitivity of the assay was determined for the purified pathogen DNAs from four strains. The mPCR detected pathogen DNA at concentrations ranging from approximately 0.45 to 0.05 pM/microl. Thus, this mPCR assay may allow for the rapid, reliable and cost-effective identification of four potentially pathogens present in the mixed bacterial communities of commercially available kimchi.

DNA Primers↗
PubMed1984

Evaluation of a two-hour method for screening pathogens from stool specimens.

The 2-h stool-screening test (SST) (API System S.A., Montalieu-Vercieu, France) was used to screen 231 organisms yielding suspicious colonies on stool differential agars for potential pathogens. All 54 salmonellae yielded correct screens. Of 14 shigellas, 9 keyed as possible Shigella spp.-Yersinia enterocolitica-rare Salmonella spp. (SYS), and 5 (all Shigella sonnei) keyed as possible S. sonnei-Y. enterocolitica-Arizona spp. (SYA). Three Arizona strains were identified as probable Salmonella spp., two were identified as SYA, and one was identified as SYS. Seven Y. enterocolitica strains keyed as SYS, and two keyed as SYA; one strain was screened out as a nonpathogen. The two Aeromonas hydrophila strains keyed as SYA, and the two Plesiomonas shigelloides strains keyed as SYS. All 63 Proteus, Morganella, Providencia, and Pseudomonas aeruginosa strains were screened out as nonpathogens. Among 80 coliforms, 37 were screened as SYA, 32 were screened as SYS, and 11 were screened as nonpathogens. On the basis of the SST, 69/80 coliforms would have been tested further. However, only 1/88 potential pathogens would have been missed, and all Proteus, Morganella, Providencia, and P. aeruginosa strains would have been excluded. The capability of the SST (when combined with a rapid identification method) of providing same-day identification of potential stool pathogens must be weighed against its inability to effectively screen out coliforms. The cost of this method is equivalent to that of a three-tube conventional screening method. The suitability of the SST for individual laboratories must be predicated on the incidence of stool pathogens and commensals in the specific setting as well as on factors related to work flow, technologist acceptability, and turnaround time.

Aeromonas↗
PubMed1990

Detection of surgical pathogens by in vitro DNA amplification. Part I. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene.

The management of candidemia and disseminated candidiasis depends on rapid, unambiguous identification of Candida. Such identification is retarded by the slow growth of Candida from clinical specimens. Administration of effective but potentially toxic antifungal therapy is often withheld pending identification. To circumvent this slow growth and thus to expedite diagnosis and therapy, the polymerase chain reaction (PCR) was used to amplify a segment of fungal DNA coding for the cytochrome P450L1A1 (lanosterol-14 alpha-demethylase) in vitro. The technique provides unambiguous evidence of C. albicans in as few as 6 hours with a detection threshold of 10 organisms in a 100 mu specimen. Clinical specimens of urine (n = 4), sputum (n = 6), wound fluid (n = 1, and blood (n = 2) were collected from patients, and C. albicans was conventionally documented at these sites; in each case, PCR was confirmed. Of 17 additional specimens that were culture negative, PCR suggested the presence of yeast in two of the specimens. PCR-based detection of surgical pathogens may have broad application in rapid screening for the presence of organisms either indigenous to a particular surgical intensive care unit or peculiar to selected patient populations.

Candida albicans↗
PubMed2006

Identification of Enterobacter sakazakii from closely related species: the use of artificial neural networks in the analysis of biochemical and 16S rDNA data.

BACKGROUND: Enterobacter sakazakii is an emergent pathogen associated with ingestion of infant formula and accurate identification is important in both industrial and clinical settings. Bacterial species can be difficult to accurately characterise from complex biochemical datasets and computer algorithms can potentially simplify the process. RESULTS: Artificial Neural Networks were applied to biochemical and 16S rDNA data derived from 282 strains of Enterobacteriaceae, including 189 E. sakazakii isolates, in order to identify key characteristics which could improve the identification of E. sakazakii. The models developed resulted in a predictive performance for blind (validation) data of 99.3 % correct discrimination between E. sakazakii and closely related species for both phenotypic and genotypic data. Three main regions of the partial rDNA sequence were found to be key in discriminating the species. Comparison between E. sakazakii and other strains also constitutively positive for expression of the enzyme alpha-glucosidase resulted in a predictive performance of 98.7 % for 16S rDNA sequence data and 100% for phenotypic data. CONCLUSION: The computationally based methods developed here show a remarkable ability in reducing data dimensionality and complexity, in order to eliminate noise from the system in order to facilitate the speed and reliability of a potential strain identification system. Furthermore, the approaches described are also able to provide valuable information regarding the population structure and distribution of individual species thus providing the foundations for novel assays and diagnostic tests for rapid identification of pathogens.

Cronobacter sakazakii↗
PubMed2002

Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole.

Candida glabrata is an increasingly important cause of opportunistic fungal infection of humans and appears to be intrinsically resistant to the triazole antifungal fluconazole. However, the mechanisms responsible for reduced susceptibility to azole drugs are not understood. Fluconazole exposure rapidly induced expression of a 169-kDa protein band in plasma membrane fractions of C. glabrata cells. Mass spectrometry of trypsin-digested peptide fragments showed that the induced protein band comprised the ATP binding cassette-type drug efflux transporter CgCdr1p. CgCdr1p was also functionally overexpressed in S. cerevisiae and similarly identified by mass spectrometry. A 61-kDa protein band in the plasma membrane fraction from C. glabrata was also induced by fluconazole exposure. Mass spectrometric peptide fingerprinting identified this band as lanosterol 14alpha-demethylase, the enzyme in the ergosterol biosynthesis pathway targeted by fluconazole. The rapid induction of a multidrug efflux pump and/or overproduction of lanosterol 14alpha-demethylase are mechanisms that could make C. glabrata appear intrinsically resistant to fluconazole. Mass spectrometric fingerprint analysis of SDS-PAGE separated plasma membrane fractions combined with heterologous hyper-expression provides a convenient method for protein identification and functional evaluation of induced proteins, even in an organism where the genome sequence database is incomplete.

Amino Acid Sequence↗
PubMed1994

An epizootic of feline herpesvirus, type 1 in a large specific pathogen-free cat colony and attempts to eradicate the infection by identification and culling of carriers.

This study describes the clinical course of an inadvertent feline herpesvirus, type 1 (FHV-1) outbreak in 2 specific pathogen-free (SPF) research and breeding colonies housing 690 cats and assesses a programme that was designed to eradicate the virus from the colonies. The clinical signs observed in these cats were milder, with more eye involvement than those previously described for FHV-1 infection and did not include abortion. FHV-1 eradication was based on the detection and elimination of both active and latent viral carriers. Carrier cats were detected by virus isolation from oral swabs before and after corticosteroid-induced reactivation of FHV-1 excretion. Four per cent of recovered cats were actively shedding virus prior to corticosteroid treatment; 21% of the virus negative cats shed virus after one corticosteroid injection, and 12% of remaining culture negative cats tested positive upon a second corticosteroid treatment 6 weeks later. The colony remained virus free for 8 months after all detectable virus carriers were culled and there was no seroconversion among new kittens. A second epizootic of FHV-1 then occurred among susceptible animals. At this time, all breeding cats that had tested negative after 2 injections of corticosteroids were treated a third time; 23% of them now tested positive for FHV-1. This study demonstrates that corticosteroid treatment can be useful in improving the rate of detection, essential as a basis for decreasing the incidence of enzootic disease, but it is unlikely to detect all possible FHV-1 carriers in large populations of cats.

Animals↗
PubMed2002

[Bacteriological risks: detection and tracing].

Advances in molecular taxonomy of bacteria have generated many tools allowing identification of any bacteria, whether culturable or not. In addition to taxonomic identification, growing knowledge on pathogenicity mechanisms allows the detection of bacteria with given virulence genes. The problem is in asking the proper questions so that the most appropriate tools can be chosen. Some rapid identification tools (gene amplification, in situ hybridisation) require a starting hypothesis. Other tools (rrs gene amplification and sequencing) can be used without prior hypothesis, but take longer. To face the bioterrorist threat, particular attention should be given to laboratory and strain dispatch (within- and between-laboratory) organisation.

Bacterial Infections↗
PubMed2006

Using a resequencing microarray as a multiple respiratory pathogen detection assay.

Simultaneous testing for detection of infectious pathogens that cause similar symptoms (e.g., acute respiratory infections) is invaluable for patient treatment, outbreak prevention, and efficient use of antibiotic and antiviral agents. In addition, such testing may provide information regarding possible coinfections or induced secondary infections, such as virally induced bacterial infections. Furthermore, in many cases, detection of a pathogen requires more than genus/species-level resolution, since harmful agents (e.g., avian influenza virus) are grouped with other, relatively benign common agents, and for every pathogen, finer resolution is useful to allow tracking of the location and nature of mutations leading to strain variations. In this study, a previously developed resequencing microarray that has been demonstrated to have these capabilities was further developed to provide individual detection sensitivity ranging from 10(1) to 10(3) genomic copies for more than 26 respiratory pathogens while still retaining the ability to detect and differentiate between close genetic neighbors. In addition, the study demonstrated that this system allows unambiguous and reproducible sequence-based strain identification of the mixed pathogens. Successful proof-of-concept experiments using clinical specimens show that this approach is potentially very useful for both diagnostics and epidemic surveillance.

Algorithms↗
PubMed1999

[Enzootic reservoir for new Ixodes ricinus-transmitted infections].

In the last decade new tick-transmitted zoonoses have emerged as threats to the public health in Europe. They are mostly due to infection by Borrelia burgdorferi s.I., Babesia divergens, B. microti and Ehrlichia sp. All these pathogens are transmitted by the rodent-feeding ticks of Ixodes persulcatus "complex". Also there is first serological information of coexistence of antibodies to B. burgdorferi, B. microti and Ehrlichia sp. in human exposured to these multiple tick-transmitted pathogens. Actually in Poland, the identification of the host species that act as zoonotical reservoir for these pathogens on which the competent vector becomes infected, is crucial to understand the dynamics of enzootic cycle of these pathogens. It is also important to assess the risk factor of infection in different habitats, especially in environmental condition where changing in farming system has left great land area under grassland or pasture, e.g. in Mazury Lakes District. Under such circumstances, susceptible rodent species, at least for two of these pathogens: B. micrnti and B. burgdorfeń s.l., increasingly became competent reservoirs. This aspect is currently receiving considerable attention in Europe.

Animals↗
PubMed2001

Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. facilitates TnphoA mutagenesis.

Burkholderia pseudomallei and Burkholderia mallei are pathogens responsible for disease in both humans and animals. Burkholderia thailandensis, while phylogenetically similar, is considered avirulent in comparison. These three species exhibit phosphatase activity when grown on media containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (XP). Tn5-OT182 mutagenesis has been utilized to isolate mutants of B. pseudomallei and B. thailandensis unable to hydrolyse XP. Sequence analysis of these mutants revealed an ORF of 1734 nucleotides demonstrating a high degree of homology to the acpA gene product of Francisella tularensis. PCR primers were designed based on the B. pseudomallei acpA gene sequence and were used to amplify an acpA homologue from B. mallei. The predicted amino acid sequence of B. pseudomallei AcpA differed from those of the predicted B. thailandensis AcpA and B. mallei AcpA by 15 and 3 amino acids, respectively. Allelic exchange was used to construct DeltaacpA mutants in each of these Burkholderia spp. These mutants were shown to be devoid of phosphatase activity and have subsequently allowed for the implementation of phoA fusion transposon mutagenesis systems. Two such systems have been successfully utilized in Burkholderia spp. for the identification of several genes encoding exported proteins.

Acid Phosphatase↗
PubMed1998

[Rhinosinusitis in children]

OBJECTIVES: To review the pathogenic mechanisms and treatment of sinusitis in children.METHODS: Mucopurulent secretion and halitosis are important signs and symptoms for the diagnose of sinusitis. The radiographic and computorized tomography investigations confirm the sinusal disease.RESULT: The identification of the main pathogens involved in the acute and chronic sinusitis is fundamental to give a better therapeutic control to these patients. CONCLUSIONS: Rhinosinusitis is one of the most reported diseases among the general population, and its prevalence has been rising in the last years. Many questions remain unanswered about its cause, pathophysiology and optimal treatment, especially in the pediatric population, generating health care costs and a high rate of morbidity.

Journal Article↗
PubMed1988

Historical perspectives and identification of Neisseria and related species.

The pathogenic Neisseria spp., N. gonorrhoeae and N. meningitidis, have been studied extensively and rapid identification procedures have been designed to distinguish these species from the commensal Neisseria and related species that are normal flora of the oro- and nasopharynx. The commensal Neisseria spp. have been largely ignored except for isolated studies. It is important that we know about these species, however, because not only may some be misidentified as pathogenic species if identified with inappropriate procedures, but also they may occasionally be isolated from unusual sites and must be correctly identified to the species level for clinical purposes.

Animals↗
PubMed1998

Arginine 302 mutations in the pyruvate dehydrogenase E1alpha subunit gene: identification of further patients and in vitro demonstration of pathogenicity.

Three further patients with mutations in the codon for arginine 302 of the E1alpha subunit of the pyruvate dehydrogenase complex have been identified. Mutations in this codon have now been found in nine patients with pyruvate dehydrogenase deficiency in seven unrelated families, in sharp contrast to the great majority of other PDH E1alpha mutations which have been described in single individuals only. Because of the relatively high frequency of this mutation and because very few PDH E1alpha mutations have been demonstrated to be causative, we have established a system for analysing the consequences of defined mutations using transfection of normal and mutant PDH E1alpha cDNA into transformed human fibroblasts which have no endogenous E1alpha mRNA or protein. Using this test system, we have demonstrated that the R302C mutation results in the production of PDH E1alpha protein which is devoid of enzymic activity.

Arginine↗
PubMed1987

Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid.

A promoter-probe plasmid suitable for use in Xanthomonas campestris pathovar campestris (causal agent of crucifer black rot) was constructed by ligating a broad host range IncQ replicon into the promoter-probe plasmid pKK232-8, which contains a promoterless chloramphenicol acetyltransferase gene. Xanthomonas chromosomal DNA fragments were 'shotgun' cloned into a restriction site in front of this gene, and the resulting library was transferred en masse into Xanthomonas. Individual transconjugants possessing DNA insertions with promoter activity in plants were identified by virtue of their ability to infect chloramphenicol-treated turnip seedlings. Of 19 transconjugants identified in this way five were chloramphenicol resistant both in turnip seedlings and on agar plates. However the remaining 14 were only chloramphenicol resistant in planta, and thus apparently contained plant-inducible promoter fragments. Resistance to chloramphenicol was correlated with increased chloramphenicol acetyltransferase activity for the transconjugants assayed. The promoter fragments were used to isolate genomic clones from a library, and the role of the genes contained in these clones in pathogenicity is being investigated.

Brassica napus↗
PubMed1996

The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani.

Four distinct laccase genes, lcc1, lcc2, lcc3 and lcc4, have been identified in the fungus Rhizoctonia solani. Both cDNA and genomic copies of these genes were isolated and characterized. Hybridization analyses indicate that each of the four laccase genes is present in a single copy in the genome. The R. solani laccases can be divided into two groups based on their protein size, intron/exon organization, and transcriptional regulation. Three of these enzymes have been expressed in the fungus Aspergillus oryzae. Two of the recombinant laccases, r-lcc1 and r-lcc4, as well as the native lcc4 enzyme were purified and characterized. The purified proteins are homodimeric, comprised of two subunits of approximately 66kDa for lcc4 and 50-100kDa for the recombinant lcc1 protein. These laccases have spectral properties that are consistent with other blue copper proteins. With syringaldazine as a substrate, lcc4 has optimal activity at pH7, whereas lcc1 has optimal activity at pH6.

Amino Acid Sequence↗
PubMed2004

Identification of Fur regulated genes in the bacterial fish pathogen Photobacterium damselae ssp. piscicida using the Fur titration assay.

Bacteria have developed a series of iron-scavenging and transport systems. The expression of many of the iron utilization genes is tightly regulated by the Fe2+ loaded Fur repressor protein. In this study, the Fur titration assay (FURTA) was used to screen for DNA fragments from a genomic DNA library of Photobacterium damselae ssp. piscicida containing potential Fe2+ Fur binding sites or iron binding-proteins which withdraw iron from Fur. One of the clones encoded a tonB gene and adjacent a functionally related exbB gene. An additional and complete tonB exbB exbD gene cluster was identified and sequenced. A gene homologous to the ferritin gene was found whose FURTA-positive phenotype may be explained by its iron-binding ability. Genes encoding a putative complete iron-regulated outer membrane transport protein and a pseudogene of a transport protein were found. The FURTA assay also revealed iron regulation of the AraC type transcriptional regulation.

Amino Acid Sequence↗