Development and Application of a gp60-Based Typing Assay for Cryptosporidium viatorum

The apicomplexan intestinal parasites of the genusCryptosporidiumtake a major toll on human and animal health and are frequent causes of waterborne outbreaks. Several species and genotypes can infect humans, includingCryptosporidium viatorum, which, to date, has only been found in humans. Molecular characterization ofCryptosporidiumspp., critical to epidemiological analyses, is commonly based on gp60 gene analysis, which appears to require bespoke species- or group-specific PCR primers due to extensive genetic diversity across the genus. In this study, we amplified, sequenced, and characterized the gp60 gene ofC. viatorumfor the first time. Moreover, we developed and validated a gp60 typing assay for this species and applied it to 27 isolates originating from Asia, Africa, and Central America. A single subtype family, XVa, was identified containing multiple alleles.

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Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

Microorganisms ◽

10.3390/microorganisms9010054 ◽

2020 ◽

Vol 9 (1) ◽

pp. 54

Author(s):

Salem Belkessa ◽

Daniel Thomas-Lopez ◽

Karim Houali ◽

Farida Ghalmi ◽

Christen Rune Stensvold

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Specific Pcr ◽

Stool Samples ◽

Pcr Targeting ◽

Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Molecular Characterization of Ciliate Diversity in Stream Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.01438-07 ◽

2008 ◽

Vol 74 (6) ◽

pp. 1740-1747 ◽

Cited By ~ 52

Author(s):

Andrew Dopheide ◽

Gavin Lear ◽

Rebecca Stott ◽

Gillian Lewis

Keyword(s):

18S Rrna ◽

Pcr Primers ◽

18S Rrna Gene ◽

Sequence Diversity ◽

Rrna Genes ◽

Rrna Gene ◽

Sequencing Analysis ◽

Specific Pcr ◽

Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

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Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines inAromatoleum aromaticumEbN1

Journal of Bacteriology ◽

10.1128/jb.00281-19 ◽

2019 ◽

Vol 201 (16) ◽

Author(s):

Georg Schmitt ◽

Martin Saft ◽

Fabian Arndt ◽

Jörg Kahnt ◽

Johann Heider

Keyword(s):

Aromatic Amines ◽

Catalytic Efficiency ◽

Growth Conditions ◽

Complete Replacement ◽

Content Type ◽

Enzyme Family ◽

First Time ◽

Α Subunits ◽

Substrate Spectrum

ABSTRACTAromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacteriumAromatoleum aromaticumEbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two hemeccofactors in their α-subunits, and exhibit extensive processing of their γ-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed an α2β2γ2composition, and had a rather broad substrate spectrum, which included 2-PEA, BAm, tyramine, and 1-butylamine. In contrast, the other enzyme was specifically induced in BAm-grown cells, showing an αβγ composition and activity only with BAm and 2-PEA. Since the former enzyme showed the highest catalytic efficiency with 2-PEA and the latter with BAm, they were designated 2-PEADH and benzylamine dehydrogenase (BAmDH). The catalytic properties and inhibition patterns of 2-PEADH and BAmDH showed considerable differences and were compared to previously characterized quinohemoproteins of the same enzyme family.IMPORTANCEThe known substrate spectrum ofA. aromaticumEbN1 is expanded toward aromatic amines, which are metabolized as sole substrates coupled to denitrification. The characterization of the two quinohemoprotein isoenzymes involved in degrading either 2-PEA or BAm expands the knowledge of this enzyme family and establishes for the first time that the necessary maturation of their quinoid CTQ cofactors does not require the presence of molecular oxygen. Moreover, the study revealed a highly interesting regulatory phenomenon, suggesting that growth with BAm leads to a complete replacement of 2-PEADH by BAmDH, which has considerably different catalytic and inhibition properties.

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In VitroCulture of Previously Uncultured Oral Bacterial Phylotypes

Applied and Environmental Microbiology ◽

10.1128/aem.02156-15 ◽

2015 ◽

Vol 81 (24) ◽

pp. 8307-8314 ◽

Cited By ~ 16

Author(s):

Hayley Thompson ◽

Alexandra Rybalka ◽

Rebecca Moazzez ◽

Floyd E. Dewhirst ◽

William G. Wade

Keyword(s):

Culture Media ◽

Pcr Primers ◽

Oral Bacteria ◽

Subgingival Plaque ◽

Community Studies ◽

Shotgun Metagenomics ◽

Content Type ◽

Specific Pcr ◽

Proteose Peptone

ABSTRACTAround a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use anin vitrobiofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA).In vitrobiofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxaBacteroidetesbacteria HOT 365 and HOT 281,Lachnospiraceaebacteria HOT 100 and HOT 500, andClostridialesbacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype,Actinomycessp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected inin vitro-cultivated biofilms.Lachnospiraceaebacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture withParvimonas micraorVeillonella dispar/parvulaafter colony hybridization-directed enrichment. The establishment ofin vitrobiofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.

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Construction and Application of a Functional Library of Cytochrome P450 Monooxygenases from the Filamentous Fungus Aspergillus oryzae

Applied and Environmental Microbiology ◽

10.1128/aem.02491-10 ◽

2011 ◽

Vol 77 (9) ◽

pp. 3147-3150 ◽

Cited By ~ 21

Author(s):

K. H. M. Nazmul Hussain Nazir ◽

Hirofumi Ichinose ◽

Hiroyuki Wariishi

Keyword(s):

Cytochrome P450 ◽

Aspergillus Oryzae ◽

Cytochrome P450 Monooxygenases ◽

Functional Screening ◽

Content Type ◽

P450 Monooxygenases ◽

Rapid Characterization ◽

Catalytic Functions ◽

First Time

ABSTRACTA functional library of cytochrome P450 monooxygenases fromAspergillus oryzae(AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase inSaccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

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Development of a Rapid, Sensitive, and Field-Deployable Razor Ex BioDetection System and Quantitative PCR Assay for Detection of Phymatotrichopsis omnivora Using Multiple Gene Targets

Applied and Environmental Microbiology ◽

10.1128/aem.03239-12 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2312-2320 ◽

Cited By ~ 23

Author(s):

M. Arif ◽

J. Fletcher ◽

S. M. Marek ◽

U. Melcher ◽

F. M. Ochoa-Corona

Keyword(s):

Rna Polymerase Ii ◽

Quantitative Pcr ◽

Target Genes ◽

Pcr Primers ◽

Internal Transcribed Spacers ◽

Diagnostic Tools ◽

Purification Method ◽

Content Type ◽

Specific Pcr ◽

Phymatotrichopsis Omnivora

ABSTRACTA validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection ofPhymatotrichopsis omnivora.This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. BecauseP. omnivorais difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based onP. omnivoranucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection ofP. omnivorain infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (∼30-min) on-site assay capability forP. omnivoradetection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.

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Fulminant Cryptosporidiosis after Near-Drowning: a Human Cryptosporidium parvum Strain Implicated in Invasive Gastrointestinal Adenocarcinoma and Cholangiocarcinoma in an Experimental Model

Applied and Environmental Microbiology ◽

10.1128/aem.06457-11 ◽

2012 ◽

Vol 78 (6) ◽

pp. 1746-1751 ◽

Cited By ~ 17

Author(s):

Gabriela Certad ◽

Sadia Benamrouz ◽

Karine Guyot ◽

Anthony Mouray ◽

Thierry Chassat ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cryptosporidium Parvum ◽

Severe Infection ◽

Digestive Cancer ◽

Content Type ◽

Near Drowning ◽

Neoplastic Process ◽

First Time

ABSTRACTIn the present work, we report the characterization of aCryptosporidium parvumstrain isolated from a patient who nearly drowned in the Deule River (Lille, France) after being discharged from the hospital where he had undergone allogeneic stem cell transplantation. After being rescued and readmitted to the hospital, he developed fulminant cryptosporidiosis. The strain isolated from the patient's stools was identified asC. parvumII2A15G2R1 (subtype linked to zoonotic exposure) and inoculated into SCID mice. In this host, this virulentC. parvumisolate induced not only severe infection but also invasive gastrointestinal and biliary adenocarcinoma. The observation of adenocarcinomas that progressed through all layers of the digestive tract to the subserosa and spread via blood vessels confirmed the invasive nature of the neoplastic process. These results indicate for the first time that a human-derivedC. parvumisolate is able to induce digestive cancer. This study is of special interest considering the exposure of a large number of humans and animals to this waterborne protozoan, which is highly tumorigenic when inoculated in a rodent model.

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Intestinal Parasites of Buffalo Calves from Romania: Molecular Characterization of Cryptoporidium spp. and Giardia Duodenalis, and the first Report of Eimeria Bareillyi

10.21203/rs.3.rs-151344/v1 ◽

2021 ◽

Author(s):

Diana Ancuța Bărburaș ◽

Vasile Cozma ◽

Angela Monica Ionică ◽

Ibrahim Abbas ◽

Remus Bărburaș ◽

...

Keyword(s):

Intestinal Parasites ◽

Giardia Duodenalis ◽

Gastrointestinal Parasites ◽

Parasitic Infections ◽

Buffalo Calves ◽

Economic Resource ◽

Toxocara Vitulorum ◽

Staining Methods ◽

First Time

Abstract Buffaloes represent an important economic resource for several regions of the world including Romania; however, no reports on parasitic infections in buffaloes from Romania are available. In the present study, we examined for the gastrointestinal parasites 104 fecal samples bimonthly collected from 38 buffalo calves (2–11 weeks old) from household rearing systems in Romania. All samples were tested using the saturated salt flotation, McMaster and modified Ziehl-Nielsen staining methods. PCR coupled with isolates sequencing methods were used to identify the Giardia duodenalis assemblages and Cryptosporidium species. Overall, 33 out of 38 examined buffalo calves were infected with different gastrointestinal parasites; 16 had single infections and 17 had mixed infections with 2 or 3 parasites. Eimeria species (32/38; 84.2%) was the most prevalent parasite; 8 species were identified according to the oocyst morphology including the pathogenic E. bareillyi which detected for the first time in buffaloes from Romania. Toxocara vitulorum (11/38; 36.8%) and Strongyloides papillosus (6/38; 15.8%) were also detected. Cryptosporidium spp. were found in 4 (10.5%) buffalo calves; 2 of them were molecularly identified as C. ryanae and another one was clustered in the same clade with C. ryanae, C. bovis, and C. xiaoi. Giardia duodenalis assemblage E was also molecularly detected in a single (2.6%) buffalo calf. The presence of other buffaloes in the same barn was identified as a risk factor for infection with T. vitulorum. Our results indicate extensive parasitic infections in buffalo calves from Northwestern Romania and underline the necessity of prophylactic treatments for T. vitulorum and E. bareillyi.

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Molecular characterization of a novel bla CTX-M-3-carrying Tn6741 transposon in Morganella morganii isolated from swine

Journal of Medical Microbiology ◽

10.1099/jmm.0.001235 ◽

2020 ◽

Vol 69 (8) ◽

pp. 1089-1094

Author(s):

Xingwei Luo ◽

Yajun Zhai ◽

Dandan He ◽

Xiaodie Cui ◽

Yingying Yang ◽

...

Keyword(s):

Type Species ◽

Susceptibility Testing ◽

Morganella Morganii ◽

Ionization Time ◽

Content Type ◽

Genetic Environment ◽

Link Type ◽

Flight Mass Spectrometry ◽

First Time

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated. Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment. Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli . Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials. Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii .

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Preparation and characterization of novel Yellow-Green fluorescent polymerizable dye and its copolymer with application of highly selective sensor for cations iron

Pigment & Resin Technology ◽

10.1108/prt-01-2021-0002 ◽

2021 ◽

Vol ahead-of-print (ahead-of-print) ◽

Author(s):

Hanieh Shaki

Keyword(s):

Metal Ions ◽

Covalent Bond ◽

Analytical Techniques ◽

Side Chain ◽

Content Type ◽

Photo Induced Electron Transfer ◽

Green Fluorescent ◽

First Time ◽

Acrylonitrile Polymer

Purpose In this work, A new 4–(2-aminoethylene) amino-N-(2-hydroxyethyl)-1,8-naphthalimide with intense green fluorescent was synthesized. This low molecular weight compound was immobilized by forming a covalent-bond with an acrylonitrile polymer containing carboxylic acid groups. The new prepared dye and self-coloured polymer were characterized by analytical techniques. Design/methodology/approach The synthesized compounds were characterized by TLC, DSC, FTIR, 1HNMR, 13CNMR, GPC, UV–visible and Fluorometery. The photophysical characteristics of the dye and polymer containing naphthalimide moiety in the side chain, were measured both in the absence and in the presence of Ag+, Cd+2, Co+2, Cr+3, Cu+2, Fe+3, Hg+2, Ni+2, Pb+2 and Zn+2 cations. Findings The results showed that the characterization of the synthesized dye and its polymer verified their structural correctness. It is shown that dye and polymer are photo-induced electron transfer (PET) fluorescent sensors which exhibit fluorescence quenching in the presence of metal ions. Among the various metal ions, both dye and polymer are more sensitive to Fe+3 cations. Originality/value This study is original. A 4–(2-aminoethylene) amino-N-(2-hydroxyethyl)-1,8-naphthalimide and its self-coloured polymer were synthesized for the first time, successfully.

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