Isolation of Potentially Novel Brucella spp. from Frogs

ABSTRACTBacterial isolates from frogs were phenotypically identified asOchrobactrum anthropi, but 16S rRNA sequencing showed up to 100% identity withBrucella inopinata. Further analysis ofrecA,omp2a,omp2b,bcsp31, and IS711and multilocus sequence analysis (MLSA) verified a close relationship withBrucella, suggesting the isolates may actually represent novel members of this growing genus of zoonotic pathogens.

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Multilocus Sequence Analysis of Phylogroup 1 and 2 Oral Treponeme Strains

Applied and Environmental Microbiology ◽

10.1128/aem.02499-16 ◽

2016 ◽

Vol 83 (3) ◽

Cited By ~ 4

Author(s):

Yong-Biao Huo ◽

Yuki Chan ◽

Donnabella C. Lacap-Bugler ◽

Sisu Mo ◽

Patrick C. Y. Woo ◽

...

Keyword(s):

Sequence Analysis ◽

16S Rrna ◽

Periodontal Diseases ◽

Genetic Relationships ◽

Species Level ◽

Multilocus Sequence Analysis ◽

Species Differentiation ◽

Treponema Denticola ◽

Rrna Gene ◽

Content Type

ABSTRACT More than 75 “species-level” phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet-uncultivated taxa or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly conserved 16S rRNA, pyrH, recA, and flaA genes. We utilized this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n = 71) of diverse geographical origins. This comprises phylogroup 1 (n = 23) and phylogroup 2 (n = 48) treponeme strains, including all relevant American Type Culture Collection reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450-bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074-nucleotide [nt]), recA (1,377-nt), and pyrH (696-nt) gene sequence data sets. Our data confirmed the species differentiation between Treponema denticola (n = 41) and Treponema putidum (n = 7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into five distinct “species-level” phylotypes. These respectively corresponded to “Treponema vincentii” (n = 11), Treponema medium (n = 1), “Treponema sinensis” (Treponema sp. IA; n = 4), Treponema sp. IB (n = 3), and Treponema sp. IC (n = 4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence. IMPORTANCE Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although Treponema denticola is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.

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Recombinant HcGAPDH Protein Expressed on Probiotic Bacillus subtilis Spores Protects Sheep from Haemonchus contortus Infection by Inducing both Humoral and Cell-Mediated Responses

mSystems ◽

10.1128/msystems.00239-20 ◽

2020 ◽

Vol 5 (3) ◽

Cited By ~ 1

Author(s):

Yi Yang ◽

Guiheng Zhang ◽

Jie Wu ◽

Xueqiu Chen ◽

Danni Tong ◽

...

Keyword(s):

Bacillus Subtilis ◽

Weight Gain ◽

16S Rrna ◽

Immune Responses ◽

Haemonchus Contortus ◽

Infected Sheep ◽

16S Rrna Sequencing ◽

Probiotic Bacteria ◽

Content Type ◽

Rrna Sequencing

ABSTRACT Probiotic Bacillales are effective in controlling pathogens. Live probiotic bacteria improve the composition of the gastrointestinal microbiota, leading to a reduction in pathogen colonization. However, it remains largely unknown how probiotics regulate the host’s immunologic responses and protect the host from parasitic infection. In this study, we addressed whether Bacillales were effective against Haemonchus contortus, a parasitic nematode that infects small ruminants worldwide. Using 16S rRNA sequencing, we found that Bacillales were largely depleted in the abomasal microbiota of sheep infected with H. contortus. We constructed a recombinant Bacillus subtilis named rBSCotB-HcG that express the glyceraldehyde-3-phosphate dehydrogenase of H. contortus (HcGAPDH) on its spore surface using the Bacillus subtilis spore coat protein B (CotB) as a carrier. Mice receiving rBSCotB-HcG orally showed strong Th1-dominated immune responses. More importantly, sheep administered BSCotB-HcG per os showed increasing proliferation of the peripheral blood mononucleates, elevated anti-HcGAPDH IgG in sera, and higher anti-HcGAPDH sIgA in the intestinal mucus than the control sheep. The average weight gain of H. contortus-infected sheep treated with rBSCotB-HcG (Hc+rBSCotB-HcG) was 48.73% greater than that of unvaccinated sheep. Furthermore, these Hc+rBSCotB-HcG sheep had fewer eggs per gram of feces by 84.1% and adult worms by 71.5%. They also demonstrated greatly lessened abomasal damage by H. contortus with an abundance of probiotic species in the abomasal microbiota. Collectively, our data unequivocally demonstrate the protective roles of CotB-HcGAPDH-expressing B. subtilis spores in against H. contortus infection and showed great potential of using probiotic-based strategy in controlling parasitic nematodes of socioeconomic importance in general. IMPORTANCE Initial analyses of the abomasal microbiota of sheep using 16S rRNA sequencing suggested that probiotic bacteria played a protective role in against H. contortus infection. A recombinant Bacillus subtilis expressing a fusion protein CotB-HcGAPDH on its spore’s surface induced strong Th1 immune response in a murine model. The same probiotic recombinant, upon only one oral application, protected sheep against H. contortus infection by reducing egg shedding and decreasing adult worm loads of the parasite and increasing body weight gain of infected sheep. Both Th1 and Th2 immune responses were evident in these immunized sheep.

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Multilocus Sequence Analysis of Clinical “Candidatus Neoehrlichia mikurensis” Strains from Europe

Journal of Clinical Microbiology ◽

10.1128/jcm.00880-15 ◽

2015 ◽

Vol 53 (10) ◽

pp. 3126-3132 ◽

Cited By ~ 11

Author(s):

Anna Grankvist ◽

Edward R. B. Moore ◽

Liselott Svensson Stadler ◽

Sona Pekova ◽

Christian Bogdan ◽

...

Keyword(s):

Czech Republic ◽

Sequence Analysis ◽

16S Rrna ◽

The Other ◽

16S Rrna Genes ◽

Rrna Genes ◽

Multilocus Sequence Analysis ◽

The Czech Republic ◽

Content Type ◽

Neoehrlichia Mikurensis

“CandidatusNeoehrlichia mikurensis” is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of “Ca. Neoehrlichia” has been studied only by comparing 16S rRNA genes andgroELoperon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical “Ca. Neoehrlichia” strains in Europe and their relatedness to other species within theAnaplasmataceaefamily. Six genes were selected:ftsZ,clpB,gatB,lipA,groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed “Ca. Neoehrlichia” infection from Sweden (n= 9), the Czech Republic (n= 2), and Germany (n= 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identicallipAsequences, while thelipAsequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in theclpBsequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA,ftsZ,gatB, andgroEL. According to the MLSA, among theAnaplasmataceae, “Ca. Neoehrlichia” is most closely related toEhrlichia ruminantium, less so toAnaplasma phagocytophilum, and least toWolbachiaendosymbionts. To conclude, three sequence types of infectious “Ca. Neoehrlichia” were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

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Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

Journal of Clinical Microbiology ◽

10.1128/jcm.01969-15 ◽

2015 ◽

Vol 53 (12) ◽

pp. 3773-3778 ◽

Cited By ~ 7

Author(s):

Camilla de Gier ◽

Lea-Ann S. Kirkham ◽

Niels Nørskov-Lauritsen

Keyword(s):

Sequence Analysis ◽

16S Rrna ◽

Haemophilus Influenzae ◽

Phylogenetic Group ◽

16S Rrna Genes ◽

Rrna Genes ◽

Multilocus Sequence Analysis ◽

Content Type ◽

Group Ii ◽

Complete Deletion

Nonhemolytic variants ofHaemophilus haemolyticusare difficult to differentiate fromHaemophilus influenzaedespite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the “fuzzy species” strains were identified asH. influenzaethat had undergone complete deletion of the fucose operon. Such strains, which are untypeable by theH. influenzaemultilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch ofH. influenzaeMLSA phylogenetic group II. We also found evidence of interspecies recombination betweenH. influenzaeandH. haemolyticuswithin the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification ofH. influenzaeis important for disease surveillance and treatment.

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Optimization of Multilocus Sequence Analysis for Identification of Species in the Genus Vibrio

Applied and Environmental Microbiology ◽

10.1128/aem.01206-14 ◽

2014 ◽

Vol 80 (17) ◽

pp. 5359-5365 ◽

Cited By ~ 20

Author(s):

Michael W. Gabriel ◽

George Y. Matsui ◽

Robert Friedman ◽

Charles R. Lovell

Keyword(s):

Sequence Analysis ◽

16S Rrna ◽

16S Rrna Gene ◽

Gene Sequence ◽

Multilocus Sequence Analysis ◽

Bootstrap Support ◽

Rrna Gene ◽

Gene Sequences ◽

Accurate Identification ◽

Content Type

ABSTRACTMultilocus sequence analysis (MLSA) is an important method for identification of taxa that are not well differentiated by 16S rRNA gene sequences alone. In this procedure, concatenated sequences of selected genes are constructed and then analyzed. The effects that the number and the order of genes used in MLSA have on reconstruction of phylogenetic relationships were examined. TherecA,rpoA,gapA, 16S rRNA gene,gyrB, andftsZsequences from 56 species of the genusVibriowere used to construct molecular phylogenies, and these were evaluated individually and using various gene combinations. Phylogenies from two-gene sequences employingrecAandrpoAin both possible gene orders were different. The addition of thegapAgene sequence, producing all six possible concatenated sequences, reduced the differences in phylogenies to degrees of statistical (bootstrap) support for some nodes. The overall statistical support for the phylogenetic tree, assayed on the basis of a reliability score (calculated from the number of nodes having bootstrap values of ≥80 divided by the total number of nodes) increased with increasing numbers of genes used, up to a maximum of four. No further improvement was observed from addition of the fifth gene sequence (ftsZ), and addition of the sixth gene (gyrB) resulted in lower proportions of strongly supported nodes. Reductions in the numbers of strongly supported nodes were also observed when maximum parsimony was employed for tree construction. Use of a small number of gene sequences in MLSA resulted in accurate identification ofVibriospecies.

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Haemophilus parainfluenzaeInfective Endocarditis Diagnosed by Direct 16S rRNA Sequencing of Vegetation

Laboratory Medicine Online ◽

10.3343/lmo.2012.2.2.111 ◽

2012 ◽

Vol 2 (2) ◽

pp. 111

Author(s):

Sung-Hee Oh ◽

Min-Chul Cho ◽

Jae-Wook Kim ◽

Dongheui An ◽

Mun-Hui Jeong ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Full-length versus short amplicon 16S rRNA sequencing: benefits, drawbacks and risks

10.26226/morressier.5f3288739a6b12016a74ff57 ◽

2020 ◽

Author(s):

Isabel Abellan-Schneyder ◽

Andrea Janina Bayer ◽

Sandra Reitmeier ◽

Klaus Neuhaus

Keyword(s):

16S Rrna ◽

Full Length ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Full-length versus short amplicon 16S rRNA sequencing: benefits, drawbacks and risks

10.26226/morressier.5f3392ca9d1718ca4c8b2f06 ◽

2020 ◽

Author(s):

Andrea Janina Bayer ◽

Sandra Reitmeier ◽

Klaus Neuhaus ◽

Isabel Abellan-Schneyder

Keyword(s):

16S Rrna ◽

Full Length ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Phenotypic and phylogenetic characterization of Lactobacillus species isolated from traditional Lighvan cheese

Food Production, Processing and Nutrition ◽

10.1186/s43014-021-00065-x ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Haleh Forouhandeh ◽

Sepideh Zununi Vahed ◽

Hossein Ahangari ◽

Vahideh Tarhriz ◽

Mohammad Saeid Hejazi

Keyword(s):

16S Rrna ◽

Random Amplified Polymorphic Dna ◽

Bacterial Species ◽

16S Rrna Sequencing ◽

Lactobacillus Species ◽

Culture Methods ◽

Phylogenetic Characterization ◽

Rrna Sequencing ◽

Specific Culture ◽

Lighvan Cheese

Abstract Lighvan cheese (Lighvan panir) is among the most famous traditional cheese in Iran for its desired aroma and flavor. Undoubtedly, the lactic acid bacteria especially the genus Lactobacillus are the critical factors in developing the aroma, flavor, and texture in Lighvan cheese. In this study, the Lactobacillus population of the main Lighvan cheese was investigated. The Lactobacillus of the main Lighvan cheese was isolated using specific culture methods according to previously published Guidelines. Then, the phylogenetic features were investigated and the phenotypic characteristics were examined using specific culture methods. Twenty-eight Gram-positive bacterial species were identified belonged to the genus Lactobacillus. According to the same sequences as each other, three groups (A, B, and C) of isolates were categorized with a high degree of similarity to L. fermentum (100%) and L. casei group (L. casei, L. paracasei, and L. rhamnosus) (99.0 to 100%). Random amplified polymorphic DNA (RAPD) fingerprint analysis manifested the presence of three clusters that were dominant in traditional Lighvan cheese. Cluster І was divided into 4 sub-clusters. By the result of carbohydrate fermentation pattern and 16S rRNA sequencing, isolates were identified as L. rhamnosus. The isolates in clusters II and III represented L. paracasei and L. fermentum, respectively as they were identified by 16S rRNA sequencing and fermented carbohydrate patterns. Our result indicated that the specific aroma and flavor of traditional Lighvan cheese can be related to its Lactobacillus population including L. fermentum, L. casei, L. paracasei, and L. rhamnosus. Graphical abstract

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Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed

Environmental Microbiome ◽

10.1186/s40793-021-00382-1 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Leena Malayil ◽

Suhana Chattopadhyay ◽

Emmanuel F. Mongodin ◽

Amy R. Sapkota

Keyword(s):

16S Rrna ◽

Irrigation Water ◽

Brackish Water ◽

Water Sources ◽

16S Rrna Sequencing ◽

Vbnc State ◽

Recycled Water ◽

Dna Labeling ◽

Rrna Sequencing ◽

Brdu Labeling

AbstractNontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, including Vibrio spp., that could be present in a viable-but-nonculturable (VBNC) state, stymieing current culture-based detection methods. To overcome this challenge, we coupled 5-bromo-2′-deoxyuridine (BrdU) labeling, enrichment techniques, and 16S rRNA sequencing to identify metabolically-active Vibrio spp. in nontraditional irrigation water (recycled water, pond water, non-tidal freshwater, and tidal brackish water). Our coupled BrdU-labeling and sequencing approach revealed the presence of metabolically-active Vibrio spp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence of V. cholerae, V. vulnificus, and V. parahaemolyticus using both methods, while V. aesturianus and V. shilonii were detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach including P. shigelloides, B. cereus and E. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted in Vibrio spp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-active Vibrio spp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.

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