Resource Development and Investigation of Novel Species from Unidentified Pathogens in NCCP using MALDI-TOF MS and 16S rRNA Gene Analysis

2016 ◽  
Vol 46 (4) ◽  
pp. 201
Author(s):  
Won Seon Yu ◽  
Kyeong Min Lee ◽  
Kyu Jam Hwang
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Novel Species ◽  
Resource Development ◽  
Gene Analysis ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

Whole-genome sequencing evaluation of MALDI-TOF MS as a species identification tool for Streptococcus suis

2021 ◽  
Author(s):  
Anna Werinder ◽  
Anna Aspán ◽  
Robert Söderlund ◽  
Annette Backhans ◽  
Marie Sjölund ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Species Identification ◽  
Gene Analysis ◽  
Rrna Gene ◽  
Whole Genome ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
The 16S Rrna Gene ◽  
Tof Ms

Streptococcus suis is an important bacterial pathogen in pigs that may also cause zoonotic disease in humans. The aim of the study was to evaluate MALDI-TOF MS identification of S. suis case isolates from diseased pigs and tonsil isolates from healthy pigs and wild boar, using sequence analysis methods. Isolates (n=348) which had been classified as S. suis by MALDI-TOF MS were whole-genome sequenced and investigated using analysis of i) the 16S rRNA gene, ii) the recN gene, and iii) whole-genome average nucleotide identity (ANI). Analysis of the 16S rRNA gene indicated that 82.8% (288 out of 348) of the isolates were S. suis , while recN -gene analysis indicated that 75.6% (263 out of 348) were S. suis . ANI analysis classified 44.3% (154 out of 348) as S. suis . In total, 44% (153 out of 348) of the investigated isolates were classified as S. suis by all of the species identification methods employed. The mean MALDI-TOF MS score was significantly higher for the S. suis case isolates compared to the tonsil isolates, however, the difference is of limited practical use. The results show that species confirmation beyond MALDI-TOF MS is needed for S. suis isolates. Since the resolution of 16S rRNA gene analysis is too low for Streptococcus spp., ANI analysis with a slightly lowered cutoff of 94% may be used instead of, or in addition to, recN -gene analysis. Supplementation of the MALDI-TOF MS reference library with mass spectra from S. orisratti , S. parasuis , S. ruminantium , and additional S. suis serotypes, should be considered in order to produce more accurate classifications.

scholarly journals Acinetobacter gandensis sp. nov. isolated from horse and cattle

2014 ◽  
Vol 64 (Pt_12) ◽  
pp. 4007-4015 ◽  
Author(s):  
Annemieke Smet ◽  
Piet Cools ◽  
Lenka Krizova ◽  
Martina Maixnerova ◽  
Ondrej Sedo ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Rrna Gene ◽  
Content Type ◽  
Dna Relatedness ◽  
Maldi Tof Ms ◽  
Link Type ◽  
Maldi Tof ◽  
Tof Ms

We previously reported the presence of an OXA-23 carbapenemase in an undescribed species of the genus Acinetobacter isolated from horse dung at the Faculty of Veterinary Medicine, Ghent University, Belgium. Here we include six strains to corroborate the delineation of this taxon by phenotypic characterization, DNA–DNA hybridization, 16S rRNA gene and rpoB sequence analysis, % G+C determination, MALDI-TOF MS and fatty acid analysis. The nearly complete 16S rRNA gene sequence of strain UG 60467T showed the highest similarities with those of the type strains of Acinetobacter bouvetii (98.4 %), Acinetobacter haemolyticus (97.7 %), and Acinetobacter schindleri (97.2 %). The partial rpoB sequence of strain UG 60467T showed the highest similarities with ‘Acinetobacter bohemicus’ ANC 3994 (88.6 %), A. bouvetii NIPH 2281 (88.6 %) and A. schindleri CIP 107287T (87.3 %). Whole-cell MALDI-TOF MS analyses supported the distinctness of the group at the protein level. The predominant fatty acids of strain UG 60467T were C12 : 0 3-OH, C12 : 0, C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). Strains UG 60467T and UG 60716 showed a DNA–DNA relatedness of 84 % with each other and a DNA–DNA relatedness with A. schindleri LMG 19576T of 17 % and 20 %, respectively. The DNA G+C content of strain UG 60467T was 39.6 mol%. The name Acinetobacter gandensis sp. nov. is proposed for the novel taxon. The type strain is UG 60467T ( = ANC 4275T = LMG 27960T = DSM 28097T).

scholarly journals Implementation of a Non-Invasive Bioprospecting Protocol for Isolation of Lactobacillus from Feces of Hens Under Foraging Conditions

2018 ◽  
Vol 14 (28) ◽  
pp. 93-111
Author(s):  
Simón Robledo-Cardona ◽  
Sabina Ramírez-Hincapié ◽  
Javier Correa-Álvarez
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Lactobacillus Species ◽  
Rrna Gene ◽  
Isolation And Identification ◽  
Maldi Tof Ms ◽  
Non Invasive ◽  
Maldi Tof ◽  
Isolation And Characterization ◽  
Tof Ms

In animal production, probiotics seek to replace the use of antibiotics, while diminishing mortality and morbidity rates to raise productivity. Probiotics constitute a natural alternative that, in contrast with antibiotics, neither produces pathogen resistance, nor leaves chemical residues in the final product. Several bacteria, including some belonging to the genus Lactobacillus have been described as probiotics with high potential. A non-invasive bioprospecting protocol aimed for the isolation and characterization of lactobacilli from chicken feces was established. Fecal samples were collected from the ground. These were diluted and cultured in LAB selective medium. Colonies were identified by three methods: Gram stain, MALDI-TOF MS and sequencing of 16S rRNA gene. An initial probiotic potential of lactobacilli isolates was determined via antagonism tests using five enteropathogen reference strains: Staphylococcus aureus, Enterococcus faecium, Candida albicans, Pseudomonas spp. and Salmonella spp. 24 isolates belonging to four Lactobacillus species were identified by MALDITOF MS. BLAST of 16S rRNA gene of eight randomly selected isolates, confirmed MALDI-TOF MS identification. Five of these eight isolates inhibited the growth of at least one of the pathogenic strains used, three isolates of Lactobacillus plantarum and two of Lactobacillus salivarius. Our protocol achieved 21 lactobacilli per 100 isolates performance, greatly surpassing the normal percentage of lactobacilli in chicken gut microbiome, that so, its implementation would facilitate the isolation and identification of new probiotic strains from feces.

scholarly journals The clinical utility of two high-throughput 16S rRNA gene sequencing workflows for taxonomic assignment of unidentifiable bacterial pathogens in MALDI-TOF MS

2021 ◽  
Author(s):  
Hiu-Yin Lao ◽  
Timothy Ting Leung Ng ◽  
Ryan Yik Lam Wong ◽  
Celia Sze Ting Wong ◽  
Chloe Toi Mei Chan ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Reference Standard ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming and low-throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests, which are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by respective built-in analysis programs (MiSeq Reporter Software and Epi2me) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a comparatively higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of optimized Nanopore 16S was 96.36%, which was identical to Sanger 16S and was better than Illumina 16S (71.52%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78h and 8.25h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

scholarly journals ‘Candidatus Burkholderia calva’ and ‘Candidatus Burkholderia nigropunctata’ as leaf gall endosymbionts of African Psychotria

2004 ◽  
Vol 54 (6) ◽  
pp. 2237-2239 ◽  
Author(s):  
Sandra Van Oevelen ◽  
Rupert De Wachter ◽  
Peter Vandamme ◽  
Elmar Robbrecht ◽  
Els Prinsen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Novel Species ◽  
Gene Analysis ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Culture Collections ◽  
Type Strains

Phylogenetic 16S rRNA gene analysis was used to assign the bacterial leaf-nodulating endosymbionts of two tropical African Psychotria species to the genus Burkholderia. The microsymbionts of the different Psychotria hosts were recognized as distinct and novel species of Burkholderia on the basis of relatively low intersequence similarities and sufficiently large evolutionary distances when compared with each other and their closest validly named neighbours. The obligate endosymbiotic nature of the bacteria prevented their in vitro cultivation and the deposition of type strains to culture collections. Therefore, the provisional status Candidatus is assigned to the bacterial partners of Psychotria calva and Psychotria nigropunctata, with the proposal of the names ‘Candidatus Burkholderia calva’ and ‘Candidatus Burkholderia nigropunctata’, respectively.

Matrix-Assisted Laser Desorption and Ionization–Time-of-Flight Mass Spectrometry, 16S rRNA Gene Sequencing, and API 32E for Identification of Cronobacter spp.: A Comparative Study

2011 ◽  
Vol 74 (12) ◽  
pp. 2182-2187 ◽  
Author(s):  
SHA ZHU ◽  
STEFAN RATERING ◽  
SYLVIA SCHNELL ◽  
RON WACKER
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Twenty-two isolates of the family Enterobacteriaceae, with focus on Cronobacter isolated from infant formula and the environment of milk powder plants, were comparatively identified using API 32E (bioMérieux, Marcy l'Etoile, France), 16S rRNA gene sequencing (Accugenix, Newark, USA), and matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry (MALDI-TOF MS; Mabritec, Riehen, Switzerland and AnagnosTec, Potsdam, Germany). With API 32E, 22% of the isolates were assigned to species, 64% were assigned to a genus, and 14% could not be discriminated at any taxonomic level. Both 16S rRNA gene sequencing and MALDI-TOF MS assigned 100% of the isolates to species, but the identifications based on MALDI-TOF MS results were more discriminating and unequivocal. Our data indicate that MALDI-TOF MS provides the most rapid and unambiguous identification of Cronobacter and closely related Enterobacteriaceae isolates.

scholarly journals The clinical utility of two high-throughput 16S rRNA gene sequencing workflows for taxonomic assignment of unidentifiable bacterial pathogens in MALDI-TOF MS

2021 ◽  
Author(s):  
Hiu-Yin Lao ◽  
Timothy Ting-Leung Ng ◽  
Ryan Yik-Lam Wong ◽  
Celia Sze-Ting Wong ◽  
Lam-Kwong Lee ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Reference Standard ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming and low-throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests, which are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by respective built-in programs (MiSeq Reporter Software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to Sanger 16S and was better than Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78h and 8.25h respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

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