scholarly journals Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

10.3791/53176 ◽  
2016 ◽  
Author(s):  
Percy Schröttner ◽  
Florian Gunzer ◽  
Jana Schüppel ◽  
Wolfram W. Rudolph
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Pathogens ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Tof Ms

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 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.

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.

Discrimination of major and minor streptococci incriminated in bovine mastitis by MALDI-TOF MS fingerprinting and 16S rRNA gene sequencing

2020 ◽  
Vol 132 ◽  
pp. 426-438 ◽  
Author(s):  
Mohamed E.A. Alnakip ◽  
Nasreddin R. Rhouma ◽  
Eman N. Abd-Elfatah ◽  
Marcos Quintela-Baluja ◽  
Karola Böhme ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bovine Mastitis ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Tof Ms

scholarly journals Research on the establishment and application of protein fingerprint spectrum database of Burkholderia pseudomallei in Hainan Province China

2020 ◽  
Author(s):  
Xu-Ming Wang ◽  
Ling-Li Liu ◽  
Hua Wu ◽  
Mei-Hui Huang
Keyword(s):  
16S Rrna ◽  
Burkholderia Pseudomallei ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Hainan Province ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Abstract Background: The aim of this study was to establish a SuperSpectrum of Burkholderia pseudomallei(B. pseudomallei) in Hainan and evaluate its application value in the rapid identification of clinical isolates of B. pseudomallei.Methods: Using a collection of 167 isolates of B. pseudomallei from June 2010 to May 2019 in different regions of Hainan province, multilocus sequence typing (MLST) was performed, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for spectrum acquisition. A SuperSpectrum was created based on the selection of 80 representative average spectra. In a second step, we validated the SuperSpectra with 137 strains of B. pseudomallei, 8 strains of Burkholderia thailandensis(B. thailandensis), 2 strains of Burkholderia cepacia(B. cepacia), 1 strain of Burkholderia cenocepacia(B. cenocepacia) and 1 strain of Burkholderia multivorans(B. multivorans), as well as 1 strain of Burkholderia gladioli(B. gladioli) identified by MLST typing and 16S rRNA gene sequencing.Results: The results showed that there was 100% agreement between the validation strains analyzed by MALDI-TOF MS and those evaluated by MLST typing and 16S rRNA gene-sequencing analysis methods. Protein fingerprints spectra showed that specific peaks occurred in B. pseudomallei from the Hainan region. The result of clustering typing indicated that B. pseudomallei and its closely related species could be well classified by MALDI-TOF MS at the protein level.Conclusions: MALDI-TOF MS is a promising, rapid, and economical method to monitor the outbreaks and spread of B. pseudomallei isolates.The establishment of an accurate and objective SuperSpectrum database can provide a new platform for the clinical rapid diagnosis of melioidosis.

scholarly journals Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

2012 ◽  
Vol 12 (1) ◽  
pp. 185 ◽  
Author(s):  
Anna Rettinger ◽  
Inke Krupka ◽  
Karola Grünwald ◽  
Viktor Dyachenko ◽  
Volker Fingerle ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Strain Identification ◽  
Rrna Gene ◽  
Maldi Tof Ms ◽  
Leptospira Spp ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Comparison of VITEK 2, MALDI-TOF MS, 16S rRNA gene sequencing, and whole-genome sequencing for identification of Roseomonas mucosa

2019 ◽  
Vol 134 ◽  
pp. 103576 ◽  
Author(s):  
Wolfram W. Rudolph ◽  
Florian Gunzer ◽  
Melanie Trauth ◽  
Boyke Bunk ◽  
Richard Bigge ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Whole Genome ◽  
Vitek 2 ◽  
Rrna Gene Sequencing ◽  
Roseomonas Mucosa ◽  
Tof Ms

scholarly journals Relative Prevalence and Antimicrobial Susceptibility of Clinical Isolates of Elizabethkingia Species Based on 16S rRNA Gene Sequencing

2016 ◽  
Vol 55 (1) ◽  
pp. 274-280 ◽  
Author(s):  
Mi-Soon Han ◽  
Hyunsoo Kim ◽  
Yangsoon Lee ◽  
Myungsook Kim ◽  
Nam Su Ku ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Antimicrobial Susceptibility ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Content Type ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Relative Prevalence

ABSTRACT Some of the previously reported clinical isolates of Elizabethkingia meningoseptica may be later named species of Elizabethkingia . We determined the accuracy of species identification (with two matrix-assisted laser desorption ionization–time of flight mass spectrometry [MALDI-TOF MS] systems and the Vitek 2 GN card), relative prevalence of three Elizabethkingia spp. in clinical specimens, and antimicrobial susceptibility of the species identified by 16S rRNA gene sequencing. Specimens for culture were collected from patients in a university hospital in Seoul, South Korea, between 2009 and 2015. All 3 Elizabethkingia spp. were detected in patients; among the 86 isolates identified by 16S rRNA gene sequencing, 17 (19.8%) were E. meningoseptica , 18 (20.9%) were Elizabethkingia miricola , and 51 (59.3%) were Elizabethkingia anophelis . Only the MALDI-TOF Vitek MS system with an amended database correctly identified all of the isolates. The majority (76.7%) of the isolates were from the lower respiratory tract, and 8 (9.3%) were from blood. Over 90% of E. meningoseptica and E. anophelis isolates were susceptible to piperacillin-tazobactam and rifampin. In contrast, all E. miricola isolates were susceptible to fluoroquinolones except ciprofloxacin. Further studies are urgently needed to determine the optimal antimicrobial agents for the treatment of infections due to each individual Elizabethkingia species.

scholarly journals Cultivable Microbial Diversity in Speleothems Using MALDI-TOF Spectrometry and DNA Sequencing from Krem Soitan, Krem Lawbah, Krem Mawpun, Khasi Hills, Meghalaya, India

2021 ◽  
Author(s):  
Mudgil Devender ◽  
Dhiraj Paul ◽  
Sushmitha Baskar ◽  
Ramanathan Baskar ◽  
Yogesh S Shouche
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Diversity ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Culture Dependent

Abstract This study reports on the culturable microbial communities in caves from the Indian sub-continent. A high bacterial diversity and a greater bacterial taxonomic diversity is reported using MALDI-TOF spectrometry and 16S rRNA gene sequencing. This approach helped to detect a number bacterial strains from the Indian caves. The microbial diversity in the Indian caves is inadequately characterized. The study aims to expand the current understanding of bacterial diversity in the speleothems from Krem Soitan, Krem Lawbah, Krem Mawpun in Khasi Hills, Meghalaya, India. High microbial enumerations were observed on dilute nutrient agar (5.3 × 103 to 8.8 × 105) followed by M9 minimal medium (4 × 104 to 1.7 × 105) and R2A medium (1.0 × 104 to 5.7 × 105). A total of 826 bacterial isolates were selected and preserved for the study. 295 bacterial isolates were identified using MALDI-TOF spectrometry and the isolates which showed no reliable peaks were further identified by 16S rRNA gene sequencing. 91% of the total bacterial diversity was dominated by Proteobacteria and Actinobacteria. The other important phyla detected include the Firmicutes (7.45%), Deinococcus-Thermus (0.33%) and Bacteroidetes (0.67%). At the genus level, Pseudomonas (55%) and Arthrobacter (23%) were ubiquitous followed by Acinetobacter, Bacillus, Brevundimonas, Deinococcus, Flavobacterium, Paenibacillus, Pseudarthrobacter. Multivariate statistical analysis indicated that the bacterial genera formed separate clusters depending on the geochemical constituents in the spring waters suitable for their growth and metabolism. A culture-dependent approach was employed for elucidating the community structure colonizing the speleothems and wall deposits in the caves using MALDI-TOF and 16S rRNA gene sequencing. To the best of our knowledge, there are no previous geomicrobiological investigations in these caves and this study is a pioneering culture dependent study of the microbial community with many cultured isolates.

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