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.

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.

Study of gut bacterial diversity of Bombyx mandarina and Bombyx mori through 16S rRNA gene sequencing

2019 ◽  
Vol 22 (2) ◽  
pp. 522-530 ◽  
Author(s):  
Dhiraj Kumar ◽  
Zhenli Sun ◽  
Guangli Cao ◽  
Renyu Xue ◽  
Xiaolong Hu ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bombyx Mori ◽  
Bacterial Diversity ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Bombyx Mandarina ◽  
Rrna Gene Sequencing

scholarly journals Bacterial Communities in Serpa Cheese by Culture Dependent Techniques, 16S rRNA Gene Sequencing and High-throughput Sequencing Analysis

2018 ◽  
Vol 83 (5) ◽  
pp. 1333-1341 ◽  
Author(s):  
Maria Teresa P. Gonçalves ◽  
María José Benito ◽  
María de Guía Córdoba ◽  
Conceição Egas ◽  
Almudena V. Merchán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Communities ◽  
High Throughput Sequencing ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Rrna Gene Sequencing ◽  
Culture Dependent

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 Differentiation between Pseudomonas and Stenotrophomonas Species Isolated from Fish Using Molecular and MALDI-TOF Method

2016 ◽  
Vol 66 (3) ◽  
pp. 304-316 ◽  
Author(s):  
Ksenija Aksentijević ◽  
Jelena Ašanin ◽  
Dušan Milivojević ◽  
Svetlana Čolović ◽  
Ana Butorac ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Intrinsic Resistance ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Pcr Test ◽  
Vitek Ms

Abstract For the purpose of precise antibiotic susceptibility testing it is necessary to clearly distinguish Pseudomonas and Stenotrophomonas genera, considering acquired resistance of Pseudomonas species, as well as the intrinsic resistance of Stenotrophomonas species. This is why in the identification of the 51 isolates originated from fish, the following methods were used: standard PCR, 16S rRNA gene sequencing, and MALDI-TOF. The results of the standard PCR test, 16S rRNA gene sequencing and MALDI-TOF analysis confirmed 35 strains to belong to the Pseudomonas genus. Standard PCR test and VITEK MS device confirmed that 10 strains belong to Stenotrophomonas maltophilia species. Three strains were positive in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Three strains were negative in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Although modern test methods that have very high specificity (PCR, 16S rRNA gene sequencing, MALDI TOF) were used in this study, precise differentiation between Pseudomonas and Stenotrophomonas species for 6 isolates could not be reached using the above mentioned methods.

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.

scholarly journals Molecular Detection and Identification of Bacteria in Urine Samples of Asymptomatic and Symptomatic Pregnant Women by 16S rRNA Gene Sequencing

2020 ◽  
Vol 15 (3) ◽  
Author(s):  
Pragya Lakshmi ◽  
Alok Bharadwaj ◽  
Ranjan Kumar Srivastava
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Tree ◽  
Pregnant Women ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Blast Analysis ◽  
Rrna Gene Sequencing

Objectives: The purpose of this study was to identify bacteria in urine samples of pregnant women of asymptomatic and symptomatic women by 16S rRNA gene sequencing. This study aims to identify different strains of microbes causing urinary tract infection (UTI). Methods: In the semi-quantitative culture technique, bacterial isolates such as Escherichia coli, Klebsiella, Pseudomonas, Staphylococcus, Coagulase-negative Staphylococcus, and Proteus were subjected to 16S rRNA gene sequencing followed by BLAST analysis and phylogenetic tree formation. The 16S rRNA gene sequencing was carried out to identify the specific strains of bacteria causing UTI. Results: According to the BLAST analysis, sample 1 revealed a 100% similarity to E. coli strain U5/41. Likewise, samples 2, 3, 4, 5 and 6 exhibited a 100% similarity to Klebsiella aerogenes strain F26, Pseudomonas entomophila strain 2014, Staphylococcus aureus strain NCTC13616, Staphylococcus saprophyticus strain FDAARGOS_355, Proteus mirabilis strain NCTC 11938, respectively. Conclusions: Six bacterial isolates were analyzed by 16S RNA gene sequencing followed by the construction of a phylogenetic tree construction up to the species level. This method was a valuable tool for cost-effective and accurate diagnosis of an array of uropathogens in both asymptomatic and symptomatic pregnant women.

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